EA041958B1 - ANTIBODIES AGAINST CD166, ACTIVATED ANTIBODIES AGAINST CD166 AND METHODS OF THEIR APPLICATION - Google Patents

Description

Related Applications

This application claims priority to U.S. Provisional Applications No. 62/156835, filed May 2015; and 62/220805, filed September 18, 2015, the contents of each of which are incorporated herein by reference.

The field of technology to which the invention belongs

The invention relates primarily to CD166-binding antibodies, activated antibodies specifically binding to CD166, and methods for making and using these anti-CD166 antibodies and activated anti-CD166 antibodies in a variety of therapeutic, diagnostic and prophylactic indications.

State of the art for the invention

Antibody-based therapies have been proven to be effective in the treatment of several diseases, but in some cases toxicity due to broad target expression limits their therapeutic efficacy. In addition, antibody-based drugs have other limitations such as rapid clearance from the circulation after administration.

In the field of small molecule drugs, methods have been developed to provide prodrugs of the active chemical compound. Such prodrugs are administered in a relatively inactive (or significantly less active) form. Following administration, the prodrug is metabolized in vivo to the active compound. Such prodrug methods can provide increased drug selectivity for its intended target and to reduce adverse effects.

Accordingly, there is an ongoing need in the field of antibody-based drugs for antibodies that mimic the desired characteristics of a small molecular weight prodrug.

The essence of the invention

The description refers to antibodies or antigen-binding fragments thereof specifically binding to CD166, also known as the 166 differentiation cluster, activated leukocyte cell adhesion molecule (ALCAM), and/or MEMD. The use of the term CD166 is intended to encompass all variants thereof, such as, but not limited to, CD-166 and/or CD 166, and all variants are used interchangeably herein.

In some embodiments, the antibody includes an antibody, or an antigen-binding fragment thereof, that specifically binds CD166. In some embodiments, the antibody or antigen-binding fragment thereof that binds CD166 is a monoclonal antibody, domain antibody, single chain, Fab fragment, F(ab')2 fragment, scFv, scAb, dAb, heavy chain single domain antibody, or antibody from a single light chain domain. In some embodiments, the CD166-binding antibody or antigen-binding fragment thereof is a mouse, other rodent, chimeric, humanized, or fully human monoclonal antibody.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 121, and 122. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence, comprising SEQ ID NO: 121 or 122. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence comprising SEQ ID NO: 121. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence, containing SEQ ID NO: 122.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 120 and 123-126. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 123-126. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region amino acid sequence comprising SEQ ID NO: 123.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 121, and 122 and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs. : 120 and 123-126.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 121 or 122 and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 123 -126. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 121 and a light chain variable region amino acid sequence selected from the group consisting of

SEQ ID NO: 123. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 122 and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 123.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to an amino acid sequence selected from the group consisting of of SEQ ID NOs: 119, 121, and 122. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence comprising SEQ ID NO: 121 or 122. In some embodiments, the antibody or antigen-binding fragment thereof contains the amino acid sequence of the heavy chain variable region at least 90, 91, 92, 93, 94, 95, 96 , 97, 98, or 99% identical to the amino acid sequence containing SEQ ID NO: 122. In some embodiments, the antibody or its antigen-binding fraction The fragment contains the amino acid sequence of the heavy chain variable region at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence containing SEQ ID NO: 121.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of from SEQ ID NOs: 120 and 123-126. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of from SEQ ID NO: 123-126. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 123.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to an amino acid sequence selected from the group consisting of of SEQ ID NOs: 119, 121, and 122, and the amino acid sequence of the light chain variable region is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence selected from the group , consisting of SEQ ID NO: 120 and 123-126.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to an amino acid sequence selected from the group consisting of of SEQ ID NO: 121 or 122, and the light chain variable region amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of from SEQ ID NO: 123-126. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence selected from SEQ ID NO. : 122 and a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 123. In some embodiments, the antibody or its antigen-binding fragment contains the amino acid sequence of the variable region of the heavy chain, at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence selected from SEQ ID NO: 121, and the amino acid sequence light chain variable region at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 123.

In some embodiments, an antibody or antigen-binding fragment thereof comprises a combination of a heavy chain variable region complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1), a heavy chain variable region complementarity determining region 2 (VH CDR2, also referred to herein) sequence. document as CDRH2), the complementarity determining sequence of variable heavy chain region 3 (CDR3 VH, also referred to herein as CDRH3), the complementarity determining sequence of variable light chain region 1 (CDR1 VL, also referred to herein as CDRL1), sequence complementarity determining region 2 of the variable light chain (CDR2 VL, also referred to herein as CDRL2) and the sequence of complementarity determining region 3 of the variable light chain (CDR3 VL, also referred to as referred to herein as CDRL3), wherein at least one complementarity-determining region sequence

- 2 041958 (CDR) is selected from the group consisting of a VH CDR1 sequence containing the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

a VH CDR2 sequence containing the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

a VH CDR3 sequence containing the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

a VL CDR1 sequence containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

a VL CDR2 sequence containing the amino acid sequence QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133); and a VL CDR3 sequence containing the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

In some embodiments, an antibody or antigen-binding fragment thereof comprises a combination of a heavy chain variable region complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1), a heavy chain variable region complementarity determining region 2 (VH CDR2, also referred to herein) sequence. document as CDRH2), the complementarity determining sequence of variable heavy chain region 3 (CDR3 VH, also referred to herein as CDRH3), the complementarity determining sequence of variable light chain region 1 (CDR1 VL, also referred to herein as CDRL1), sequence complementarity determining region 2 of the variable light chain (CDR2 VL, also referred to herein as CDRL2) and the sequence of complementarity determining region 3 of the variable light chain (CDR3 VL, also referred to as referred to herein as CDRL3), wherein at least one complementarity determining region (CDR) sequence is selected from the group consisting of a VH CDR1 sequence containing the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

a VH CDR2 sequence containing the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

a VH CDR3 sequence containing the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

a VL CDR1 sequence containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130);

a VL CDR2 sequence containing the amino acid sequence QMSNLAS (SEQ ID NO: 132); and a VL CDR3 sequence containing the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

In some embodiments, the antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a CDR1 sequence. a VH containing a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the CDR1 sequence of a VH containing the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

a VH CDR2 sequence comprising a sequence at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VH CDR2 sequence comprising the amino acid sequence NIWWWSEDKH (SEQ ID NO: 128);

a VH CDR3 sequence containing a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VH CDR3 sequence containing the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

a VL CDR1 sequence containing a sequence at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VL CDR1 sequence containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

a VL CDR2 sequence containing a sequence at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VL CDR2 sequence containing the amino acid sequence QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133); and a VL CDR3 sequence containing a sequence at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VL CDR3 sequence containing the amino acid sequence AQNLELPYT (SEQ ID NO: 134) .

In some embodiments, the antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a CDR3 sequence

- 3 041958

VH, VL CDR1 sequences, VL CDR2 sequences, and VL CDR3 sequences, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence containing a sequence of at least 90, 91, 92, 93,

94, 95, 96, 97, 98, 99% or more identical to the VH CDR1 sequence containing the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

a VH CDR2 sequence comprising a sequence at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VH CDR2 sequence comprising the amino acid sequence NIWWWSEDKH (SEQ ID NO: 128);

a VH CDR3 sequence containing a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VH CDR3 sequence containing the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

a VL CDR1 sequence containing a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VL CDR1 sequence containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130);

a VL CDR2 sequence containing a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VL CDR2 sequence containing the amino acid sequence QMSNLAS (SEQ ID NO: 132); and a VL CDR3 sequence containing a sequence at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VL CDR3 sequence containing the amino acid sequence AQNLELPYT (SEQ ID NO: 134) .

In some embodiments, the antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence contains the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127) ;

the VH CDR2 sequence contains the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

the CDR3 VH sequence contains the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

the VL CDR1 sequence contains the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

the VL CDR2 sequence contains the amino acid sequence QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133); and the VL CDR3 sequence contains the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

In some embodiments, the antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence contains the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127) ;

the VH CDR2 sequence contains the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

the CDR3 VH sequence contains the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

the VL CDR1 sequence contains the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130);

the CDR2 VL sequence contains the amino acid sequence QMSNLAS (SEQ ID NO: 132); and the VL CDR3 sequence contains the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

In some embodiments, the antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence contains at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the amino acid sequence of GFSLSTYGMGVG (SEQ ID NO: 127);

the VH CDR2 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of NIWWSEDKH (SEQ ID NO: 128);

the VH CDR3 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

- 4 041958 the CDR1 VL sequence contains the sequence at least 90, 91, 92, 93, 94,

95, 96, 97, 98, 99% or more identical to the amino acid sequence of RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

the VL CDR2 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133); and the VL CDR3 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AQNLELPYT (SEQ ID NO: 134).

In some embodiments, the antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence contains at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the amino acid sequence of GFSLSTYGMGVG (SEQ ID NO: 127);

the VH CDR2 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of NIWWSEDKH (SEQ ID NO: 128);

the VH CDR3 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

the VL CDR1 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130);

the VL CDR2 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of QMSNLAS (SEQ ID NO: 132); and the VL CDR3 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AQNLELPYT (SEQ ID NO: 134).

In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 119, 121, and 122. In some embodiments, implementation the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence containing a nucleic acid sequence encoding a heavy chain amino acid sequence containing an amino acid sequence selected from the group consisting of SEQ ID NO: 121 or 122. In some embodiments, the antibody or antigen-binding fragment thereof is encoded by the sequence nucleic acid containing a nucleic acid sequence encoding a heavy chain amino acid sequence containing an amino acid sequence selected from SEQ ID NO: 122. In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a heavy chain amino acid sequence comprising an amino acid sequence selected from SEQ ID NO: 121.

In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a light chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 120 and 123-126. In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a light chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 123-126. In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 123.

In some embodiments, an antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 121, and 122, and a nucleic acid sequence, a light chain amino acid coding sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 120 and 123-126.

- 5 041958

In some embodiments, an antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 121 and 122 and a nucleic acid sequence encoding an amino acid a light chain sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 123-126.

In some embodiments, an antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 122 and a nucleic acid sequence encoding a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 123. In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a heavy chain amino acid sequence comprising the amino acid sequence from SEQ ID NO: 121 and a nucleic acid sequence encoding a light chain amino acid sequence containing the amino acid sequence of SEQ ID NO: 123.

In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence containing a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence encoding the amino acid a heavy chain sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 121 or 122. In some embodiments, an antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence comprising a nucleic acid sequence of at least 90, 91, 92 , 93, 94, 95, 96, 97, 98, or 99% identical to a nucleic acid sequence encoding a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 121 or 122. In some embodiments, an antibody or its antigen-binding fragment encoded by a nucleic acid sequence containing a nucleic acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to a nucleic acid sequence encoding a heavy chain amino acid sequence containing the amino acid sequence from SEQ ID NO: 122. In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence containing a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence a nucleic acid encoding a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 121.

In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence containing a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence encoding the amino acid a light chain sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 120 and 123-126. In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence containing a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence encoding the amino acid a light chain sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 123-126. In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence containing a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence encoding the amino acid a light chain sequence containing the amino acid sequence of SEQ ID NO: 123.

In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence containing a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence encoding the amino acid a heavy chain sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 121, and 122, and a nucleic acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the nucleic acid sequence encoding the amino acid sequence of the light chain, containing the amino acid sequence selected from the group consisting of SEQ ID NO: 120 and 123-126.

In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence containing a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence.

- 6 041958 a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 121 or 122 and a nucleic acid sequence of at least 90, 91, 92, 93, 94, 95 , 96, 97, 98 or 99% identical to the nucleic acid sequence encoding the amino acid sequence of the light chain containing the amino acid sequence selected from the group consisting of SEQ ID NO: 123-126. In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence containing a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence encoding the amino acid a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 122 and a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence encoding the amino acid a light chain sequence comprising the amino acid sequence of SEQ ID NO: 123. In some embodiments, an antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence comprising a nucleic acid sequence of at least 90, 91, 92, 93, 94, 95, 96 , 97, 98 or 99% identical to the nucleic acid sequence encoding a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 121 and a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence, encoding a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 123.

In some embodiments, the antibody, or antigen-binding fragment thereof, is included in a multispecific antibody, or antigen-binding fragment thereof, wherein at least one arm of the multispecific antibody, or antigen-binding fragment thereof, specifically binds CD166. In some embodiments, the antibody or antigen-binding fragment thereof is included in a bispecific antibody or antigen-binding fragment thereof, wherein at least one arm of the bispecific antibody or antigen-binding fragment thereof specifically binds CD166.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOS: 119, 121, and 122. In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a heavy chain variable region amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 121 or 122. In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, contains an amino acid sequence of the variable region a heavy chain containing the amino acid sequence of SEQ ID NO: 122. In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g. SEQ ID NO: 121.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 120 and 123-126. In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 123-126. In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a light chain variable region amino acid sequence comprising the amino acid sequence of SEQ ID NO: 123.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a heavy chain variable region amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 121, and 122, and a light chain variable region amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 120 and 123-126.

In some embodiments, at least one arm of the multispecific anal

- 7 041958 of the title or its antigen-binding fragment, for example, a bispecific antibody or its antigen-binding fragment, contains the amino acid sequence of the variable region of the heavy chain, containing the amino acid sequence selected from the group consisting of SEQ ID NO: 121 or 122, and the amino acid sequence of the variable region of the light chain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 123-126. In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a heavy chain variable region amino acid sequence comprising the amino acid sequence from SEQ ID NO: 122 and a light chain variable region amino acid sequence, containing the amino acid sequence from SEQ ID NO: 123. The bispecific antibody or antigen-binding fragment thereof contains the amino acid sequence of the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 121, and the amino acid sequence of the light chain variable region containing the amino acid sequence of SEQ ID NO: 123.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a heavy chain variable region amino acid sequence of at least 90, 91, 92, 93, 94, 95, 96, 97%, 98%, or 99% amino acid sequence identity containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 121, and 122. In some embodiments, at least one arm of a multispecific antibody or antigen-binding fragment thereof, e.g., a bispecific antibody or the antigen-binding fragment, contains the amino acid sequence of the variable region of the heavy chain, at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence containing the amino acid sequence selected from the group consisting of SEQ ID NO: 121 or 122. Some In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, contains the amino acid sequence of the heavy chain variable region at least 90, 91, 92, 93, 94, 95, 96, 97 , 98% or 99% identical to the amino acid sequence comprising the amino acid sequence of SEQ ID NO: 122. In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, contains the amino acid sequence of the heavy chain variable region , at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence containing the amino acid sequence of SEQ ID NO: 121.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, contains a light chain variable region amino acid sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence, containing an amino acid sequence selected from the group consisting of SEQ ID NO: 120 and 123-126. In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, contains a light chain variable region amino acid sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence containing an amino acid sequence selected from the group consisting of SEQ ID NO: 123-126. In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, contains a light chain variable region amino acid sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence containing the amino acid sequence of SEQ ID NO: 123.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a heavy chain variable region amino acid sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid sequence identity, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 121, and 122, and an amino acid sequence of a light chain variable region by at least 90, 91, 92, 93, 94 , 95, 96, 97, 98 or 99% identical to the amino acid sequence containing an amino acid sequence selected from the group consisting of SEQ ID NO: 120 and 123-126.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, contains the amino acid sequence of the heavy variable region

- 8 041958 chain, at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence, containing an amino acid sequence selected from the group consisting of SEQ ID NO: 121 or 122 , and the amino acid sequence of the light chain variable region is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to an amino acid sequence containing an amino acid sequence selected from the group consisting of SEQ ID NO: 123-126.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a heavy chain variable region amino acid sequence of at least 90, 91, 92, 93, 94, 95, 96, 97%, 98%, or 99% amino acid sequence identity containing the amino acid sequence of SEQ ID NO: 122 and the amino acid sequence of the light chain variable region by at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence containing the amino acid sequence of SEQ ID NO: 123.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a heavy chain variable region amino acid sequence of at least 90, 91, 92, 93, 94, 95, 96, 97%, 98%, or 99% identical amino acid sequence containing the amino acid sequence of SEQ ID NO: 121 and the amino acid sequence of the light chain variable region by at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence containing the amino acid sequence of SEQ ID NO: 123.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a combination of a heavy chain variable region complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1), a complementarity determining sequence complementarity of heavy chain variable region region 2 (VH CDR2, also referred to herein as CDRH2), heavy chain variable region complementarity determining region 3 sequence (VH CDR3, also referred to herein as CDRH3), light variable region complementarity determining region 1 sequence chain (CDR1 VL, also referred to herein as CDRL1), the light chain variable region complementarity determining region 2 sequence (CDR2 VL, also referred to herein as CDRL2), and a light chain variable region complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3), wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence containing the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

a VH CDR2 sequence containing the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

a VH CDR3 sequence containing the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

a VL CDR1 sequence containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

a VL CDR2 sequence containing the amino acid sequence QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133); and a VL CDR3 sequence containing the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a combination of a heavy chain variable region complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1), a complementarity determining sequence complementarity of heavy chain variable region region 2 (VH CDR2, also referred to herein as CDRH2), heavy chain variable region complementarity determining region 3 sequence (VH CDR3, also referred to herein as CDRH3), light variable region complementarity determining region 1 sequence chain (CDR1 VL, also referred to herein as CDRL1), the light chain variable region complementarity determining region 2 sequence (CDR2 VL, also referred to herein as CDRL2), and a light chain variable region complementarity determining region 3 sequence (CDR3 VL, also referred to herein as CDRL3), wherein at least one CDR sequence is selected from the group consisting of

- 9 041958 sequence CDR1 VH containing the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

a VH CDR2 sequence containing the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

a VH CDR3 sequence containing the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

a VL CDR1 sequence containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130);

a VL CDR2 sequence containing the amino acid sequence QMSNLAS (SEQ ID NO: 132); and a VL CDR3 sequence containing the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where at least one CDR sequence is selected from the group consisting of the sequence

A VH CDR1 comprising a sequence at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VH CDR1 sequence containing the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127) ;

a VH CDR2 sequence comprising a sequence at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the VH CDR2 sequence, the amino acid sequence of NIVWSEDKH (SEQ ID NO: 128) ;

a VH CDR3 sequence comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR3 IDyGnDYAFTY sequence (SEQ ID NO: 129);

a VL CDR1 sequence comprising a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VL CDR1 sequence containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130 ) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

a VL CDR2 sequence comprising a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VL CDR2 sequence comprising the amino acid sequence QMSNLAS (SEQ ID NO: 132 ) or QMSNRAS (SEQ ID NO: 133); and a VL CDR3 sequence comprising a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VL CDR3 sequence comprising the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence including a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence CDR1 VH containing the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

a VH CDR2 sequence comprising a sequence at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the VH CDR2 sequence, the amino acid sequence of NIVWSEDKH (SEQ ID NO: 128) ;

a VH CDR3 sequence comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR3 IDYGNDYAFTY sequence (SEQ ID NO: 129);

a VL CDR1 sequence comprising a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VL CDR1 sequence containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130 );

a VL CDR2 sequence comprising a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VL CDR2 sequence comprising the amino acid sequence QMSNLAS (SEQ ID NO: 132 ); and a VL CDR3 sequence comprising a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VL CDR3 sequence comprising the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

In some embodiments, at least one arm of a multispecific antibody or antigen-binding fragment thereof, e.g., a bispecific antibody or antigen-binding fragment thereof, comprises a combination of a VH CDR1 sequence, a

VH CDR2 sequences, VH CDR3 sequences, VL CDR1 sequences, VL CDR2 sequences, and VL CDR3 sequences, wherein the VH CDR1 sequence contains the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

the VH CDR2 sequence contains the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

the CDR3 VH sequence contains the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

the VL CDR1 sequence contains the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

the VL CDR2 sequence contains the amino acid sequence QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133); and the VL CDR3 sequence contains the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where the CDR1 VH sequence contains the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

the VH CDR2 sequence contains the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

the CDR3 VH sequence contains the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

the VL CDR1 sequence contains the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130);

the CDR2 VL sequence contains the amino acid sequence QMSNLAS (SEQ ID NO: 132); and the VL CDR3 sequence contains the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where the VH CDR1 sequence contains a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the amino acid sequence of GFSLSTYGMGVG (SEQ ID NO: 127);

the VH CDR2 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of NIWWSEDKH (SEQ ID NO: 128);

the VH CDR3 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

the VL CDR1 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

the VL CDR2 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133); and the VL CDR3 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AQNLELPYT (SEQ ID NO: 134).

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where the CDR1 VH sequence contains the sequence at least 90, 91, 92, 93, 94,

- 11 041958

95, 96, 97, 98, 99% or more identical to the amino acid sequence of GFSLSTYGMGVG (SEQ ID NO: 127);

the CDR2 VH sequence contains the sequence at least 90, 91, 92, 93, 94,

95, 96, 97, 98, 99% or more identical to the amino acid sequence of NIWWSEDKH (SEQ ID NO: 128);

the VH CDR3 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

the VL CDR1 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130);

the VL CDR2 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of QMSNLAS (SEQ ID NO: 132); and the VL CDR3 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AQNLELPYT (SEQ ID NO: 134).

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a heavy chain or a heavy chain variable region comprising or derived from an amino acid sequence selected from the group consisting of variable region amino acid sequences heavy chain shown in table. 12. In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a light chain or a light chain variable region comprising or derived from an amino acid sequence selected from the group consisting of amino acid sequences variable region of the light chain shown in table. 12. In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a heavy chain or a heavy chain variable region comprising or derived from an amino acid sequence selected from the group consisting of variable sequences. the heavy chain region shown in Table. 12 and a light chain or light chain variable region comprising or derived from an amino acid sequence selected from the group consisting of the light chain variable region amino acid sequences shown in Table 12. 12.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a heavy chain variable region amino acid sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence selected from the group consisting of the sequences of the variable region of the heavy chain shown in table. 12. In some embodiments, at least one arm of a multispecific antibody or antigen-binding fragment thereof, e.g., a bispecific antibody or antigen-binding fragment thereof, contains an amino acid sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence selected from the group consisting of the sequences of the variable region of the light chain shown in table. 12. In some embodiments, at least one arm of a multispecific antibody or antigen-binding fragment thereof, e.g., a bispecific antibody or antigen-binding fragment thereof, contains an amino acid sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence selected from the group consisting of the sequence of the variable region of the heavy chain shown in table. 12 and a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of light chain variable region sequences, shown in table. 12.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a combination of a heavy chain variable region complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1), a complementarity determining sequence complementarity of heavy chain variable region region 2 (VH CDR2, also referred to herein as CDRH2), heavy chain variable region complementarity determining region 3 sequence (VH CDR3, also referred to herein as CDRH3), light variable region complementarity determining region 1 sequence chain (CDR1 VL, also referred to in this

- 12 041958 as CDRL1), a light chain variable region complementarity determining region 2 (CDR2 VL, also referred to herein as CDRL2) and a light chain variable region complementarity determining region 3 (CDR3 VL, also referred to herein as CDRL3) sequence. ), where at least one sequence

The CDR is selected from the group consisting of the VH CDR1 sequence shown in Table 1. 13;

sequence CDR2 VH shown in table. 13;

sequence CDR3 VH shown in table. 13;

sequence CDR1 VL shown in table. 13;

sequence CDR2 VL shown in table. 13; and sequence CDR3 VL shown in table. 13.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence including a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence CDR1 VH, shown in table. 13;

sequence CD2 VH, including a sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the CDR2 VH sequence shown in table. 13;

sequence CDR3 VH, including a sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the CDR3 VH sequence shown in table. 13;

sequence CDR1 VL, including a sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the CDR1 VL sequence shown in table. 13;

sequence CDR2 VL, including a sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the CDR2 VL sequence shown in table. 13; and a VL CDR3 sequence comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR3 sequence shown in Table 1. 13.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where the combination is a combination of six CDR sequences (CDR1 VH, CDR2 VH, CDR3 VH, CDR1 VL, CDR2 VL and CDR3 VL) shown in one line in table. 13.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a light chain variable region comprising a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where the combination is a combination of three sequences of light chain CDRs (CDR1 VL, CDR2 VL, CDR3 VL) shown in one line in table. 13.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a heavy chain variable region comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, where the combination is a combination of the three heavy chain CDR sequences (CDR1 VH, CDR2 VH, CDR3 VH) shown on the same line in Table 1. 13.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where each CDR sequence in the combination contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the corresponding CDR sequence in the combination of six CDR sequences (CDR1 VH, CDR2 VH, CDR3 VH, CDR1 VL, CDR2 VL and CDR3 VL), shown in one line in the table. 13.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a heavy chain variable region comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein each CDR sequence in combination contains the sequence at least 90, 91, 92, 93,

- 13 041958

94, 95, 96, 97, 98, 99% or more identical to the corresponding CDR sequence in a combination of three heavy chain CDR sequences (CDR1 VH, CDR2 VH, CDR3 VH) shown in one row in Table. 13.

In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., the bispecific antibody or antigen-binding fragment thereof, comprises a light chain variable region comprising a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein each CDR sequence in combination contains a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the corresponding CDR sequence in a combination of three light chain CDR sequences (CDR1 VL, CDR2 VL, CDR3 VL ) shown in one row in Table. 13.

Suitable anti-CD166 antibodies as described herein also include an antibody, or an antigen-binding fragment thereof, that binds the same epitope on human CD166 and/or cynomolgus CD166 as an anti-CD166 antibody comprising a heavy chain variable region amino acid sequence selected from the group, consisting of SEQ ID NOs: 119, 121 and 122, and an amino acid sequence of a light chain variable region selected from the group consisting of SEQ ID NOs: 120 and 123-126.

Suitable anti-CD166 antibodies of this specification also include an antibody or antigen-binding fragment thereof that binds the same epitope on human CD166 and/or cynomolgus CD166 as an anti-CD166 antibody comprising a CDR1 VH sequence comprising the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

a VH CDR2 sequence containing the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

a VH CDR3 sequence containing the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

a VL CDR1 sequence containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

a VL CDR2 sequence containing the amino acid sequence QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133); and a VL CDR3 sequence containing the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

Suitable anti-CD166 antibodies of this specification also include an antibody or antigen-binding fragment thereof that binds the same epitope on human CD166 and/or cynomolgus CD166 as an anti-CD166 antibody comprising a CDR1 VH sequence comprising the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

a VH CDR2 sequence containing the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

a VH CDR3 sequence containing the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

a VL CDR1 sequence containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130);

a VL CDR2 sequence containing the amino acid sequence QMSNLAS (SEQ ID NO: 132); and a VL CDR3 sequence containing the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

Suitable anti-CD166 antibodies as described herein also include an antibody or antigen-binding fragment thereof that cross-competes for binding to human CD166 and/or cynomolgus monkey CD166 with an anti-CD166 antibody comprising a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 121 and 122, and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 120 and 123-126.

Suitable anti-CD166 antibodies as described herein also include an antibody or antigen-binding fragment thereof that cross-competes for binding to human CD166 and/or cynomolgus monkey CD166 with an anti-CD166 antibody comprising a heavy chain variable region amino acid sequence comprising SEQ ID NO: 122, and a light chain variable region amino acid sequence comprising SEQ ID NO: 123. Suitable anti-CD166 antibodies as described herein also include an antibody or antigen-binding fragment thereof that cross-competes for binding to human CD166 and/or cynomolgus CD166 with an anti-CD166 antibody, containing the amino acid sequence of the heavy chain variable region containing SEQ ID NO: 121 and the amino acid sequence of the light chain variable region containing SEQ ID NO: 123.

Suitable anti-CD166 antibodies as described herein also include an antibody or antigen-binding fragment thereof that cross-competes for binding to human CD166 and/or

Cynomolgus monkey CD166 with an anti-CD166 antibody containing a VH CDR1 sequence containing the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

a VH CDR2 sequence containing the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

a VH CDR3 sequence containing the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

a VL CDR1 sequence containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

a VL CDR2 sequence containing the amino acid sequence QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133); and a VL CDR3 sequence containing the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

Suitable anti-CD166 antibodies as described herein also include an antibody or antigen-binding fragment thereof that cross-competes for binding to human CD166 and/or cynomolgus monkey CD166 with an anti-CD166 antibody containing a CDR1 VH sequence containing the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127 );

a VH CDR2 sequence containing the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

a VH CDR3 sequence containing the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

a VL CDR1 sequence containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130);

a VL CDR2 sequence containing the amino acid sequence QMSNLAS (SEQ ID NO: 132); and a VL CDR3 sequence containing the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

The description also relates to activated antibodies comprising an antibody or antigen-binding fragment thereof specifically binding to CD166 attached to a masking group (MM) such that the addition of MM reduces the ability of the antibody or antigen-binding fragment to bind CD166.

In some embodiments, the MM is attached via a sequence containing a substrate for a protease, such as a protease that is active in diseased tissue and/or a protease co-localized with CD166 at a treatment site in a subject. The activated anti-CD166 antibodies provided herein, also referred to herein interchangeably as activated anti-CD166 antibodies or activated anti-CD166 antibodies, are stable in the circulation, activated at the sites targeted for therapy and/or diagnosis, but not in the normal, for example, healthy, tissue or other tissue not targeted for treatment and/or diagnosis and, when activated, have CD166 binding at least comparable to the corresponding unmodified antibody, also referred to herein as the parent antibody.

The invention also relates to methods for treating, preventing and/or delaying the onset or progression of, or alleviating a symptom associated with altered expression and/or activity of CD166 in a subject using activated antibodies that bind CD166, in particular activated antibodies that bind and neutralize or otherwise in a manner that inhibits at least one type of CD166 biological activity and/or CD166-mediated signaling.

The invention also relates to methods for treating, preventing and/or delaying the onset or progression, or alleviating a symptom associated with the presence, growth, proliferation, metastasis and/or activity of cells expressing CD166 or altered expression of CD166 in a subject, using activated antibodies that bind CD166, in particular activated antibodies that bind, target, neutralize, kill or otherwise inhibit at least one biological activity of cells expressing or altered expression of CD166.

The invention also relates to methods for treating, preventing and/or delaying the onset or progression, or alleviating a symptom associated with the presence, growth, proliferation, metastasis and/or activity of CD166-expressing cells in a subject using activated CD166-binding antibodies, in particular activated antibodies that bind, target, neutralize, destroy, or otherwise inhibit at least one

- 15 041958 type of biological activity of cells expressing CD 166.

The invention also relates to methods for treating, preventing and/or delaying the onset or progression, or alleviating a symptom associated with the presence, growth, proliferation, metastasis and/or activity of cells with altered CD166 expression in a subject using activated CD166 binding antibodies, in particular activated antibodies that bind, target, neutralize, destroy, or otherwise inhibit at least one biological activity of cells with altered CD166 expression.

Activated antibodies in the activated state bind CD166 and include (i) an antibody or antigen-binding fragment (AB) thereof that specifically binds CD166;

(ii) a masking group (MM) which, when the activated antibody is in an uncleaved state, inhibits AB binding to CD166; and (iii) a cleavable group (CM) attached to AB, where CM is a polypeptide that functions as a substrate for a protease.

In some embodiments, the activated antibody in the uncleaved state has the following N-terminal to C-terminal structural arrangement: MM-CM-AB or AB-CM-MM.

In some embodiments, the implementation of the activated antibody contains a binding peptide between MM and SM.

In some embodiments, the activated antibody comprises a binding peptide between the CM and AB.

In some embodiments, the activated antibody comprises a first binding peptide (LP1) and a second binding peptide (LP2), and wherein the activated antibody in an uncleaved state has the following N-terminal to C-terminal structural arrangement: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the two binding peptides need not be identical to each other.

In some embodiments, at least one of LP1 or LP2 contains an amino acid sequence selected from the group consisting of (GS) _n , (GGS) _n , (GSGGS) _n (SEQ ID NO: 1) and (GGGS) _n (SEQ ID NO: 2), where n is an integer, at least one.

In some embodiments, at least one of LP1 or LP2 contains an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7) and GSSSG (SEQ ID NO: 8).

In some embodiments, LP1 contains the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10), GSSGGSGGSGGS (SEQ ID NO: 11), GSSGGSGGSGGSGGGS (SEQ ID NO: 12), GSSGGSGGSG (SEQ ID NO: 12), 13) or GSSGGSGGSGS (SEQ ID NO: 14).

In some embodiments, LP2 contains the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 15), GSSGT (SEQ ID NO: 16), or GSSG (SEQ ID NO: 17).

In some embodiments, the AB has a dissociation constant of about 100 nM or less for binding to CD166.

In some embodiments, the AB has a dissociation constant of about 100 nM or less for binding to mammalian CD166. In some embodiments, the AB has a dissociation constant of about 10 nM or less to bind to mammalian CD166. In some embodiments, the AB has a dissociation constant of about 5 nM or less for binding to CD166. In some embodiments, the AB has a dissociation constant of about 1 nM or less for binding to CD166. In some embodiments, the AB has a dissociation constant of about 0.5 nM or less for binding to CD166. In some embodiments, the AB has a dissociation constant of about 0.1 nM or less for binding to CD166. In some embodiments, the AB has a dissociation constant of 0.01-100, 0.01-10, 0.01-5, 0.01-1, 0.01-0.5, 0.01-0.1, 0, 01-0.05, 0.05-100, 0.05-10, 0.05-5, 0.05-1, 0.05-0.5, 0.05-0.1, 0.1- 100, 0.1-10, 0.1-5, 0.1-1, 0.1-0.5, 0.5-100, 0.5-10, 0.5-5, 0.5- 1, 1-100, 1-10, 1-5, 5-100, 5-10, or 10-100 nM for binding to mammalian CD166.

In some embodiments, the activated antibody includes an antibody or an antigen-binding fragment (AB) thereof that specifically binds CD166. In some embodiments, the CD166-binding antibody or antigen-binding fragment thereof is a monoclonal antibody, domain antibody, single chain, Fab fragment, F(ab') ₂ fragment, scFv, scAb, dAb, heavy chain single domain antibody, or a single light chain domain. In some embodiments, the CD166-binding antibody or antigen-binding fragment thereof is a mouse, other rodent, chimeric, humanized, or fully human monoclonal antibody.

In some embodiments, the activated antibody in the uncleaved state specifically binds mammalian CD166 with a dissociation constant less than or equal to 1 nM, less than or equal to 5 nM, less than or equal to 10 nM, less than or equal to 15 nM, less than or equal to

- 16 041958 nM less than or equal to 25 nM, less than or equal to 50 nM, less than or equal to 100 nM, less than or equal to 150 nM, less than or equal to 250 nM, less than or equal to 500 nM, less than or equal to

750 nM, less than or equal to 1000 nM and/or less than or equal to 2000 nM.

In some embodiments, the activated antibody in the uncleaved state specifically binds mammalian CD166 with a dissociation constant greater than or equal to 1 nM, greater than or equal to 5 nM, greater than or equal to 10 nM, greater than or equal to 15 nM, greater than or equal to 20 nM, greater than or equal to 25 nM greater than or equal to 50 nM greater than or equal to 100 nM greater than or equal to 150 nM greater than or equal to 250 nM greater than or equal to 500 nM greater than or equal to 750 nM greater than or equal to 1000 nM and/or less than or equal 2000 nM.

In some embodiments, the activated antibody in the uncleaved state specifically binds mammalian CD166 with a dissociation constant in the range of 1-2000, 1-1000, 1-750, 1-500, 1-250, 1-150, 1-100, 1-50, 1-25, 1-15, 1-10, 1-5, 5-2000, 5-1000, 5-750, 5-500, 5-250, 5-150, 5-100, 5-50, 5- 25, 5-15, 5-10, 10-2000, 10-1000, 10-750, 10-500, 10-250, 10-150, 10-100, 10-50, 10-25, 10-15, 15-2000, 15-1000, 15-750, 15-500, 15-250, 15-150, 15-100, 15-50, 15-25, 25-2000, 25-1000, 25-750, 25- 500, 25-250, 25-150, 25-100, 25-50, 50-2000, 50-1000, 50-750, 50-500, 50-250, 50-150, 50-100, 100-2000, 100-1000, 100-750, 100-500, 100-250, 100-150, 150-2000, 150-1000, 150-750, 150-500, 150-250, 250-2000, 250-1000, 250- 750, 250-500, 500-2000, 500-1000, 500-750, 500-500, 500-250, 500-150, 500-100, 500-50, 750-2000, 750-1000 or 1000-2000 nM .

In some embodiments, an activated antibody in the activated state specifically binds mammalian CD166 with a dissociation constant less than or equal to 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 nM.

In some embodiments, an activated antibody in the activated state specifically binds mammalian CD166 with a dissociation constant greater than or equal to 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 nM.

In some embodiments, an activated antibody in the activated state specifically binds mammalian CD166 with a dissociation constant in the range of 0.01-100, 0.01-10, 0.01-5, 0.01-1, 0.01-0.5, 0.01-0.1, 0.01-0.05, 0.05-100, 0.05-10, 0.05-5, 0.05-1, 0.05-0.5, 0, 05-0.1, 0.1-100, 0.1-10, 0.1-5, 0.1-1, 0.1-0.5, 0.5-100, 0.5-10, 0.5-5, 0.5-1, 1-100, 1-10, 1-5, 5-100, 5-10, or 10-100 nM.

In some embodiments, mammalian CD166 is selected from the group consisting of human CD166 and cynomolgus CD166. In some embodiments, the AB specifically binds human CD166 or cynomolgus monkey CD166 with a dissociation constant of less than 1 nM. In some embodiments, the mammalian CD166 is human CD166. In some embodiments, the mammalian CD166 is cynomolgus monkey CD166.

In some embodiments, the AV has one or more of the following characteristics:

(a) AB specifically binds human CD166; and (b) AB specifically binds human CD166 and cynomolgus monkey CD166.

In some embodiments, the AV has one or more of the following characteristics:

(a) AB specifically binds human CD166 and cynomolgus monkey CD166;

(b) AB inhibits the binding of mammalian CD6 to mammalian CD166;

(c) AB inhibits the binding of human CD6 to human CD166; and (d) AB inhibits the binding of cynomolgus monkey CD6 to cynomolgus monkey CD166.

In some embodiments, the AV blocks the ability of a natural ligand or receptor to bind mammalian CD166 with an EC50 less than or equal to 5 nM, less than or equal to 10 nM, less than or equal to 50 nM, less than or equal to 100 nM, less than or equal to 500 nM, and/or less or equal to 1000 nM.

In some embodiments, AV blocks the ability of mammalian CD6 to bind mammalian CD166 with an EC50 less than or equal to 5 nM, less than or equal to 10 nM, less than or equal to 50 nM, less than or equal to 100 nM, less than or equal to 500 nM, and/or less than or equal to 1000 nM. In some embodiments, the natural ligand or receptor for CD166 is CD6.

In some embodiments, the AB blocks the ability of a natural ligand to bind mammalian CD166 to EC50 5-1000, 5-500, 5-100 5-50, 5-10, 10-1000, 10-500, 10-100, 10-50, 50 -1000, 50-500, 50-100, 100-1000, 100-500, 500-1000 nM. In some embodiments, the AB blocks the ability of mammalian CD6 to bind mammalian CD166 to EC50 5-1000, 5-500, 5-100, 5-50, 5-10, 10-1000, 10-500, 10-100, 10-50, 50-1000, 50-500, 50-100, 100-1000, 100-500, 500-1000 nM. In some embodiments, the natural ligand or receptor for CD166 is CD6.

In some embodiments, the AB of the present disclosure inhibits or reduces the growth, proliferation, and/or metastasis of mammalian CD166-expressing cells. Without intending to be bound by any theory, the AB of the present disclosure may inhibit or reduce the growth, proliferation, and/or metastasis of cells expressing mammalian CD166 by specifically binding to CD166 and inhibiting, blocking, and/or preventing natural ligand binding or receptor with mammalian CD166. In some embodiments, the natural mammalian CD166 ligand or receptor is mammalian CD6.

In some embodiments, the activated antibody comprises a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 121, and 122. In some embodiments, the activated antibody comprises a heavy chain variable region amino acid sequence comprising SEQ ID NO: 121 or 122.

In some embodiments, the activatable antibody comprises a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 120 and 123126. In some embodiments, the activatable antibody comprises a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 123-126.

In some embodiments, the activated antibody comprises a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 121, and 122 and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 120 and 123-126.

In some embodiments, the activated antibody comprises a heavy chain variable region amino acid sequence comprising SEQ ID NOs: 121 or 122 and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 123-126.

In some embodiments, the activated antibody comprises a heavy chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 121, and 122. In some embodiments, the activated antibody comprises a heavy chain variable region amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence, containing SEQ ID NO: 121 or 122.

In some embodiments, the activated antibody comprises a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 120 and 123-126. In some embodiments, the activated antibody comprises a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 123-126.

In some embodiments, the activated antibody comprises a heavy chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 121 and 122, and a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 120 and 123-126.

In some embodiments, the activated antibody comprises a heavy chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence containing SEQ ID NO: 121 or 122 , and the amino acid sequence of the light chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 123-126 .

In some embodiments, the activated antibody comprises a combination of a heavy chain variable region complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1), a heavy chain variable region complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence. ), variable heavy chain region 3 complementarity determining sequence (VH CDR3, also referred to herein as CDRH3), light chain variable region complementarity determining region 1 sequence (VL CDR1, also referred to herein as CDRL1), region complementarity determining sequence 2 light chain variable region (CDR2 VL, also referred to herein as CDRL2) and the complementarity determining region 3 variable light chain region (CDR3 VL, also referred to herein mente as CDRL3), wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence containing the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

a VH CDR2 sequence containing the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

- 18 041958 sequence CDR3 VH containing the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

a VL CDR1 sequence containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

a VL CDR2 sequence containing the amino acid sequence QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133); and a VL CDR3 sequence containing the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence comprising including a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence containing the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

a VH CDR2 sequence comprising a sequence at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VH CDR2 sequence comprising the amino acid sequence NIWWSEDKH (SEQ ID NO: 128 );

a VH CDR3 sequence comprising a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VH CDR3 sequence containing the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129 );

a VL CDR1 sequence comprising a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VL CDR1 sequence containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130 ) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

a VL CDR2 sequence comprising a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VL CDR2 sequence comprising the amino acid sequence QMSNLAS (SEQ ID NO: 132 ) or QMSNRAS (SEQ ID NO: 133); and a VL CDR3 sequence comprising a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to a VL CDR3 sequence comprising the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

the VH CDR2 sequence contains the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

the CDR3 VH sequence contains the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

the VL CDR1 sequence contains the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

the VL CDR2 sequence contains the amino acid sequence QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133); and the VL CDR3 sequence contains the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence contains a sequence of at least 90, 91, 92, 93 , 94, 95, 96, 97, 98, 99% or more identical to the amino acid sequence of GFSLSTYGMGVG (SEQ ID NO: 127);

the VH CDR2 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of NIWWSEDKH (SEQ ID NO: 128);

the VH CDR3 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

the VL CDR1 sequence contains a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

the CDR2 VL sequence contains the sequence at least 90, 91, 92, 93,

- 19 041958

94, 95, 96, 97, 98, 99% or more identical to the amino acid sequence of QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133); and the CDR3 VL sequence contains the sequence at least 90, 91, 92, 93, 94,

95, 96, 97, 98, 99% or more identical to the amino acid sequence of AQNLELPYT (SEQ ID NO: 134).

In some embodiments, the AV-activated anti-CD166 antibody comprises a heavy chain variable region amino acid sequence selected from the group consisting of the heavy chain variable region sequences shown in Table 1. 12. In some embodiments, the AV-activated anti-CD166 antibody comprises a light chain variable region amino acid sequence selected from the group consisting of the light chain variable region sequences shown in Table 12. 12. In some embodiments, the AV-activated anti-CD166 antibody comprises a heavy chain variable region amino acid sequence selected from the group consisting of the heavy chain variable region sequences shown in Table 12. 12 and a light chain variable region amino acid sequence selected from the group consisting of the light chain variable region sequences shown in Table 12. 12.

In some embodiments, an AV-activated anti-CD166 antibody comprises a heavy chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of from the sequences of the variable region of the heavy chain shown in table. 12. In some embodiments, an AV-activated anti-CD166 antibody contains an amino acid sequence of a light chain variable region that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group , consisting of the sequences of the variable region of the light chain shown in table. 12. In some embodiments, an AV-activated anti-CD166 antibody contains an amino acid sequence of a heavy chain variable region that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group , consisting of the sequences of the variable region of the heavy chain shown in table. 12 and a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of light chain variable region sequences, shown in table. 12.

In some embodiments, the activated antibody comprises a combination of a heavy chain variable region complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1), a heavy chain variable region complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence. ), variable heavy chain region 3 complementarity determining sequence (VH CDR3, also referred to herein as CDRH3), light chain variable region complementarity determining region 1 sequence (VL CDR1, also referred to herein as CDRL1), region complementarity determining sequence 2 light chain variable region (CDR2 VL, also referred to herein as CDRL2) and the complementarity determining region 3 variable light chain region (CDR3 VL, also referred to herein mente as CDRL3), where at least one CDR sequence is selected from the group consisting of the VH CDR1 sequence shown in table. 13;

sequence CDR2 VH shown in table. 13;

sequence CDR3 VH shown in table. 13;

sequence CDR1 VL shown in table. 13;

sequence CDR2 VL shown in table. 13; and sequence CDR3 VL shown in table. 13.

In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence comprising includes a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR1 sequence shown in Table 1. 13;

sequence CDR2 VH, including a sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the CDR2 VH sequence shown in table. 13;

sequence CDR3 VH, including a sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the CDR3 VH sequence shown in table. 13;

sequence CDR1 VL, including a sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the CDR1 VL sequence shown in table. 13;

sequence CDR2 VL, including the sequence, at least 90, 91,

- 20 041958

92, 93, 94, 95, 96, 97, 98, 99% or more identical to the VL CDR2 sequence shown in table. 13;

and a VL CDR3 sequence comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR3 sequence shown in Table 1. 13.

In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where the combination is a combination of six CDR sequences (CDR1 VH, CDR2 VH, CDR3 VH , CDR1 VL, CDR2 VL and CDR3 VL), shown in one line in the table. 13.

In some embodiments, the activated antibody comprises a heavy chain variable region comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein the combination is a combination of three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in one line in the table. 13.

In some embodiments, the activated antibody comprises a light chain variable region comprising a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination is a combination of the three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) shown in one line in the table. 13.

In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where each CDR sequence in combination contains a sequence of at least 90, 91, 92 , 93, 94, 95, 96, 97, 98, 99% or more identical to the corresponding CDR sequence in the combination of the six CDR sequences (CDR1 VH, CDR2 VH, CDR3 VH, CDR1 VL, CDR2 VL, and CDR3 VL) shown in one line in the table. 13.

In some embodiments, the activated antibody comprises a heavy chain variable region comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, where each CDR sequence in the combination contains a sequence of at least 90, 91, 92, 93, 94, 95 , 96, 97, 98, 99% or more identical to the corresponding CDR sequence in a combination of three heavy chain CDR sequences (CDR1 VH, CDR2 VH, CDR3 VH) shown in one line in table. 13.

In some embodiments, the activated antibody comprises a light chain variable region comprising a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where each CDR sequence in combination contains a sequence of at least 90, 91, 92, 93, 94, 95 , 96, 97, 98, 99% or more identical to the corresponding CDR sequence in a combination of the three light chain CDR sequences (CDR1 VL, CDR2 VL, CDR3 VL) shown in one line in table. 13.

In some embodiments, the implementation of the activated antibody may contain one or more polypeptides, including a combination of sequences in a given line of the table. A, or any combination of a masking sequence (MM), a substrate sequence (SM), a light chain variable sequence or light chain variable CDR sequences, and a heavy chain variable sequence or heavy chain variable CDR sequences from Table 1. IN.

In some embodiments, antibody drug conjugates (ADC) and activated antibody drug conjugates (AADC) may comprise one or more polypeptides comprising a combination of a light chain sequence or a light chain variable domain sequence and a heavy chain or heavy chain variable domain sequence. chain, linker and toxin in this line from the table. C, or any combination of a light chain sequence or a light chain variable domain sequence, and a heavy chain sequence or a heavy chain variable domain sequence, a linker and a toxin from Table WITH.

In some embodiments, the MM has a dissociation constant for binding to AB that is greater than the dissociation constant of AB from CD166.

In some embodiments, the implementation of the MM has a dissociation constant for binding to AB, which is not greater than the dissociation constant of AB from CD166.

In some embodiments, the implementation of the MM has a dissociation constant for binding to AB, which is less than the dissociation constant of AB from CD166.

In some embodiments, the dissociation constant (Kd) of MM with respect to AB is no more than 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, 50000, 100000 , 500000, 1000000, 5000000, 10000000, 50000000 times or more, or 1-5, 5-10, 10-100, 10-1000, 10-10000, 10-100000, 101000000, 10-10000000, 0.0 100-10000, 100-100000, 100-1000000, 100-10000000, 1000-10000, 1000100000, 1000-1000000, 1000-10000000, 10000-100000, 1000-1000000, 10000-10000000, 100000-1000000 or 100000-10000000 times or more than the dissociation constant AB with respect to the target.

- 21 041958

In some embodiments, MM does not interfere with or compete with AV binding to

CD166 when the activated antibody is in a cleaved state.

In some embodiments, MM is a polypeptide of approximately 2-40 amino acids in length. In some embodiments, MM is a polypeptide up to about 40 amino acids in length.

In some embodiments, the MM polypeptide sequence is different from the CD166 sequence. In some embodiments, the MM polypeptide sequence is no more than 50% identical to any natural AB binding partner. In some embodiments, the MM polypeptide sequence differs from the CD166 sequence and is no more than 40%, 30%, 25%, 20%, 15%, or 10% identical to any natural AB binding partner.

In some embodiments, attachment of MM to AB reduces the ability of AB to bind CD166 such that the dissociation constant (Kd) of AB to CD166 with MM attached is at least twice the Kd of AB to CD166 without MM attachment.

In some embodiments, attachment of MM to AB reduces the ability of AB to bind CD166 such that the dissociation constant (Kd) of AB to CD166 with MM attached is at least five times the Kd of AB to CD166 without MM attachment.

In some embodiments, attachment of MM to AB reduces the ability of AB to bind CD166 such that the dissociation constant (Kd) of AB to CD166 with MM attached is at least 10 times the Kd of AB to CD166 without MM attachment.

In some embodiments, attachment of MM to AB reduces the ability of AB to bind CD166 such that the dissociation constant (Kd) of AB to CD166 with MM attached is at least 20 times the Kd of AB to CD166 without MM attachment.

In some embodiments, attachment of MM to AB reduces the ability of AB to bind CD166 such that the dissociation constant (Kd) of AB to CD166 with MM attached is at least 40 times the Kd of AB to CD166 without MM attachment.

In some embodiments, attachment of MM to AB reduces the ability of AB to bind CD166 such that the dissociation constant (Kd) of AB to CD166 with MM attached is at least 100 times the Kd of AB to CD166 without MM attachment.

In some embodiments, attachment of MM to AB reduces the ability of AB to bind CD166 such that the dissociation constant (Kd) of AB to CD166 with MM attached is at least 1000 times the Kd of AB to CD166 without MM attachment.

In some embodiments, attachment of MM to AB reduces the ability of AB to bind CD166 such that the dissociation constant (Kd) of AB to CD166 with MM attached is at least 10,000 times the Kd of AB to CD166 without MM attachment.

In some embodiments, in the presence of CD166, MM reduces the ability of an AB to bind CD166 by at least 90% when CM is uncleaved compared to when CM is cleaved, when assayed in vitro using a target displacement assay, such as for example described in PCT Publication No. WO 2010/081173, the entire content of which is incorporated herein by reference.

In some embodiments, the implementation of the MM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 135-238.

In some embodiments, the CM-cleaving protease is active, e.g., up-regulated or otherwise unregulated in diseased tissue, and the protease cleaves CM in the activated antibody when the activated antibody is exposed to the protease.

In some embodiments, the protease is co-localized with CD166 in tissue and the protease cleaves the CM in the activated antibody when the activated antibody is exposed to the protease.

In some embodiments, the SM is located in the activated antibody such that when the activated antibody is in an uncleaved state, the binding of the activated antibody to CD166 is reduced to occur at a dissociation constant at least twice the dissociation constant of unmodified AB binding to CD166 , while in the cleaved state (ie, when the antibody to be activated is in the cleaved state), AB binds CD166.

In some embodiments, the implementation of the SM is located in the activated antibody such that when the activated antibody is in the uncleaved state, the binding of the activated antibody to CD166 is reduced so that it occurs with a dissociation constant at least five times the dissociation constant of the binding of unmodified AB to CD166 , while in the cleaved state (ie, when the antibody to be activated is in the cleaved state), AB binds CD166.

In some embodiments, the implementation of the CM is located in the activated antibody in such a way that

- 22 041958 that, when the activated antibody is in the uncleaved state, the binding of the activated antibody to CD166 is reduced so that it occurs with a dissociation constant at least times the dissociation constant of unmodified AB binding to CD166, while in the cleaved state (t i.e. when the activated antibody is in a cleaved state)

AB binds CD166.

In some embodiments, the implementation of the CM is located in the activated antibody such that when the activated antibody is in the uncleaved state, the binding of the activated antibody to CD166 is reduced so that it occurs with a dissociation constant at least 20 times the dissociation constant of binding of unmodified AB to CD166 , while in the cleaved state (ie, when the antibody to be activated is in the cleaved state), AB binds CD166.

In some embodiments, the implementation of the CM is located in the activated antibody such that when the activated antibody is in the uncleaved state, the binding of the activated antibody to CD166 is reduced so that it occurs with a dissociation constant of at least 40 times the dissociation constant of the binding of unmodified AB to CD166 , while in the cleaved state AB binds CD166.

In some embodiments, the implementation of the SM is located in the activated antibody such that when the activated antibody is in the uncleaved state, the binding of the activated antibody to CD166 is reduced so that it occurs with a dissociation constant of at least 50 times the dissociation constant of binding of unmodified AB to CD166 , while in the cleaved state AB binds CD166.

In some embodiments, the implementation of the CM is located in the activated antibody such that when the activated antibody is in the uncleaved state, the binding of the activated antibody to CD166 is reduced so that it occurs with a dissociation constant of at least 100 times the dissociation constant of binding of unmodified AB to CD166 , while in the cleaved state AB binds CD166.

In some embodiments, the implementation of the SM is located in the activated antibody such that when the activated antibody is in the uncleaved state, the binding of the activated antibody to CD166 is reduced so that it occurs with a dissociation constant at least 200 times the dissociation constant of binding of unmodified AB to CD166 , while in the cleaved state AB binds CD166.

In some embodiments, the CM is a polypeptide up to 15 amino acids in length.

In some embodiments, the CM is a polypeptide that contains a first cleavable group (CM1) that is a substrate for at least one matrix metalloproteinase (MMP) and a second cleavable group (CM2) that is a substrate for at least one serine protease (SP ). In some embodiments, the substrate sequence CM1 of the substrate sequence and the substrate sequence CM2 of the substrate CM1-CM2 are each independently a polypeptide up to 15 amino acids in length.

In some embodiments, CM is a substrate for at least one protease that is or is believed to be upregulated or otherwise unregulated in the cancer. In some embodiments, SM is a substrate for at least one protease that is, or is believed to be, upregulated in inflammation. In some embodiments, CM is a substrate for at least one protease that is, or is believed to be, upregulated in autoimmunity.

In some embodiments, SM is a substrate for at least one protease selected from the group consisting of matrix metalloproteinase (MMP), thrombin, neutrophil elastase, cysteine protease, legumain, and a serine protease such as matriptase (MT-SP1) and urokinase ( uPA). Without wishing to be bound by theory, these proteases are believed to be upregulated or otherwise unregulated in at least one case of cancer, inflammation, and/or autoimmunity.

Illustrative substrates include, but are not limited to, substrates cleaved by one or more of the following enzymes or proteases listed in Table. 4.

In some embodiments, the SM is chosen for use with a particular protease, for example, a protease known to be co-localized with the target of an activated antibody.

In some embodiments, the SM is a substrate for at least one MMP. Examples of MMPs include the MMPs listed in Table. 4. In some embodiments, CM is a substrate for a protease selected from the group consisting of MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. In some embodiments, SM is a substrate for MMP9. In some embodiments, SM is a substrate for MMP14.

In some embodiments, the SM is a substrate containing the sequence TGRGPSWV (SEQ ID NO: 18); SARGPSRW (SEQ ID NO: 19); TARGPSFK (SEQ ID NO: 20); LSGRSDNH (SEQ ID NO: 21); GGWHTGRN (SEQ ID NO: 22); HTGRSGAL (SEQ ID NO: 23); PLTGRSGG (SEQ ID NO: 24); AARGPAIH (SEQ ID NO: 25); RGPAFNPM (SEQ ID NO: 26); SSRGPAYL (SEQ ID NO: 27); RGPATPIM (SEQ ID NO: 28); RGPA (SEQ ID NO: 29); GGQPSGMWGW (SEQ ID NO: 30); FPRPLGITGL (SEQ ID NO: 31); VHMPLGFLGP (SEQ ID NO: 32); SPLTGRSG (SEQ ID NO: 33); SAGFSLPA (SEQ ID NO: 34); LAPLGLQRR (SEQ ID NO: 35); SGGPLGVR (SEQ ID NO: 36); PLGL (SEQ ID NO: 37); LSGRSGNH (SEQ ID NO: 318); SGRSANPRG (SEQ ID NO: 319); LSGRSDDH (SEQ ID NO: 320);

LSGRSDIH (SEQ ID NO: 321); LSGRSDQH (SEQ ID NO: 322); LSGRSDTH (SEQ ID NO:323);

LSGRSDYH (SEQ ID NO: 324); LSGRSDNP (SEQ ID NO: 325); LSGRSANP (SEQ ID NO:326);

LSGRSANI (SEQ ID NO: 327); LSGRSDNI (SEQ ID NO: 328); MIAPVAYR (SEQ ID NO:329);

RPSPMWAY (SEQ ID NO: 330); WATPRPMR (SEQ ID NO: 331); FRLLDWQW (SEQ ID NO: 332); ISSGL (SEQ ID NO: 333); ISSGLLS (SEQ ID NO: 334); and/or ISSGLL (SEQ ID NO: 335).

In some embodiments, the implementation of the SM contains the amino acid sequence LSGRSDNH (SEQ ID NO: 21). In some embodiments, the implementation of the CM contains the amino acid sequence TGRGPSWV (SEQ ID NO: 18). In some embodiments, the implementation of the CM contains the amino acid sequence PLTGRSGG (SEQ ID NO: 24). In some embodiments, the implementation of the SM contains the amino acid sequence GGQPSGMWGW (SEQ ID NO: 30). In some embodiments, the implementation of the SM contains the amino acid sequence FPRPLGITGL (SEQ ID NO: 31). In some embodiments, the implementation of the SM contains the amino acid sequence VHMPLGFLGP (SEQ ID NO: 32). In some embodiments, the implementation of the SM contains the amino acid sequence PLGL (SEQ ID NO: 37). In some embodiments, the implementation of the SM contains the amino acid sequence SARGPSRW (SEQ ID NO: 19). In some embodiments, the implementation of the CM contains the amino acid sequence TARGPSFK (SEQ ID NO: 20). In some embodiments, the implementation of the SM contains the amino acid sequence GGWHTGRN (SEQ ID NO: 22). In some embodiments, the implementation of the SM contains the amino acid sequence HTGRSGAL (SEQ ID NO: 23). In some embodiments, the implementation of the CM contains the amino acid sequence AARGPAIH (SEQ ID NO: 25). In some embodiments, the implementation of the SM contains the amino acid sequence RGPAFNPM (SEQ ID NO: 26). In some embodiments, the implementation of the SM contains the amino acid sequence SSRGPAYL (SEQ ID NO: 27). In some embodiments, the implementation of the CM contains the amino acid sequence RGPATPIM (SEQ ID NO: 28). In some embodiments, the implementation of the SM contains the amino acid sequence RGPA (SEQ ID NO: 29). In some embodiments, the implementation of the CM contains the amino acid sequence LSGRSGNH (SEQ ID NO: 318). In some embodiments, the implementation of the CM contains the amino acid sequence SGRSANPRG (SEQ ID NO: 319). In some embodiments, the implementation of the CM contains the amino acid sequence LSGRSDDH (SEQ ID NO: 320). In some embodiments, the implementation of the SM contains the amino acid sequence LSGRSDIH (SEQ ID NO: 321). In some embodiments, the implementation of the CM contains the amino acid sequence LSGRSDQH (SEQ ID NO: 322). In some embodiments, the implementation of the SM contains the amino acid sequence LSGRSDTH (SEQ ID NO: 323). In some embodiments, the implementation of the CM contains the amino acid sequence LSGRSDYH (SEQ ID NO: 324). In some embodiments, the implementation of the SM contains the amino acid sequence LSGRSDNP (SEQ ID NO: 325). In some embodiments, the implementation of the SM contains the amino acid sequence LSGRSANP (SEQ ID NO: 326). In some embodiments, the implementation of the CM contains the amino acid sequence LSGRSANI (SEQ ID NO: 327). In some embodiments, the implementation of the CM contains the amino acid sequence LSGRSDNI (SEQ ID NO: 328). In some embodiments, the implementation of the SM contains the amino acid sequence MIAPVAYR (SEQ ID NO: 329). In some embodiments, the implementation of the SM contains the amino acid sequence RPSPMWAY (SEQ ID NO: 330). In some embodiments, the implementation of the SM contains the amino acid sequence WATPRPMR (SEQ ID NO: 331). In some embodiments, the implementation of the CM contains the amino acid sequence FRLLDWQW (SEQ ID NO: 332). In some embodiments, the implementation of the SM contains the amino acid sequence ISSGL (SEQ ID NO: 333). In some embodiments, the implementation of the CM contains the amino acid sequence ISSGLLS (SEQ ID NO: 334). In some embodiments, the implementation of the SM contains the amino acid sequence ISSGLL (SEQ ID NO: 335).

In some embodiments, the implementation of the CM is a substrate for MMP and contains the sequence ISSGLSS (SEQ ID NO: 38); QNQALRMA (SEQ ID NO: 39); AQNLLGMV (SEQ ID NO: 40); STPFGMF (SEQ ID NO: 41); PVGYTSSL (SEQ ID NO: 42); DWLYWPGI (SEQ ID NO: 43), ISSGLLSS (SEQ ID NO: 44), LKAAPRWA (SEQ ID NO: 45); GPSHLVLT (SEQ ID NO: 46); LPGGLSPW (SEQ ID NO: 47); MGLFSEAG (SEQ ID NO: 48); SPLPLRVP (SEQ ID NO: 49); RMHLRSLG (SEQ ID NO: 50); LAAPLGLL (SEQ ID NO: 51); AVGLAPP (SEQ ID NO: 52); LLAPSHRA (SEQ ID NO: 53); and/or PAGLWLDP (SEQ ID NO: 54).

In some embodiments, the implementation of the CM contains the amino acid sequence ISSGLSS (SEQ ID NO: 38). In some embodiments, the CM contains an amino acid

- 24 041958 sequence QNQALRMA (SEQ ID NO: 39). In some embodiments, the implementation of the CM contains the amino acid sequence AQNLLGMV (SEQ ID NO: 40). In some embodiments, the implementation of the CM contains the amino acid sequence STFPFGMF (SEQ ID NO: 41). In some embodiments, the implementation of the SM contains the amino acid sequence PVGYTSSL (SEQ ID NO: 42). In some embodiments, the implementation of the SM contains the amino acid sequence DWLYWPGI (SEQ ID NO: 43). In some embodiments, the implementation of the SM contains the amino acid sequence ISSGLLSS (SEQ ID NO: 44). In some embodiments, the implementation of the CM contains the amino acid sequence LKAAPRWA (SEQ ID NO: 45). In some embodiments, the implementation of the CM contains the amino acid sequence GPSHLVLT (SEQ ID NO: 46). In some embodiments, the implementation of the SM contains the amino acid sequence LPGGLSPW (SEQ ID NO: 47). In some embodiments, the implementation of the SM contains the amino acid sequence MGLFSEAG (SEQ ID NO: 48). In some embodiments, the implementation of the SM contains the amino acid sequence SPLPLRVP (SEQ ID NO: 49). In some embodiments, the implementation of the SM contains the amino acid sequence RMHLRSLG (SEQ ID NO: 50). In some embodiments, the implementation of the SM contains the amino acid sequence LAAPLGLL (SEQ ID NO: 51). In some embodiments, the implementation of the SM contains the amino acid sequence AVGLLAPP (SEQ ID NO: 52). In some embodiments, the implementation of the CM contains the amino acid sequence LLAPSHRA (SEQ ID NO: 53). In some embodiments, the implementation of the CM contains the amino acid sequence PAGLWLDP (SEQ ID NO: 54).

In some embodiments, SM is a substrate for thrombin. In some embodiments, the implementation of the SM is a substrate for thrombin and contains the sequence GPRSFGL (SEQ ID NO: 55) or GPRSFG (SEQ ID NO: 56). In some embodiments, the implementation of the SM contains the amino acid sequence of GPRSFGL (SEQ ID NO: 57). In some embodiments, the implementation of the CM contains the amino acid sequence GPRSFG (SEQ ID NO: 58).

In some embodiments, the implementation of the SM contains an amino acid sequence selected from the group consisting of NTLSGRSENHSG (SEQ ID NO: 59); NTLSGRSGNHGS (SEQ ID NO: 60); TSTSGRSANPRG (SEQ ID NO: 61); TSGRSANP (SEQ ID NO: 62); VAGRSMRP (SEQ ID NO: 63); WPEGRRS (SEQ ID NO: 64); ILPRSPAF (SEQ ID NO: 65); MVLGRSLL (SEQ ID NO: 66); QGRAITFI (SEQ ID NO: 67); SPRSIMLA (SEQ ID NO: 68); and SMLRSMPL (SEQ ID NO: 69).

In some embodiments, the implementation of the CM contains the amino acid sequence NTLSGRSENHSG (SEQ ID NO: 59). In some embodiments, the implementation of the SM contains the amino acid sequence NTLSGRSGNHGS (SEQ ID NO: 60). In some embodiments, the implementation of the CM contains the amino acid sequence TSTSGRSANPRG (SEQ ID NO: 61). In some embodiments, the implementation of the CM contains the amino acid sequence TSGRSANP (SEQ ID NO: 62). In some embodiments, the implementation of the CM contains the amino acid sequence of VAGRSMRP (SEQ ID NO: 63). In some embodiments, the implementation of the SM contains the amino acid sequence WPEGRRS (SEQ ID NO: 64). In some embodiments, the implementation of the CM contains the amino acid sequence of ILPRSPAF (SEQ ID NO: 65). In some embodiments, the implementation of the SM contains the amino acid sequence MVLGRSLL (SEQ ID NO: 66). In some embodiments, the implementation of the CM contains the amino acid sequence QGRAITFI (SEQ ID NO: 67). In some embodiments, the implementation of the SM contains the amino acid sequence SPRSIMLA (SEQ ID NO: 68). In some embodiments, the implementation of the CM contains the amino acid sequence SMLRSMPL (SEQ ID NO: 69).

In some embodiments, SM is a substrate for neutrophil elastase. In some embodiments, SM is a substrate for a serine protease. In some embodiments, SM is a substrate for uPA. In some embodiments, SM is a substrate for legumain. In some embodiments, CM is a substrate for matriptase. In some embodiments, SM is a substrate for a cysteine protease. In some embodiments, SM is a substrate for a cysteine protease such as cathepsin.

In some embodiments, the CM is a CM1-CM2 substrate and contains the sequence

ISSGLLSGRSDNH (SEQ ID NO: 70), also referred to herein as substrate 2001;

ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 71);

AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 72);

TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 73);

VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 74);

TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 75);

AVGLLAPPGGLSGRSDNH (SEQ ID NO: 76), also referred to herein as substrate 3001;

LSGRSDNHGGAVGLLAPP (SEQ ID NO: 77);

- 25 041958

VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 78);

LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 79);

LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 80);

LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 81);

ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 82);

LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 83);

QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 84);

LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 85);

QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 86);

ISSGLLSGRSGNH (SEQ ID NO: 87);

GLSGRSDNHGGAVGLLAPP (SEQ ID NO: 336);

GLSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 337);

ISSGLLSGRSANPRG (SEQ ID NO: 338), also referred to herein as substrate 2003;

AVGLLAPPTSGRSANPRG (SEQ ID NO: 339), also referred to herein as substrate 2004;

AVGLLAPPSGRSANPRG (SEQ ID NO: 340), also referred to herein as substrate 2005;

ISSGLLSGRSDDH (SEQ ID NO: 341), also referred to herein as substrate 2006;

ISSGLLSGRSDIH (SEQ ID NO: 342), also referred to herein as substrate 2007;

ISSGLLSGRSDQH (SEQ ID NO: 343), also referred to herein as substrate 2008;

ISSGLLSGRSDTH (SEQ ID NO: 344), also referred to herein as substrate 2009;

ISSGLLSGRSDYH (SEQ ID NO: 345), also referred to herein as substrate 2010;

ISSGLLSGRSDNP (SEQ ID NO: 346), also referred to herein as substrate 2011;

ISSGLLSGRSANP (SEQ ID NO: 347), also referred to herein as substrate 2012;

ISSGLLSGRSANI (SEQ ID NO: 348), also referred to herein as substrate 2013;

AVGLLAPPGGLSGRSDDDH (SEQ ID NO: 349), also referred to herein as substrate 3006;

AVGLLAPPGGLSGRSDIH (SEQ ID NO: 350), also referred to herein as substrate 3007;

AVGLLAPPGGLSGRSDQH (SEQ ID NO: 351), also referred to herein as substrate 3008;

AVGLLAPPGGLSGRSDTH (SEQ ID NO: 352), also referred to herein as substrate 3009;

AVGLLAPPGGLSGRSDYH (SEQ ID NO: 353), also referred to herein as substrate 3010;

AVGLLAPPGGLSGRSDNP (SEQ ID NO: 354), also referred to herein as substrate 3011;

AVGLLAPPGGLSGRSANP (SEQ ID NO: 355), also referred to herein as substrate 3012;

AVGLLAPPGGLSGRSANI (SEQ ID NO: 356), also referred to herein as substrate 3013;

ISSGLLSGRSDNI (SEQ ID NO: 357), also referred to herein as substrate 2014; and/or

AVGLLAPPGGLSGRSDNI (SEQ ID NO: 358), also referred to herein as substrate 3014.

In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSDNH (SEQ ID NO: 70). In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 71). In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 72). In some embodiments, the CM1-CM2 substrate contains the sequence TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 73). In some embodiments, the CM1-CM2 substrate contains the sequence VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 74). In some embodiments, the CM1-CM2 substrate contains the sequence TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 75). In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGLSGRSDNH (SEQ ID NO: 76). In some embodiments, the CM1-CM2 substrate contains the sequence LSGRSDNHGGAVGLLAPP (SEQ ID NO: 77). In some embodiments, the CM1-CM2 substrate contains the sequence VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 78). In some embodiments, the CM1-CM2 substrate contains the sequence LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 79). In some embodiments, the CM1-CM2 substrate contains the sequence LSGRSDNHGGSGGSISSGLSS (SEQ ID NO: 80). In some embodiments, the implementation of the substrate CM1-CM2 contains

- 26 041958 sequence LSGRSGNHGGSGGSISSGLSS (SEQ ID NO: 81). In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 82). In some embodiments, the CM1-CM2 substrate contains the sequence LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 83). In some embodiments, the CM1-CM2 substrate contains the sequence QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 84). In some embodiments, the CM1-CM2 substrate contains the sequence LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 85). In some embodiments, the CM1-CM2 substrate contains the sequence QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 86). In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSGNH (SEQ ID NO: 87). In some embodiments, the CM1-CM2 substrate contains the sequence GLSGRSDNHGGAVGLLAPP (SEQ ID NO: 336). In some embodiments, the CM1-CM2 substrate contains the sequence and/or GLSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 337). In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSANPRG (SEQ ID NO: 338). In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPTSGRSANPRG (SEQ ID NO: 339). In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPSGRSANPRG (SEQ ID NO: 340). In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSDDH (SEQ ID NO: 341). In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSDIH (SEQ ID NO: 342). In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSDQH (SEQ ID NO: 343). In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSDTH (SEQ ID NO: 344). In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSDYH (SEQ ID NO: 345). In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSDNP (SEQ ID NO: 346). In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSANP (SEQ ID NO: 347). In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSANI (SEQ ID NO: 348). In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGLSGRSDDDH (SEQ ID NO: 349). In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGLSGRSDIH (SEQ ID NO: 350). In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGLSGRSDQH (SEQ ID NO: 351). In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGLSGRSDTH (SEQ ID NO: 352). In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGLSGRSDYH (SEQ ID NO: 353). In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGLSGRSDNP (SEQ ID NO: 354). In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGLSGRSANP (SEQ ID NO: 355). In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGLSGRSANI (SEQ ID NO: 356), ISSGLLSGRSDNI (SEQ ID NO: 357). In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGLSGRSDNI (SEQ ID NO: 358).

In some embodiments, the SM is a substrate for at least two proteases. In some embodiments, each protease is selected from the group consisting of the proteases shown in Table. 4. In some embodiments, CM is a substrate for at least two proteases, wherein one of the proteases is selected from the group consisting of MMP, thrombin, neutrophil elastase, cysteine protease, uPA, legumain, and matriptase, and the other protease is selected from the group consisting of from the proteases shown in table. 4. In some embodiments, SM is a substrate for at least two proteases selected from the group consisting of MMP, thrombin, neutrophil elastase, cysteine protease, uPA, legumain, and matriptase.

In some embodiments, the activated antibody comprises at least a first CM and a second CM. In some embodiments, each of the first CM and the second CM are polypeptides no more than 15 amino acids in length. In some embodiments, the first CM and the second CM in the activated antibody in the uncleaved state have the following N-terminal to C-terminal structural arrangement: MM-CM1-CM2-AB or AB-CM2-CM1-MM. In some embodiments, at least one of the first CM and the second CM is a polypeptide that functions as a substrate for a protease selected from the group consisting of MMP, thrombin, neutrophil elastase, cysteine protease, uPA, legumain, and matriptase. In some embodiments, the first CM is cleaved with a first cleaving agent selected from the group consisting of MMP, thrombin, neutrophil elastase, cysteine protease, uPA, legumain, and matriptase in the target tissue, and the second CM is cleaved with a second cleaving agent in the target tissue . In some embodiments, the implementation of another protease is selected from the group consisting of the proteases shown in table. 4. In some embodiments, the first cleaving agent and the second cleaving agent are the same protease selected from the group consisting of MMP, thrombin, neutrophil elastase, cysteine protease, uPA, legumain, and matriptase, and the first CM and the second CM are a variety of substrates for fer

- 27 041958 ment. In some embodiments, the first cleaving agent and the second cleaving agent are the same protease selected from the group consisting of the proteases shown in Table 1. 4. In some embodiments, the first cleaving agent and the second cleaving agent are different proteases. In some embodiments, the first disintegrating agent and the second disintegrating agent are localized together in the target tissue. In some embodiments, the first CM and the second CM are cleaved by at least one cleaving agent in the target tissue.

In some embodiments, the activated antibody is exposed to a protease and cleaved by the protease such that, in the activated or cleaved state, the activated antibody comprises a light chain amino acid sequence containing at least a portion of the LP2 and/or CM sequence after cleavage by the CM protease.

Suitable activated CD166 antibodies as described herein also include an antibody or antigen-binding fragment thereof that binds the same epitope on human CD166 and/or cynomolgus CD166 as an anti-CD166 antibody comprising a heavy chain variable region amino acid sequence selected from the group , consisting of SEQ ID NO: 119, 121 and 122, and the amino acid sequence of the variable region of the light chain selected from the group consisting of SEQ ID NO: 120 and 123-126.

Suitable activated anti-CD166 antibodies as described herein also include an antibody or antigen-binding fragment thereof that binds the same epitope on human CD166 and/or cynomolgus CD166 as an anti-CD166 antibody comprising a VH CDR1 sequence comprising the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

a VH CDR2 sequence containing the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

a VH CDR3 sequence containing the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

a VL CDR1 sequence containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

a VL CDR2 sequence containing the amino acid sequence QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133); and a VL CDR3 sequence containing the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

Suitable activated CD166 antibodies as described herein also include an antibody or antigen-binding fragment thereof that binds the same epitope on human CD166 and/or cynomolgus CD166 as an anti-CD166 antibody comprising a heavy chain variable region amino acid sequence selected from the group , consisting of the sequences of the variable region of the heavy chain shown in table. 12 and a light chain variable region amino acid sequence selected from the group consisting of the light chain variable region sequences shown in Table 12. 12.

Suitable activated anti-CD166 antibodies of this disclosure also include an antibody or antigen-binding fragment thereof binding the same epitope on human CD166 and/or cynomolgus CD166 as an anti-CD166 antibody comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, a CDR3 VH, CDR1 VL sequences, CDR2 VL sequences, and CDR3 VL sequences, where the combination is a combination of the six CDR sequences (CDR1 VH, CDR2 VH, CDR3 VH, CDR1 VL, CDR2 VL, and CDR3 VL) shown on the same line in tab. 13.

Suitable activated anti-CD166 antibodies of this disclosure also include an antibody or antigen-binding fragment thereof that cross-competes for binding to human CD166 and/or cynomolgus monkey CD166 with an anti-CD166 antibody comprising a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 121 and 122, and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 120 and 123-126.

Suitable activated anti-CD166 antibodies as described herein also include an antibody or antigen-binding fragment thereof that cross-competes for binding to human CD166 and/or cynomolgus monkey CD166 with an anti-CD166 antibody comprising a CDR1 VH sequence comprising the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

a VH CDR2 sequence containing the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

a VH CDR3 sequence containing the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

CDR1 VL sequence containing the amino acid sequence RSSKSLLHSN- 28 041958

GITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

a VL CDR2 sequence containing the amino acid sequence QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133); and a VL CDR3 sequence containing the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

Suitable activated anti-CD166 antibodies of this disclosure also include an antibody or antigen-binding fragment thereof that cross-competes for binding to human CD166 and/or cynomolgus monkey CD166 with an anti-CD166 antibody comprising a heavy chain variable region amino acid sequence selected from the group consisting of sequences of the variable region of the heavy chain shown in table. 12 and a light chain variable region amino acid sequence selected from the group consisting of the light chain variable region sequences shown in Table 12. 12.

Suitable activated CD166 antibodies as described herein also include an antibody or antigen-binding fragment thereof that cross-competes for binding to human CD166 and/or cynomolgus CD166 with an anti-CD166 antibody comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, CDR1 VL sequences, CDR2 VL sequences, and CDR3 VL sequences, where the combination is a combination of six CDR sequences (CDR1 VH, CDR2 VH, CDR3 VH, CDR1 VL, CDR2 VL, and CDR3 VL) shown on the same row in Table 13.

In some embodiments, the activated antibody also contains an AB-conjugated agent. In some embodiments, the agent conjugated to an AV or AV-activated antibody is a drug. In some embodiments, the agent is an antineoplastic agent. In some embodiments, the agent is a toxin or a fragment thereof. As used herein, a toxin fragment is one that retains toxic activity. In some embodiments, the agent is AB-conjugated via a cleavable linker. In some embodiments, the agent is AB-conjugated via a linker containing at least one CM1-CM2 substrate sequence. In some embodiments, the agent is conjugated to AB via a non-cleavable linker. In some embodiments, the agent is AV-conjugated via a linker that is cleavable in the intracellular or lysosomal environment. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent, a DNA cleaving agent, a DNA crosslinking agent, a DNA intercalating agent, or another DNA damaging agent. In some embodiments, the implementation of the tool is a tool selected from the group listed in table. 5. In some embodiments, the agent is dolastatin. In some embodiments, the agent is auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is pyrrolobenzodiazepine. In some embodiments, the agent is a dimer of pyrrolobenzodiazepine.

In some embodiments, the activated antibody is conjugated to one or more equivalents of the agent. In some embodiments, the activated antibody is conjugated to one equivalent of the agent. In some embodiments, the activated antibody is conjugated to two, three, four, five, six, seven, eight, nine, ten, or more than ten equivalents of the agent. In some embodiments, the activated antibody is part of a mixture of activated antibodies having the same number of conjugate agent equivalents. In some embodiments, the activated antibody is part of a mixture of activated antibodies having a different number of conjugate agent equivalents. In some embodiments, the mixture of activated antibodies is such that the average number of agents conjugated to each activated antibody is between zero and one, between one and two, between two and three, between three and four, between four and five, between five and six between six and seven, between seven and eight, between eight and nine, between nine and ten, and ten or more. In some embodiments, the mixture of activated antibodies is such that the average number of agents conjugated to each activated antibody is one, two, three, four, five, six,

- 29 041958 seven, eight, nine, ten or more. In some embodiments, the activated antibody contains one or more site-specific amino acid sequence modifications such that the number of lysine and/or cysteine residues is increased or decreased relative to the original amino acid sequence of the activated antibody, thus, in some embodiments, respectively increasing or decreasing the amount agents that can be conjugated to an activated antibody, or In some embodiments, by limiting the conjugation of agents to an activated antibody in a site-specific manner. In some embodiments, the modified activated antibody is modified with one or more non-natural amino acids in a site-specific manner, thus limiting the conjugation of agents to only non-natural amino acid regions in some embodiments.

In some embodiments, the agent is an anti-inflammatory agent.

In some embodiments, the implementation of the activated antibody also contains a detectable group. In some embodiments, the detectable group is a diagnostic tool.

In some embodiments, the activated antibody also contains a signal peptide. In some embodiments, the signal peptide is conjugated to an activated antibody via a spacer. In some embodiments, the spacer is conjugated to an activated antibody in the absence of a signal peptide. In some embodiments, a spacer is attached directly to the MM of the antibody to be activated. In some embodiments, a spacer is attached directly to the MM of the antibody to be activated in an N-terminal to C-terminal structural arrangement of spacer-MM-CM-AB. An example of a spacer attached directly to the N-terminus of an MM of an activated antibody is QGQSGQ (SEQ ID NO: 88). Other examples of a spacer attached directly to the N-terminus of the MM of the activated antibody include QGQSGQG (SEQ ID NO: 305), QGQSG (SEQ ID NO: 306), QGQS (SEQ ID NO: 307), QGQ (SEQ ID NO: 308), QG (SEQ ID NO: 309) and Q. Other examples of a spacer attached directly to the N-terminus of the MM of an activated antibody include GQSGQG (SEQ ID NO: 359), QSGQG (SEQ ID NO: 360), SGQG (SEQ ID NO: 361), GQG (SEQ ID NO: 362), and G. In some embodiments, a spacer is not attached to the N-terminus of the MM. In some embodiments, the spacer contains at least the amino acid sequence QGQSGQ (SEQ ID NO: 88). In some embodiments, the spacer contains at least the amino acid sequence QGQSGQG (SEQ ID NO: 305). In some embodiments, the spacer contains at least the amino acid sequence QGQSG (SEQ ID NO: 306). In some embodiments, the spacer contains at least the amino acid sequence QGQS (SEQ ID NO: 307). In some embodiments, the spacer contains at least the amino acid sequence QGQ (SEQ ID NO: 308). In some embodiments, the implementation of the spacer contains at least the amino acid sequence QG (SEQ ID NO: 309). In some embodiments, the spacer contains at least the amino acid residue Q. In some embodiments, the spacer contains at least the amino acid sequence GQSGQG (SEQ ID NO: 359). In some embodiments, the spacer contains at least the amino acid sequence QSGQG (SEQ ID NO: 360). In some embodiments, the spacer contains at least the amino acid sequence SGQG (SEQ ID NO: 361). In some embodiments, the spacer contains at least the amino acid sequence GQG (SEQ ID NO: 362). In some embodiments, the implementation of the spacer contains at least the amino acid sequence G. In some embodiments, the implementation of the spacer is missing.

In some embodiments, the AB of the activated antibody naturally contains one or more disulfide bonds. In some embodiments, the implementation of the AB can be designed to include one or more disulfide bonds.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOS: 119, 121, and 122. In some embodiments, the activated antibody is encoded by a nucleic acid sequence containing a nucleic acid sequence encoding the amino acid sequence of the variable region of the heavy chain, containing SEQ ID NO: 121 or 122.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 120 and 123-126. In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 123-126.

- 30 041958

In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 121, and 122, and a nucleic acid sequence encoding the variable region amino acid sequence a light chain selected from the group consisting of SEQ ID NOs: 120 and 123-126.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 121 and 122 and a nucleic acid sequence encoding a light chain variable region amino acid sequence. selected from the group consisting of SEQ ID NO: 123-126.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence encoding the variable region amino acid sequence. heavy chain containing SEQ ID NO: 121 or 122. In some embodiments, the activated antibody is encoded by a nucleic acid sequence containing a nucleic acid sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the nucleic acid sequence encoding the amino acid sequence of the heavy chain variable region containing SEQ ID NO: 121 or 122.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence encoding the variable region amino acid sequence. a light chain selected from the group consisting of SEQ ID NOs: 120 and 123-126. In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence encoding the variable region amino acid sequence. a light chain selected from the group consisting of SEQ ID NOs: 123-126.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence encoding the variable region amino acid sequence. heavy chain selected from the group consisting of SEQ ID NOs: 119, 121 and 122 and a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence a nucleic acid encoding a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 120 and 123-126.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence encoding the variable region amino acid sequence. heavy chain containing SEQ ID NO: 121 or 122, and a nucleic acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the nucleic acid sequence encoding the amino acid sequence of the variable a light chain region selected from the group consisting of SEQ ID NOs: 123-126.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a heavy chain variable region amino acid sequence selected from the group consisting of the heavy chain variable region sequences shown in Table 1. 12. In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a light chain variable region amino acid sequence selected from the group consisting of the light chain variable region sequences shown in Table 12. 12. In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a heavy chain variable region amino acid sequence selected from the group consisting of the heavy chain variable region sequences shown in Table 12. 12 and a nucleic acid sequence encoding a light chain variable region amino acid sequence selected from the group consisting of the light chain variable region sequences shown in Table 12. 12.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence containing a nucleic acid sequence encoding an amino acid.

- 31 041958 lot sequence of the variable region of the heavy chain, at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence selected from the group consisting of sequences of the variable region of the heavy chain, shown in table. 12. In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a light chain variable region amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence selected from the group consisting of the sequences of the variable region of the light chain shown in table. 12. In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a heavy chain variable region amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence selected from the group consisting of the sequences of the variable region of the heavy chain shown in table. 12 and a nucleic acid sequence comprising a nucleic acid sequence encoding a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from group consisting of the sequences of the variable region of the light chain shown in table. 12.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a combination of a heavy chain variable region complementarity determining region 1 (CDR1 VH, also referred to herein as CDRH1) sequence, a heavy chain variable region complementarity determining region 2 sequence ( CDR2 VH, also referred to herein as CDRH2), the complementarity determining sequence of variable heavy chain region 3 (CDR3 VH, also referred to herein as CDRH3), the complementarity determining sequence of variable light chain region 1 (CDR1 VL, also referred to in herein as CDRL1) the complementarity-determining sequence of variable light chain region 2 (CDR2 VL, also referred to herein as CDRL2), and the c-determining sequence complementarity of light chain variable region 3 (VL CDR3, also referred to herein as CDRL3), wherein at least one CDR sequence is selected from the group consisting of the VH CDR1 sequence shown in Table 1. 13;

sequence CDR2 VH shown in table. 13;

sequence CDR3 VH shown in table. 13;

sequence CDR1 VL shown in table. 13;

sequence CDR2 VL shown in table. 13; and sequence CDR3 VL shown in table. 13.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence selected from the group consisting of a VH CDR1 sequence including a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR1 sequence shown in Table 1. 13;

sequence CD2 VH, including a sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the CDR2 VH sequence shown in table. 13;

sequence CDR3 VH, including a sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the CDR3 VH sequence shown in table. 13;

sequence CDR1 VL, including a sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the CDR1 VL sequence shown in table. 13;

sequence CDR2 VL, including a sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the CDR2 VL sequence shown in table. 13; and a VL CDR3 sequence comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR3 sequence shown in Table 1. 13.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where the combination is a combination of six CDR sequences (CDR1 VH, CDR2 VH, CDR3 VH, CDR1 VL, CDR2 VL and CDR3 VL), shown in one line in the table. 13.

In some embodiments, the activated antibody is encoded by the sequence

- 32 041958 nucleic acid containing a nucleic acid sequence encoding a light chain variable region containing a combination of the CDR1 VL sequence, the sequence

CDR2 VL and CDR3 VL sequences, where the combination is a combination of three light chain CDR sequences (CDR1 VL, CDR2 VL, CDR3 VL) shown on the same row in Table 1. 13.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a heavy chain variable region comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein the combination is a combination of three heavy chain CDR sequences (CDR1 VH , CDR2 VH, CDR3 VH), shown in one line in the table. 13.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein each CDR sequence in combination contains the sequence , at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the corresponding CDR sequence in a combination of six CDR sequences (CDR1 VH, CDR2 VH, CDR3 VH, CDR1 VL, CDR2 VL and CDR3 VL), shown in one line in the table. 13.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a heavy chain variable region comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, where each CDR sequence in combination contains a sequence of at least 90 , 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the corresponding CDR sequence in the combination of the three heavy chain CDR sequences (CDR1 VH, CDR2 VH, CDR3 VH) shown in one row in Table . 13.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a light chain variable region comprising a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where each CDR sequence in combination contains a sequence of at least 90 , 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the corresponding CDR sequence in the combination of the three light chain CDR sequences (CDR1 VL, CDR2 VL, CDR3 VL) shown on the same row in Table . 13.

In some embodiments, the implementation of the activated antibody contains one or more polypeptides containing a combination of sequences in a given line of the table. A or any combination of a masking sequence (MM), a substrate sequence (SM), a light chain variable sequence or light chain variable CDR sequences and a heavy chain variable sequence or heavy chain variable CDR sequences from Table 1. IN.

Table A

Combinations of activated anti-CD 166 antibodies

no. combinations Masking sequence (MM) Substrate sequence (CM) CDR VL, SEQ ID NO CDR VH, SEQ ID NO 1 LCHPLVLSAWESCSS (SEQ ID NO: 219) LSGRSDNH (SEQ ID NO: 21) 130, 132, 134 127, 128, 129 2 LCHPLVLSAWESCSS (SEQ ID NO: 219) ISSGLLSS (SEQ ID NO: 44) 130, 132, 134 127, 128, 129 3 LCHPLVLSAWESCSS (SEQ ID NO: 219) LSGRSGNH (SEQ ID NO: 318) 130, 132, 134 127, 128, 129 4 LCHPLVLSAWESCSS (SEQ ID NO: 219) AVGLAPP (SEQ ID NO: 52) 130, 132, 134 127, 128, 129 5 LCHPLVLSAWESCSS (SEQ ID NO: 219) VHMPLGFLGP (SEQ ID NO: 32) 130, 132, 134 127, 128, 129 6 LCHPLVLSAWESCSS (SEQ ID NO: 219) TSTSGRSANPRG (SEQ ID NO: 61) 130, 132, 134 127, 128, 129

- 33 041958

7 LCHPLVLSAWESCSS (SEQ ID NO: 219) QNQALRMA (SEQ ID NO: 39) 130, 132, 134 127, 128, 129 8 LCHPLVLSAWESCSS (SEQ ID NO: 219) ISSGLLSGRSDNH (SEQ ID NO : 7 0) 130, 132, 134 127, 128, 129 9 LCHPLVLSAWESCSS (SEQ ID NO: 219) ISSGLLSGRSGNH (SEQ ID NO: 87) 130, 132, 134 127, 128, 129 10 LCHPLVLSAWESCSS (SEQ ID NO: 219) ISSGLLSGRSANPRG (SEQ ID NO: 338) 130, 132, 134 127, 128, 129 eleven LCHPLVLSAWESCSS (SEQ ID NO: 219) AVGLLAPPTSGRSANPRG (SEQ ID NO: 339) 130, 132, 134 127, 128, 129 12 LCHPLVLSAWESCSS (SEQ ID NO: 219) AVGLLAPPSGRSANPRG (SEQ ID NO: 340) 130, 132, 134 127, 128, 129 13 LCHPLVLSAWESCSS (SEQ ID NO: 219) ISSGLLSGRSDDH (SEQ ID NO: 341) 130, 132, 134 127, 128, 129 14 LCHPLVLSAWESCSS (SEQ ID NO: 219) ISSGLLSGRSDDIH (SEQ ID NO: 342) 130, 132, 134 127, 128, 129 15 LCHPLVLSAWESCSS (SEQ ID NO: 219) ISSGLLSGRSDQH (SEQ ID NO: 343) 130, 132, 134 127, 128, 129 16 LCHPLVLSAWESCSS (SEQ ID NO: 219) ISSGLLSGRSDDIH (SEQ ID NO: 344) 130, 132, 134 127, 128, 129 17 LCHPLVLSAWESCSS (SEQ ID NO: 219) ISSGLLSGRSDYH (SEQ ID NO: 345) 130, 132, 134 127, 128, 129 18 LCHPLVLSAWESCSS (SEQ ID NO: 219) ISSGLLSGRSDNP (SEQ ID NO: 346) 130, 132, 134 127, 128, 129 19 LCHPLVLSAWESCSS (SEQ ID NO: 219) ISSGLLSGRSANP (SEQ ID NO: 347) 130, 132, 134 127, 128, 129 20 LCHPLVLSAWESCSS (SEQ ID NO: 219) ISSGLLSGRSANI (SEQ ID NO: 348) 130, 132, 134 127, 128, 129 21 LCHPLVLSAWESCSS (SEQ ID NO: 219) ISSGLLSGRSDNI (SEQ ID NO: 357) 130, 132, 134 127, 128, 129 22 LCHPLVLSAWESCSS (SEQ ID NO: 219) AVGLLAPPGGLSGRSDNH (SEQ ID NO: 76) 130, 132, 134 127, 128, 129 23 LCHPLVLSAWESCSS (SEQ ID NO: 219) AVGLLAPPGGLSGRSDDDH (SEQ ID NO: 349) 130, 132, 134 127, 128, 129 24 LCHPLVLSAWESCSS (SEQ ID NO: 219) AVGLLAPPGGLSGRSDIH (SEQ ID NO: 350) 130, 132, 134 127, 128, 129 25 LCHPLVLSAWESCSS (SEQ ID NO: 219) AVGLLAPPGGLSGRSDQH (SEQ ID NO: 351) 130, 132, 134 127, 128, 129 26 LCHPLVLSAWESCSS (SEQ ID NO: 219) AVGLLAPPGGLSGRSDIH (SEQ ID NO: 352) 130, 132, 134 127, 128, 129 27 LCHPLVLSAWESCSS (SEQ ID NO: 219) AVGLLAPPGGLSGRSDYH (SEQ ID NO: 353) 130, 132, 134 127, 128, 129 28 LCHPLVLSAWESCSS (SEQ ID NO: 219) AVGLLAPPGGLSGRSDNP (SEQ ID NO: 354) 130, 132, 134 127, 128, 129 29 LCHPLVLSAWESCSS (SEQ ID NO: 219) AVGLLAPPGGLSGRSANP (SEQ ID NO: 355) 130, 132, 134 127, 128, 129 thirty LCHPLVLSAWESCSS (SEQ ID NO: 219) AVGLLAPPGGLSGRSANI (SEQ ID NO: 356) 130, 132, 134 127, 128, 129 31 LCHPLVLSAWESCSS (SEQ ID NO: 219) AVGLLAPPGGLSGRSDNI (SEQ ID NO: 358) 130, 132, 134 127, 128, 129

- 34 041958

32 LCHPLVLSAWESCSS (SEQ ID NO: 219) ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 71) 130, 132, 134 127, 128, 129 33 LCHPAVLSAWESCSSS (SEQ ID NO: 222) LSGRSDNH (SEQ ID NO: 21) 130, 132, 134 127, 128, 129 34 LCHPAVLSAWESCSSS (SEQ ID NO: 222) ISSGLLSS (SEQ ID NO: 44) 130, 132, 134 127, 128, 129 35 LCHPAVLSAWESCSSS (SEQ ID NO: 222) LSGRSGNH (SEQ ID NO: 318) 130, 132, 134 127, 128, 129 36 LCHPAVLSAWESCSSS (SEQ ID NO: 222) AVGLAPP (SEQ ID NO: 52) 130, 132, 134 127, 128, 129 37 LCHPAVLSAWESCSSS (SEQ ID NO: 222) VHMPLGFLGP (SEQ ID NO: 32) 130, 132, 134 127, 128, 129 38 LCHPAVLSAWESCSSS (SEQ ID NO: 222) TSTSGRSANPRG (SEQ ID NO: 61) 130, 132, 134 127, 128, 129 39 LCHPAVLSAWESCSSS (SEQ ID NO: 222) QNQALRMA (SEQ ID NO: 39) 130, 132, 134 127, 128, 129 40 LCHPAVLSAWESCSSS (SEQ ID NO: 222) ISSGLLSGRSDNH (SEQ ID NO : 7 0) 130, 132, 134 127, 128, 129 41 LCHPAVLSAWESCSSS (SEQ ID NO: 222) ISSGLLSGRSGNH (SEQ ID NO: 87) 130, 132, 134 127, 128, 129 42 LCHPAVLSAWESCSSS (SEQ ID NO: 222) ISSGLLSGRSANPRG (SEQ ID NO: 338) 130, 132, 134 127, 128, 129 43 LCHPAVLSAWESCSSS (SEQ ID NO: 222) AVGLLAPPTSGRSANPRG (SEQ ID NO: 339) 130, 132, 134 127, 128, 129 44 LCHPAVLSAWESCSSS (SEQ ID NO: 222) AVGLLAPPSGRSANPRG (SEQ ID NO: 340) 130, 132, 134 127, 128, 129 45 LCHPAVLSAWESCSSS (SEQ ID NO: 222) ISSGLLSGRSDDH (SEQ ID NO: 341) 130, 132, 134 127, 128, 129 46 LCHPAVLSAWESCSSS (SEQ ID NO: 222) ISSGLLSGRSDDIH (SEQ ID NO: 342) 130, 132, 134 127, 128, 129 47 LCHPAVLSAWESCSSS (SEQ ID NO: 222) ISSGLLSGRSDQH (SEQ ID NO: 343) 130, 132, 134 127, 128, 129 48 LCHPAVLSAWESCSSS (SEQ ID NO: 222) ISSGLLSGRSDDIH (SEQ ID NO: 344) 130, 132, 134 127, 128, 129 49 LCHPAVLSAWESCSSS (SEQ ID NO: 222) ISSGLLSGRSDYH (SEQ ID NO: 345) 130, 132, 134 127, 128, 129 50 LCHPAVLSAWESCSSS (SEQ ID NO: 222) ISSGLLSGRSDNP (SEQ ID NO: 346) 130, 132, 134 127, 128, 129 51 LCHPAVLSAWESCSSS (SEQ ID NO: 222) ISSGLLSGRSANP (SEQ ID NO: 347) 130, 132, 134 127, 128, 129 52 LCHPAVLSAWESCSSS (SEQ ID NO: 222) ISSGLLSGRSANI (SEQ ID NO: 348) 130, 132, 134 127, 128, 129 53 LCHPAVLSAWESCSSS (SEQ ID NO: 222) ISSGLLSGRSDNI (SEQ ID NO: 357) 130, 132, 134 127, 128, 129 54 LCHPAVLSAWESCSSS (SEQ ID NO: 222) AVGLLAPPGGLSGRSDNH (SEQ ID NO: 76) 130, 132, 134 127, 128, 129 55 LCHPAVLSAWESCSSS (SEQ ID NO: 222) AVGLLAPPGGLSGRSDDDH (SEQ ID NO: 349) 130, 132, 134 127, 128, 129 56 LCHPAVLSAWESCSSS (SEQ ID NO: 222) AVGLLAPPGGLSGRSDIH (SEQ ID NO: 350) 130, 132, 134 127, 128, 129

- 35 041958

57 LCHPAVLSAWESCSSS (SEQ ID NO: 222) AVGLLAPPGGLSGRSDQH (SEQ ID NO: 351) 130, 132, 134 127, 128, 129 58 LCHPAVLSAWESCSSS (SEQ ID NO: 222) AVGLLAPPGGLSGRSDTH (SEQ ID NO: 352) 130, 132, 134 127, 128, 129 59 LCHPAVLSAWESCSSS (SEQ ID NO: 222) AVGLLAPPGGLSGRSDYH (SEQ ID NO: 353) 130, 132, 134 127, 128, 129 60 LCHPAVLSAWESCSSS (SEQ ID NO: 222) AVGLLAPPGGLSGRSDNP (SEQ ID NO: 354) 130, 132, 134 127, 128, 129 61 LCHPAVLSAWESCSSS (SEQ ID NO: 222) AVGLLAPPGGLSGRSANP (SEQ ID NO: 355) 130, 132, 134 127, 128, 129 62 LCHPAVLSAWESCSSS (SEQ ID NO: 222) AVGLLAPPGGLSGRSANI (SEQ ID NO: 356) 130, 132, 134 127, 128, 129 63 LCHPAVLSAWESCSSS (SEQ ID NO: 222) AVGLLAPPGGLSGRSDNI (SEQ ID NO: 358) 130, 132, 134 127, 128, 129 64 LCHPAVLSAWESCSSS (SEQ ID NO: 222) ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 71) 130, 132, 134 127, 128, 129 65 LCHPLVASAWESCSS (SEQ ID NO: 224) LSGRSDNH (SEQ ID NO: 21) 130, 132, 134 127, 128, 129 66 LCHPLVASAWESCSS (SEQ ID NO: 224) ISSGLLSS (SEQ ID NO: 44) 130, 132, 134 127, 128, 129 67 LCHPLVASAWESCSS (SEQ ID NO: 224) LSGRSGNH (SEQ ID NO: 318) 130, 132, 134 127, 128, 129 68 LCHPLVASAWESCSS (SEQ ID NO: 224) AVGLAPP (SEQ ID NO: 52) 130, 132, 134 127, 128, 129 69 LCHPLVASAWESCSS (SEQ ID NO: 224) VHMPLGFLGP (SEQ ID NO: 32) 130, 132, 134 127, 128, 129 70 LCHPLVASAWESCSS (SEQ ID NO: 224) TSTSGRSANPRG (SEQ ID NO: 61) 130, 132, 134 127, 128, 129 71 LCHPLVASAWESCSS (SEQ ID NO: 224) QNQALRMA (SEQ ID NO: 39) 130, 132, 134 127, 128, 129 72 LCHPLVASAWESCSS (SEQ ID NO: 224) ISSGLLSGRSDNH (SEQ ID NO : 7 0) 130, 132, 134 127, 128, 129 73 LCHPLVASAWESCSS (SEQ ID NO: 224) ISSGLLSGRSGNH (SEQ ID NO : 8 7) 130, 132, 134 127, 128, 129 74 LCHPLVASAWESCSS (SEQ ID NO: 224) ISSGLLSGRSANPRG (SEQ ID NO: 338) 130, 132, 134 127, 128, 129 75 LCHPLVASAWESCSS (SEQ ID NO: 224) AVGLLAPPTSGRSANPRG (SEQ ID NO: 339) 130, 132, 134 127, 128, 129 76 LCHPLVASAWESCSS (SEQ ID NO: 224) AVGLLAPPSGRSANPRG (SEQ ID NO: 340) 130, 132, 134 127, 128, 129 77 LCHPLVASAWESCSS (SEQ ID NO: 224) ISSGLLSGRSDDH (SEQ ID NO: 341) 130, 132, 134 127, 128, 129 78 LCHPLVASAWESCSS (SEQ ID NO: 224) ISSGLLSGRSDDIH (SEQ ID NO: 342) 130, 132, 134 127, 128, 129 79 LCHPLVASAWESCSS (SEQ ID NO: 224) ISSGLLSGRSDQH (SEQ ID NO: 343) 130, 132, 134 127, 128, 129 80 LCHPLVASAWESCSS (SEQ ID NO: 224) ISSGLLSGRSDDIH (SEQ ID NO: 344) 130, 132, 134 127, 128, 129 81 LCHPLVASAWESCSS (SEQ ID NO: 224) ISSGLLSGRSDYH (SEQ ID NO: 345) 130, 132, 134 127, 128, 129

- 36 041958

82 LCHPLVASAWESCSS (SEQ ID NO: 224) ISSGLLSGRSDNP (SEQ ID NO: 346) 130, 132, 134 127, 128, 129 83 LCHPLVASAWESCSS (SEQ ID NO: 224) ISSGLLSGRSANP (SEQ ID NO: 347) 130, 132, 134 127, 128, 129 84 LCHPLVASAWESCSS (SEQ ID NO: 224) ISSGLLSGRSANI (SEQ ID NO: 348) 130, 132, 134 127, 128, 129 85 LCHPLVASAWESCSS (SEQ ID NO: 224) ISSGLLSGRSDNI (SEQ ID NO: 357) 130, 132, 134 127, 128, 129 86 LCHPLVASAWESCSS (SEQ ID NO: 224) AVGLLAPPGGLSGRSDNH (SEQ ID NO: 76) 130, 132, 134 127, 128, 129 87 LCHPLVASAWESCSS (SEQ ID NO: 224) AVGLLAPPGGLSGRSDDDH (SEQ ID NO: 349) 130, 132, 134 127, 128, 129 88 LCHPLVASAWESCSS (SEQ ID NO: 224) AVGLLAPPGGLSGRSDIH (SEQ ID NO: 350) 130, 132, 134 127, 128, 129 89 LCHPLVASAWESCSS (SEQ ID NO: 224) AVGLLAPPGGLSGRSDQH (SEQ ID NO: 351) 130, 132, 134 127, 128, 129 90 LCHPLVASAWESCSS (SEQ ID NO: 224) AVGLLAPPGGLSGRSDTH (SEQ ID NO: 352) 130, 132, 134 127, 128, 129 91 LCHPLVASAWESCSS (SEQ ID NO: 224) AVGLLAPPGGLSGRSDYH (SEQ ID NO: 353) 130, 132, 134 127, 128, 129 92 LCHPLVASAWESCSS (SEQ ID NO: 224) AVGLLAPPGGLSGRSDNP (SEQ ID NO: 354) 130, 132, 134 127, 128, 129 93 LCHPLVASAWESCSS (SEQ ID NO: 224) AVGLLAPPGGLSGRSANP (SEQ ID NO: 355) 130, 132, 134 127, 128, 129 94 LCHPLVASAWESCSS (SEQ ID NO: 224) AVGLLAPPGGLSGRSANI (SEQ ID NO: 356) 130, 132, 134 127, 128, 129 95 LCHPLVASAWESCSS (SEQ ID NO: 224) AVGLLAPPGGLSGRSDNI (SEQ ID NO: 358) 130, 132, 134 127, 128, 129 96 LCHPLVASAWESCSS (SEQ ID NO: 224) ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 71) 130, 132, 134 127, 128, 129 97 LEGWCLHPLCLWGAG (SEQ ID NO: 230) LSGRSDNH (SEQ ID NO: 21) 130, 132, 134 127, 128, 129 98 LEGWCLHPLCLWGAG (SEQ ID NO: 230) ISSGLLSS (SEQ ID NO: 44) 130, 132, 134 127, 128, 129 99 LEGWCLHPLCLWGAG (SEQ ID NO: 230) LSGRSGNH (SEQ ID NO: 318) 130, 132, 134 127, 128, 129 100 LEGWCLHPLCLWGAG (SEQ ID NO: 230) AVGLAPP (SEQ ID NO: 52) 130, 132, 134 127, 128, 129 101 LEGWCLHPLCLWGAG (SEQ ID NO: 230) VHMPLGFLGP (SEQ ID NO: 32) 130, 132, 134 127, 128, 129 102 LEGWCLHPLCLWGAG (SEQ ID NO: 230) TSTSGRSANPRG (SEQ ID NO: 61) 130, 132, 134 127, 128, 129 103 LEGWCLHPLCLWGAG (SEQ ID NO: 230) QNQALRMA (SEQ ID NO: 39) 130, 132, 134 127, 128, 129 104 LEGWCLHPLCLWGAG (SEQ ID NO: 230) ISSGLLSGRSDNH (SEQ ID NO : 7 0) 130, 132, 134 127, 128, 129 105 LEGWCLHPLCLWGAG (SEQ ID NO: 230) ISSGLLSGRSGNH (SEQ ID NO: 87) 130, 132, 134 127, 128, 129 106 LEGWCLHPLCLWGAG (SEQ ID NO: 230) ISSGLLSGRSANPRG (SEQ ID NO: 338) 130, 132, 134 127, 128, 129

- 37 041958

107 LEGWCLHPLCLWGAG (SEQ ID NO: 230) AVGLLAPPTSGRSANPRG (SEQ ID NO: 339) 130, 132, 134 127, 128, 129 108 LEGWCLHPLCLWGAG (SEQ ID NO: 230) AVGLLAPPSGRSANPRG (SEQ ID NO: 340) 130, 132, 134 127, 128, 129 109 LEGWCLHPLCLWGAG (SEQ ID NO: 230) ISSGLLSGRSDDH (SEQ ID NO: 341) 130, 132, 134 127, 128, 129 110 LEGWCLHPLCLWGAG (SEQ ID NO: 230) ISSGLLSGRSDDIH (SEQ ID NO: 342) 130, 132, 134 127, 128, 129 111 LEGWCLHPLCLWGAG (SEQ ID NO: 230) ISSGLLSGRSDQH (SEQ ID NO: 343) 130, 132, 134 127, 128, 129 112 LEGWCLHPLCLWGAG (SEQ ID NO: 230) ISSGLLSGRSDDIH (SEQ ID NO: 344) 130, 132, 134 127, 128, 129 113 LEGWCLHPLCLWGAG (SEQ ID NO: 230) ISSGLLSGRSDYH (SEQ ID NO: 345) 130, 132, 134 127, 128, 129 114 LEGWCLHPLCLWGAG (SEQ ID NO: 230) ISSGLLSGRSDNP (SEQ ID NO: 346) 130, 132, 134 127, 128, 129 115 LEGWCLHPLCLWGAG (SEQ ID NO: 230) ISSGLLSGRSANP (SEQ ID NO: 347) 130, 132, 134 127, 128, 129 116 LEGWCLHPLCLWGAG (SEQ ID NO: 230) ISSGLLSGRSANI (SEQ ID NO: 348) 130, 132, 134 127, 128, 129 117 LEGWCLHPLCLWGAG (SEQ ID NO: 230) ISSGLLSGRSDNI (SEQ ID NO: 357) 130, 132, 134 127, 128, 129 118 LEGWCLHPLCLWGAG (SEQ ID NO: 230) AVGLLAPPGGLSGRSDNH (SEQ ID NO: 76) 130, 132, 134 127, 128, 129 119 LEGWCLHPLCLWGAG (SEQ ID NO: 230) AVGLLAPPGGLSGRSDDDH (SEQ ID NO: 349) 130, 132, 134 127, 128, 129 120 LEGWCLHPLCLWGAG (SEQ ID NO: 230) AVGLLAPPGGLSGRSDIH (SEQ ID NO: 350) 130, 132, 134 127, 128, 129 121 LEGWCLHPLCLWGAG (SEQ ID NO: 230) AVGLLAPPGGLSGRSDQH (SEQ ID NO: 351) 130, 132, 134 127, 128, 129 122 LEGWCLHPLCLWGAG (SEQ ID NO: 230) AVGLLAPPGGLSGRSDIH (SEQ ID NO: 352) 130, 132, 134 127, 128, 129 123 LEGWCLHPLCLWGAG (SEQ ID NO: 230) AVGLLAPPGGLSGRSDYH (SEQ ID NO: 353) 130, 132, 134 127, 128, 129 124 LEGWCLHPLCLWGAG (SEQ ID NO: 230) AVGLLAPPGGLSGRSDNP (SEQ ID NO: 354) 130, 132, 134 127, 128, 129 125 LEGWCLHPLCLWGAG (SEQ ID NO: 230) AVGLLAPPGGLSGRSANP (SEQ ID NO: 355) 130, 132, 134 127, 128, 129 126 LEGWCLHPLCLWGAG (SEQ ID NO: 230) AVGLLAPPGGLSGRSANI (SEQ ID NO: 356) 130, 132, 134 127, 128, 129 127 LEGWCLHPLCLWGAG (SEQ ID NO: 230) AVGLLAPPGGLSGRSDNI (SEQ ID NO: 358) 130, 132, 134 127, 128, 129 128 LEGWCLHPLCLWGAG (SEQ ID NO: 230) ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 71) 130, 132, 134 127, 128, 129

- 38 041958

Table B

Components of activated antibodies against CD 166

Masking sequence (MM) Substrate sequence (CM) VL or CDR VL VH or CDR VF LCHPLVLSAWESCSS (SEQ ID NO: 219) LSGRSDNH (SEQ ID NO: 21) SEQ ID NO: 130, 132, 134 SEQID NO: 127, 128, 129 LCAPLVLSAWESCSS (SEQ ID NO: 220) TGRGPSWV (SEQ ID NO: 18) SEQID NO: 123 SEQID NO: 121 LCHALVLSAWESCSSS (SEQ ID NO: 221) PLTGRSGG (SEQ ID NO: 24) SEQID NO: 124 SEQID NO: 122 LCHPAVLSAWESCSSS (SEQ ID NO: 222) TARGPSFK (SEQ ID NO: 20) SEQID NO: 125 LCHPLALSAWESCSS (SEQ ID NO: 223) NTLSGRSENHSG (SEQ ID NO: 59) SEQID NO: 126 LCHPLVASAWESCSS (SEQ ID NO: 224) NTLSGRSGNHGS (SEQ ID NO: 60) SEQ ID NO: 131, 133, 134 LCHPLVLSAAESCSS (SEQ ID NO: 225) TSTSGRSANPRG (SEQ ID NO: 61) LCHPLVLSAWASSCSS (SEQ ID NO: 226) TSGRSANP (SEQ ID NO: 62) HPLVL (SEQ ID NO: 228) VHMPLGFLGP (SEQ ID NO: 32) HPL (SEQ ID NO: 229) AVGLAPP (SEQ ID NO: 52) LEGWCLHPLCLWGAG (SEQ ID NO: 230) AQNLLGMV (SEQ ID NO: 40) LEGACLHPLCLWGAG (SEQ ID NO: 231) QNQALRMA (SEQ ID NO: 39) LEGWCAHPLCLWGAG (SEQ ID NO: 232) LAAPLGLL (SEQ ID NO: 51) LEGWCLAPLCLWGAG (SEQ ID NO: 233) STPFGMF (SEQ ID NO: 41) LEGWCLHALCLWGAG (SEQ ID NO: 234) ISSGLLSS (SEQ ID NO: 44) LEGWCLHPACLWGAG (SEQ ID NO: 235) PAGLWLDP (SEQ ID NO: 54) LEGWCLHPLCAWGAG (SEQ ID NO: 236) VAGRSMRP (SEQ ID NO: 63) LEGWCLHPLCLAGAG (SEQ ID NO: 237) VVPEGRRS (SEQ ID NO: 64) CLHPLC (SEQ ID NO: 238) ILPRSPAF (SEQ ID NO: 65) MVLGRSLL (SEQ ID NO: 66) QGRAITFI (SEQ ID NO: 67) SPRSIMLA (SEQ ID NO: 68) SMLRSMPL (SEQ ID NO: 69) ISSGLLSGRSDNH (SEQ ID NO: 70) AVGLLAPPGGLSGRSDNH (SEQ ID NO: 76) ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 71) LSGRSGNH (SEQ ID NO: 318)

- 39 041958

SGRSANPRG (SEQ ID NO: 319) LSGRSDDH (SEQ ID NO: 320) LSGRSDIH (SEQ ID NO: 321) LSGRSDQH (SEQ ID NO: 322) LSGRSDTH (SEQ ID NO: 323) LSGRSDYH (SEQ ID NO: 324) LSGRSDNP (SEQ ID NO: 325) LSGRSANP (SEQ ID NO: 326) LSGRSANI (SEQ ID NO: 327) LSGRSDNI (SEQ ID NO: 328) MIAPVAYR (SEQ ID NO: 329) RPSPMWAY (SEQ ID NO: 330) WATPRPMR (SEQ ID NO: 331) FRLLDWQW (SEQ ID NO: 332) ISSGL (SEQ ID NO: 333) ISSGLLS (SEQ ID NO: 334) ISSGLL (SEQ ID NO: 335) ISSGLLSGRSANPRG (SEQ ID NO: 338) AVGLLAPPTSGRSANPRG (SEQ ID NO: 339) AVGLLAPPSGRSANPRG (SEQ ID NO: 340) ISSGLLSGRSDDH (SEQ ID NO: 341) ISSGLLSGRSDDIH (SEQ ID NO: 342) ISSGLLSGRSDQH (SEQ ID NO: 343) ISSGLLSGRSDTH (SEQ ID NO: 344) ISSGLLSGRSDYH (SEQ ID NO: 345) ISSGLLSGRSDNP (SEQ ID NO: 346) ISSGLLSGRSANP (SEQ ID NO: 347) ISSGLLSGRSANI (SEQ ID NO: 348) AVGLLAPPGGLSGRSDDDH (SEQ ID NO: 349) AVGLLAPPGGLSGRSDIH (SEQ ID NO: 350) AVGLLAPPGGLSGRSDQH (SEQ ID NO: 351) AVGLLAPPGGLSGRSDTH (SEQ ID NO: 352) AVGLLAPPGGLSGRSDYH (SEQ ID NO: 353) AVGLLAPPGGLSGRSDNP (SEQ ID NO: 354) AVGLLAPPGGLSGRSANP (SEQ ID NO: 355) AVGLLAPPGGLSGRSANI (SEQ ID NO: 356)

ISSGLLSGRSDNI (SEQ ID NO: 357) AVGLLAPPGGLSGRSDNI (SEQ ID NO: 358) GLSGRSDNHGGAVGLLAPP (SEQ ID NO: 336) GLSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 337)

In some embodiments, the implementation of the activated antibody according to the present description contains one or more polypeptides containing a combination of sequences selected from table. A or B, where the polypeptide contains a combination of a masking sequence selected from the column entitled Masking sequence (MM) from table. A or B, the substrate sequence from the column entitled Substrate sequence (SM), from the table. A or B, light chain variable domain or light chain CDR from the column headed VL or CDR VL or CDR VL, SEQ ID NO, from Table. A or B, and the heavy chain variable domain or heavy CDR

- 40 041958 chains from the column entitled VH or CDR VH or CDR VH, SEQ ID NO, from the table. A or B. For example, an activated antibody of the present disclosure may comprise amino acid sequences from combination #54, including a masking sequence from SEQ ID NO: 222, a substrate sequence from SEQ ID NO: 76, a light chain variable domain including the sequences VL CDRs of SEQ ID NOs: 130, 132, and 134, and a heavy chain variable domain comprising the sequences of VH CDRs of SEQ ID NOs: 127, 128, and 129. Thus, an activated antibody comprising at least a combination of sequences in any this line of the table. A is described in this document. Similarly, any combination of a masking sequence (MM), a substrate sequence (SM), a light chain variable sequence or light chain variable CDR sequences, and a heavy chain variable sequence or heavy chain variable CDR sequences from Table B, described in this document. An activated antibody comprising at least any combination of a masking sequence, a substrate sequence, a heavy chain variable region or a heavy chain variable region CDR, and a light chain variable region or a light chain variable region CDR selected from the appropriate columns of Table 1. A or B is also described herein. In some exemplary embodiments, an activated antibody comprising at least a combination of sequences in any given row of Table A or any combination of a masking sequence (MM), a substrate sequence (SM), a light chain variable sequence or light chain variable CDR sequences, and a heavy chain variable sequence or heavy chain variable CDR sequences from Table 1. B, may be combined with one or more toxins, including dolastatin or a derivative thereof, auristatin or a derivative thereof, maytansinoid or a derivative thereof, duocarmycin or a derivative thereof, calicheamicin or a derivative thereof, or pyrrolobenzodiazepine or a derivative thereof. In some exemplary embodiments, an activated antibody comprising at least a combination of sequences in any given row of Table A or any combination of a masking sequence (MM), a substrate sequence (SM), a light chain variable sequence or light chain variable CDR sequences, and a heavy chain variable sequence or heavy chain variable CDR sequences from Table 1. B, can be combined with one or more toxins, including auristatin E, monomethylauristatin F (MMAF), monomethylauristatin E (MMAE), monomethylauristatin D (MMAD), maytansinoid DM4, maytansinoid DM1, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer and/or duocarmycin.

Any of the combinations in Table. A or B, as described above, can be combined with human immunoglobulin constant regions to produce fully human IgGs, including IgG1, IgG2, IgG4, or with mutant constant regions to produce human IgGs with altered functions, such as IgG1 N297A, IgG1 N297Q, or IgG4 S228P. The combinations described in Table. A or B are not limited to the specific combinations shown in any given row, and thus may include any masking sequence from column 2 of Table 2. A (or column 1 of Table B) combined with any substrate sequence from column 3 of Table B. A (or column 2 of Table B) combined with any VL sequence or set of VL CDR sequences from column 4 of Table B. A (or column 3 of Table B) combined with any VH sequence or set of VH CDR sequences from column 5 of Table B. A (or column 4 from Table B). In addition to the masking sequences described in column 2 of Table. A or column 1 from the table. B, any masking sequence described herein may be used in combination. In addition to the substrate sequences described in column 3 of Table. A or column 2 from the table. B, any CM described herein may be used in combination. In addition to the light chain variable region sequence or light chain CDR sequences described in column 4 of Table A or column 3 from the table. B, any light chain variable region sequence or light chain CDR sequences described herein may be used in combination. In addition to the heavy chain variable region sequence or heavy chain CDR sequences described in column 5 of Table A or column 4 from the table. B, any heavy chain variable region sequence or heavy chain CDR sequences described herein may be used in combination.

In some embodiments, antibody drug conjugates (ADC) and activated antibody drug conjugates (AADC) may comprise one or more polypeptides comprising a combination of a light chain sequence or a light chain variable domain sequence and a heavy chain or heavy chain variable domain sequence. chain, linker and toxin in this line from the table. C or any combination of a light chain sequence or a light chain variable domain sequence and a heavy chain sequence or a heavy chain variable domain sequence, a linker and a toxin from Table. WITH.

- 41 041958

Table C

Combinations of Anti-CD166 ADC Antibodies and Activated Anti-CD166 ADC Antibodies

no. combination Heavy chain (HC) or variable region HC, SEQ ID NO. Light chain (LC) or variable region LC, SEQ ID NO. Linker Toxin 1 122 123 VC MMAD 2 122 123 PEG2-VC MMAD 3 122 123 VC MMAE 4 122 123 VC duocarmycin 5 122 123 spdb DM4 6 239 240 VC MMAD 7 239 240 PEG2-VC MMAD 8 239 240 VC MMAE 9 239 240 VC duocarmycin 10 239 240 spdb DM4 eleven 239 242 VC MMAD 12 239 242 PEG2-VC MMAD 13 239 242 VC MMAE 14 239 242 VC duocarmycin 15 239 242 spdb DM4 16 239 310 VC MMAD 17 239 310 PEG2-VC MMAD 18 239 310 VC MMAE 19 239 310 VC duocarmycin 20 239 310 spdb DM4 21 122 363 VC MMAD 22 122 363 PEG2-VC MMAD 23 122 363 VC MMAE 24 122 363 VC duocarmycin 25 122 363 spdb DM4 26 122 364 VC MMAD 27 122 364 PEG2-VC MMAD 28 122 364 VC MMAE 29 122 364 VC duocarmycin thirty 122 364 spdb DM4 31 239 244 VC MMAD 32 239 244 PEG2-VC MMAD 33 239 244 VC MMAE 34 239 244 VC duocarmycin

- 42 041958

35 239 244 spdb DM4 36 239 312 VC MMAD 37 239 312 PEG2-VC MMAD 38 239 312 VC MMAE 39 239 312 VC duocarmycin 40 239 312 spdb DM4 41 122 365 VC MMAD 42 122 365 PEG2-VC MMAD 43 122 365 VC MMAE 44 122 365 VC duocarmycin 45 122 365 spdb DM4 46 122 366 VC MMAD 47 122 366 PEG2-VC MMAD 48 122 366 VC MMAE 49 122 366 VC duocarmycin 50 122 366 spdb DM4 51 239 246 VC MMAD 52 239 246 PEG2-VC MMAD 53 239 246 VC MMAE 54 239 246 VC duocarmycin 55 239 246 spdb DM4 56 239 314 VC MMAD 57 239 314 PEG2-VC MMAD 58 239 314 VC MMAE 59 239 314 VC duocarmycin 60 239 314 spdb DM4 61 122 367 VC MMAD 62 122 367 PEG2-VC MMAD 63 122 367 VC MMAE 64 122 367 VC duocarmycin 65 122 367 spdb DM4 66 122 368 VC MMAD 67 122 368 PEG2-VC MMAD 68 122 368 VC MMAE 69 122 368 VC duocarmycin 70 122 368 spdb DM4 71 239 303 VC MMAD 72 239 303 PEG2-VC MMAD 73 239 303 VC MMAE 74 239 303 VC duocarmycin 75 239 303 spdb DM4 76 239 316 VC MMAD 77 239 316 PEG2-VC MMAD 78 239 316 VC MMAE 79 239 316 VC duocarmycin 80 239 316 spdb DM4 81 122 369 VC MMAD 82 122 369 PEG2-VC MMAD 83 122 369 VC MMAE

- 43 041958

84 122 369 VC duocarmycin 85 122 369 spdb DM4 86 122 370 VC MMAD 87 122 370 PEG2-VC MMAD 88 122 370 VC MMAE 89 122 370 VC duocarmycin 90 122 370 spdb DM4 91 239 387 VC MMAD 92 239 387 PEG2-VC MMAD 93 239 387 VC MMAE 94 239 387 VC duocarmycin 95 239 387 spdb DM4 96 239 388 VC MMAD 97 239 388 PEG2-VC MMAD 98 239 388 VC MMAE 99 239 388 VC duocarmycin 100 239 388 spdb DM4 101 122 389 VC MMAD 102 122 389 PEG2-VC MMAD 103 122 389 VC MMAE 104 122 389 VC duocarmycin 105 122 389 spdb DM4 106 122 390 VC MMAD 107 122 390 PEG2-VC MMAD 108 122 390 VC MMAE 109 122 390 VC duocarmycin 110 122 390 spdb DM4 111 239 391 VC MMAD 112 239 391 PEG2-VC MMAD 113 239 391 VC MMAE 114 239 391 VC duocarmycin 115 239 391 spdb DM4 116 239 392 VC MMAD 117 239 392 PEG2-VC MMAD 118 239 392 VC MMAE 119 239 392 VC duocarmycin 120 239 392 spdb DM4 121 122 393 VC MMAD 122 122 393 PEG2-VC MMAD 123 122 393 VC MMAE 124 122 393 VC duocarmycin 125 122 393 spdb DM4 126 122 394 VC MMAD 127 122 394 PEG2-VC MMAD 128 122 394 VC MMAE 129 122 394 VC duocarmycin 130 122 394 spdb DM4 131 239 395 VC MMAD 132 239 395 PEG2-VC MMAD

- 44 041958

133 239 395 VC ΜΜΆΕ 134 239 395 VC duocarmycin 135 239 395 spdb DM4 136 239 396 VC MMAD 137 239 396 PEG2-VC MMAD 138 239 396 VC ΜΜΆΕ 139 239 396 VC duocarmycin 140 239 396 spdb DM4 141 122 397 VC MMAD 142 122 397 PEG2-VC MMAD 143 122 397 VC ΜΜΆΕ 144 122 397 VC duocarmycin 145 122 397 spdb DM4 146 122 398 VC MMAD 147 122 398 PEG2-VC MMAD 148 122 398 VC ΜΜΆΕ 149 122 398 VC duocarmycin 150 122 398 spdb DM4 151 239 399 VC MMAD 152 239 399 PEG2-VC MMAD 153 239 399 VC ΜΜΆΕ 154 239 399 VC duocarmycin 155 239 399 spdb DM4 156 239 400 VC MMAD 157 239 400 PEG2-VC MMAD 158 239 400 VC ΜΜΆΕ 159 239 400 VC duocarmycin 160 239 400 spdb DM4 161 122 401 VC MMAD 162 122 401 PEG2-VC MMAD 163 122 401 VC ΜΜΆΕ 164 122 401 VC duocarmycin 165 122 401 spdb DM4 166 122 402 VC MMAD 167 122 402 PEG2-VC MMAD 168 122 402 VC ΜΜΆΕ 169 122 402 VC duocarmycin 170 122 402 spdb DM4 171 239 427 VC MMAD 172 239 427 PEG2-VC MMAD 173 239 427 VC ΜΜΆΕ 174 239 427 VC duocarmycin 175 239 427 spdb DM4 176 239 428 VC MMAD 177 239 428 PEG2-VC MMAD 178 239 428 VC ΜΜΆΕ 179 239 428 VC duocarmycin 180 239 428 spdb DM4 181 122 429 VC MMAD

- 45 041958

182 122 429 PEG2-vc MMAD 183 122 429 VC ΜΜΆΕ 184 122 429 VC duocarmycin 185 122 429 spdb DM4 186 122 430 VC MMAD 187 122 430 PEG2-VC MMAD 188 122 430 VC ΜΜΆΕ 189 122 430 VC duocarmycin 190 122 430 spdb DM4 191 239 431 VC MMAD 192 239 431 PEG2-VC MMAD 193 239 431 VC ΜΜΆΕ 194 239 431 VC duocarmycin 195 239 431 spdb DM4 196 239 432 VC MMAD 197 239 432 PEG2-VC MMAD 198 239 432 VC ΜΜΆΕ 199 239 432 VC duocarmycin 200 239 432 spdb DM4 201 122 433 VC MMAD 202 122 433 PEG2-VC MMAD 203 122 433 VC ΜΜΆΕ 204 122 433 VC duocarmycin 205 122 433 spdb DM4 206 122 434 VC MMAD 207 122 434 PEG2-VC MMAD 208 122 434 VC ΜΜΆΕ 209 122 434 VC duocarmycin 210 122 434 spdb DM4 211 239 435 VC MMAD 212 239 435 PEG2-VC MMAD 213 239 435 VC ΜΜΆΕ 214 239 435 VC duocarmycin 215 239 435 spdb DM4 216 239 436 VC MMAD 217 239 436 PEG2-VC MMAD 218 239 436 VC ΜΜΆΕ 219 239 436 VC duocarmycin 220 239 436 spdb DM4 221 122 437 VC MMAD 222 122 437 PEG2-VC MMAD 223 122 437 VC ΜΜΆΕ 224 122 437 VC duocarmycin 225 122 437 spdb DM4 226 122 438 VC MMAD 227 122 438 PEG2-VC MMAD 228 122 438 VC ΜΜΆΕ 229 122 438 VC duocarmycin 230 122 438 spdb DM4

- 46 041958

231 239 439 VC MMAD 232 239 439 PEG2-VC MMAD 233 239 439 VC MMAE 234 239 439 VC duocarmycin 235 239 439 spdb DM4 236 239 440 VC MMAD 237 239 440 PEG2-VC MMAD 238 239 440 VC MMAE 239 239 440 VC duocarmycin 240 239 440 spdb DM4 241 122 441 VC MMAD 242 122 441 PEG2-VC MMAD 243 122 441 VC MMAE 244 122 441 VC duocarmycin 245 122 441 spdb DM4 246 122 442 VC MMAD 247 122 442 PEG2-VC MMAD 248 122 442 VC MMAE 249 122 442 VC duocarmycin 250 122 442 spdb DM4 251 239 451 VC MMAD 252 239 451 PEG2-VC MMAD 253 239 451 VC MMAE 254 239 451 VC duocarmycin 255 239 451 spdb DM4 256 239 452 VC MMAD 257 239 452 PEG2-VC MMAD 258 239 452 VC MMAE 259 239 452 VC duocarmycin 260 239 452 spdb DM4 261 122 453 VC MMAD 262 122 453 PEG2-VC MMAD 263 122 453 VC MMAE 264 122 453 VC duocarmycin 265 122 453 spdb DM4 266 122 454 VC MMAD 267 122 454 PEG2-VC MMAD 268 122 454 VC MMAE 269 122 454 VC duocarmycin 270 122 454 spdb DM4 271 239 455 VC MMAD 272 239 455 PEG2-VC MMAD 273 239 455 VC MMAE 274 239 455 VC duocarmycin 275 239 455 spdb DM4 276 239 456 VC MMAD 277 239 456 PEG2-VC MMAD 278 239 456 VC MMAE 279 239 456 VC duocarmycin

- 47 041958

280 239 456 spdb DM4 281 122 457 VC MMAD 282 122 457 PEG2-VC MMAD 283 122 457 VC MMAE 284 122 457 VC duocarmycin 285 122 457 spdb DM4 286 122 458 VC MMAD 287 122 458 PEG2-VC MMAD 288 122 458 VC MMAE 289 122 458 VC duocarmycin 290 122 458 spdb DM4 291 239 459 VC MMAD 292 239 459 PEG2-vc MMAD 293 239 459 VC MMAE 294 239 459 VC duocarmycin 295 239 459 spdb DM4 296 239 460 VC MMAD 297 239 460 PEG2-VC MMAD 298 239 460 VC MMAE 299 239 460 VC duocarmycin 300 239 460 spdb DM4 301 122 461 VC MMAD 302 122 461 PEG2-VC MMAD 303 122 461 VC MMAE 304 122 461 VC duocarmycin 305 122 461 spdb DM4 306 122 462 VC MMAD 307 122 462 PEG2-VC MMAD 308 122 462 VC MMAE 309 122 462 VC duocarmycin 310 122 462 spdb DM4 311 239 463 VC MMAD 312 239 463 PEG2-VC MMAD 313 239 463 VC MMAE 314 239 463 VC duocarmycin 315 239 463 spdb DM4 316 239 464 VC MMAD 317 239 464 PEG2-VC MMAD 318 239 464 VC MMAE 319 239 464 VC duocarmycin 320 239 464 spdb DM4 321 122 465 VC MMAD 322 122 465 PEG2-VC MMAD 323 122 465 VC MMAE 324 122 465 VC duocarmycin 325 122 465 spdb DM4 326 122 466 VC MMAD 327 122 466 PEG2-VC MMAD 328 122 466 VC MMAE

- 48 041958

329 122 466 VC duocarmycin 330 122 466 spdb DM4 331 239 467 VC MMAD 332 239 467 PEG2-VC MMAD 333 239 467 VC MMAE 334 239 467 VC duocarmycin 335 239 467 spdb DM4 336 239 468 VC MMAD 337 239 468 PEG2-VC MMAD 338 239 468 VC MMAE 339 239 468 VC duocarmycin 340 239 468 spdb DM4 341 122 469 VC MMAD 342 122 469 PEG2-VC MMAD 343 122 469 VC MMAE 344 122 469 VC duocarmycin 345 122 469 spdb DM4 346 122 470 VC MMAD 347 122 470 PEG2-VC MMAD 348 122 470 VC MMAE 349 122 470 VC duocarmycin 350 122 470 spdb DM4 351 239 471 VC MMAD 352 239 471 PEG2-VC MMAD 353 239 471 VC MMAE 354 239 471 VC duocarmycin 355 239 471 spdb DM4 356 239 472 VC MMAD 357 239 472 PEG2-VC MMAD 358 239 472 VC MMAE 359 239 472 VC duocarmycin 360 239 472 spdb DM4 361 122 473 VC MMAD 362 122 473 PEG2-VC MMAD 363 122 473 VC MMAE 364 122 473 VC duocarmycin 365 122 473 spdb DM4 366 122 474 VC MMAD 367 122 474 PEG2-VC MMAD 368 122 474 VC MMAE 369 122 474 VC duocarmycin 370 122 474 spdb DM4

An antibody drug conjugate (ADC) as described herein, or an activated antibody drug conjugate (AADC) as described herein, may comprise one or more polypeptides comprising a combination of amino acid sequences, a linker, and a toxin listed in this row of Table 1. C. Thus, an antibody drug conjugate (ADC) as described herein, or an activated antibody drug conjugate (AADC) as described herein, comprising a combination of the amino acid sequences, linker, and toxin listed on this line or presented in the form of a specific combinations described in this document. For example, an activated antibody drug conjugate of the present disclosure may comprise amino acid sequences from combination #55, including a heavy chain containing the amino acid sequence of SEQ ID NO: 239, a light chain containing the amino acid sequence of SEQ ID NO: 246, and linker-toxin spdb-DM4. In another example of the AADCs disclosed and described herein, an activated antibody drug conjugate of the present disclosure may contain amino acid sequences from the combination of no. 33, including a heavy chain containing the amino acid sequence of SEQ ID NO: 239, a light chain containing the amino acid sequence of SEQ ID NO: 244, and a vc-MMAE toxin linker.

Any of the combinations in the table. C, which lists the variable regions of the heavy chain and light chain

- 49 041958 chains can be combined with human immunoglobulin constant regions to produce fully human IgG, including IgG1, IgG2, IgG4, or with mutant constant regions to produce human IgG with altered functions, such as IgG1 N297A, IgG1 N297Q or IgG4 S228P. The combinations described in Table. C are not limited to the specific combinations shown in any given row, and thus may include any heavy chain sequence or heavy chain variable region sequence from column 2 of Table 1. C combined with any light chain sequence or light chain variable region sequence from column 3 of Table C, combined with any linker from column 4, combined with any toxin from column 5. In addition to the heavy chain or heavy chain variable region sequences listed in column 2, any heavy chain or heavy chain variable region sequence described herein document can be used in combination. In addition to the light chain or light chain variable region sequences listed in column 3, any light chain or light chain variable region sequence described herein may be used in combination. In addition to the linkers listed in column 4, any linker described herein can be used in combination. In addition to the toxins listed in column 5, any toxin described herein may be used in combination.

In some embodiments, the serum half-life of the activated antibody is longer than that of the corresponding antibody; for example, the pK of the activated antibody is longer than that of the corresponding antibody. In some embodiments, the serum half-life of the activated antibody is similar to that of the corresponding antibody. In some embodiments, the serum half-life of the activated antibody is at least 15 days after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 12 days after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 11 days after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 10 days after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 9 days after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 8 days after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 7 days after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 6 days after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 5 days after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 4 days after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 3 days after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 2 days after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 24 hours after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 20 hours after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 18 hours after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 16 hours after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 14 hours after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 12 hours after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 10 hours after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 8 hours after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 6 hours after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 4 hours after administration to the body. In some embodiments, the serum half-life of the activated antibody is at least 3 hours after administration to the body.

In some embodiments, the CD166 activated antibody and/or the conjugated CD166 activated antibody is monospecific. In some embodiments, the CD166 activated antibody and/or the conjugated CD166 activated antibody is multispecific, e.g., as a non-limiting example, the bispecificity

- 50 041958 calic or trifunctional. In some embodiments, an activated anti-CD166 antibody and/or a conjugated activated anti-CD166 antibody are in the form of part of a bispecific T-cell-attracting activator (BITE) promolecule. In some embodiments, an activated CD166 antibody and/or a conjugated activated CD166 antibody is formulated as part of a chimeric antigen proreceptor (CAR), a modified T cell, or other engineered receptor.

In some embodiments, the activated antibody or antigen-binding fragment thereof is included in a multispecific activated antibody or antigen-binding fragment thereof, wherein at least one arm of the multispecific activated antibody specifically binds CD166. In some embodiments, the activated antibody or antigen-binding fragment thereof is included in a bispecific antibody or antigen-binding fragment thereof, wherein at least one arm of the bispecific activated antibody specifically binds CD166.

In some embodiments, the anti-CD166 antibodies, conjugated anti-CD166 antibodies, activated anti-CD166 antibodies, and/or conjugated activated anti-CD166 antibodies described herein are used in combination with one or more additional agents, or a combination of additional agents. Suitable additional agents include modern pharmaceutical and/or surgical therapeutic agents for the intended use, such as against cancer. For example, anti-CD166 antibodies, conjugated anti-CD166 antibodies, activated anti-CD166 antibodies, and/or conjugated activated anti-CD166 antibodies can be used in combination with an additional chemotherapeutic or antineoplastic agent.

In some embodiments, the additional agent(s) is a chemotherapeutic agent, such as a chemotherapeutic agent selected from the group consisting of docetaxel, paclitaxel, abraxane (i.e., albumin-conjugated paclitaxel), doxorubicin, oxaliplatin, carboplatin, cisplatin, irinotecan and gemcitabine.

In some embodiments, the additional agent(s) is(are) a checkpoint inhibitor, a kinase inhibitor, inhibitors of targeting the agent in the tumor microenvironment, and/or a T cell or NK agonist. In some embodiments, the additional agent(s) is(are) radiotherapy, alone or in combination with other additional agent(s), such as a chemotherapeutic agent or an antineoplastic agent. In some embodiments, the additional agent(s) is(are) a vaccine, an oncovirus, and/or a DC activating agent, such as, as a non-limiting example, a toll-like receptor (TLR) agonist and/or a-CD40 . In some embodiments, the additional agent(s) is a tumor-targeting antibody designed to kill a tumor by ADCC or by direct conjugation to a toxin (eg, an antibody drug conjugate (ADC).

In some embodiments, the checkpoint inhibitor is a target inhibitor selected from the group consisting of CTLA-4, LAG-3, PD-1, CD166, TIGIT, TIM-3, B7H4, and Vista. In some embodiments, the kinase inhibitor is selected from the group consisting of B-RAFi, MEKi, and Btk inhibitors, such as ibrutinib. In some embodiments, the kinase inhibitor is crizotinib. In some embodiments, the tumor microenvironment inhibitor is selected from the group consisting of an IDO inhibitor, an α-CSF1R inhibitor, an α-CCR4 inhibitor, TGF-beta, a myeloid-derived suppressor cell, or a regulatory T cell. In some embodiments, the agonist is selected from the group consisting of Ox40, GITR, CD137, ICOS, CD27, and HVEM.

In some embodiments, the inhibitor is a CTLA-4 inhibitor. In some embodiments, the inhibitor is a LAG-3 inhibitor. In some embodiments, the inhibitor is a PD-1 inhibitor. In some embodiments, the inhibitor is a CD166 inhibitor. In some embodiments, the inhibitor is a TIGIT inhibitor. In some embodiments, the inhibitor is TIM-3. In some embodiments, the inhibitor is a B7H4 inhibitor. In some embodiments, the inhibitor is a Vista inhibitor. In some embodiments, the inhibitor is a B-RAFi inhibitor. In some embodiments, the inhibitor is a MEKi inhibitor. In some embodiments, the inhibitor is a Btk inhibitor. In some embodiments, the inhibitor is ibrutinib. In some embodiments, the inhibitor is crizotinib. In some embodiments, the inhibitor is an IDO inhibitor. In some embodiments, the inhibitor is an a-CSF1R inhibitor. In some embodiments, the implementation of the inhibitor is an inhibitor of a-CCR4. In some embodiments, the inhibitor is TGF-beta. In some embodiments, the inhibitor is a suppressor cell of myeloid origin. In some embodiments,

- 51 041958 ion inhibitor is a regulatory T-cell.

In some embodiments, the agonist is Ox40. In some embodiments, the agonist is GITR. In some embodiments, the agonist is CD137. In some embodiments, the agonist is an ICOS. In some embodiments, the agonist is CD27. In some embodiments, the agonist is HVEM.

In some embodiments, the antibody, conjugated antibody, activated antibody, and/or conjugated CD166 activated antibody is administered during and/or after treatment in combination with one or more additional agents, such as, for example, a chemotherapeutic agent, an anti-inflammatory agent, and/or an immunosuppressive agent. means. In some embodiments, an anti-CD166 antibody, a conjugated anti-CD166 antibody, an activated anti-CD166 antibody, and/or a conjugated activated anti-CD166 antibody and an additional agent are constituted in one therapeutic composition, and an anti-CD166 antibody, an anti-CD166 conjugated antibody, an activated anti-CD166 antibody and/ or a conjugated anti-CD166 activated antibody and an additional agent are administered simultaneously. Alternatively, the anti-CD166 antibody, conjugated anti-CD166 antibody, activated anti-CD166 antibody and/or conjugated activated anti-CD166 antibody and the additional agent are separated from each other, e.g., each constituted in a separate therapeutic composition, and the anti-CD166 antibody, conjugated anti-CD166 antibody, Activated anti-CD166 antibody and/or conjugated activated anti-CD166 antibody and an additional agent are administered simultaneously, or Anti-CD166 antibody, conjugated anti-CD166 antibody, activated anti-CD166 antibody and/or conjugated activated anti-CD166 antibody and an additional agent are administered at different times during the course of treatment regimen. For example, anti-CD166 antibody, conjugated anti-CD166 antibody, activated anti-CD166 antibody, and/or conjugated activated anti-CD166 antibody is administered prior to administration of the additional agent, anti-CD166 antibody, conjugated anti-CD166 antibody, activated anti-CD166 antibody, and/or conjugated activated anti-CD166 antibody administered after administration of the additional agent, or an anti-CD166 antibody, a conjugated anti-CD166 antibody, an activated anti-CD166 antibody, and/or a conjugated activated anti-CD166 antibody, and the additional agent is administered in an alternating fashion. As described herein, an anti-CD166 antibody, a conjugated anti-CD166 antibody, an activated anti-CD166 antibody and/or a conjugated activated anti-CD166 antibody, and an additional agent are administered in single doses or in multiple doses.

In some embodiments, the anti-CD166 antibody, conjugated anti-CD166 antibody, activated anti-CD166 antibody, and/or conjugated activated anti-CD166 antibody, and additional agent(s) are administered simultaneously. For example, an anti-CD166 antibody, a conjugated anti-CD166 antibody, an activated anti-CD166 antibody and/or a conjugated activated anti-CD166 antibody, and additional agent(s) may be formulated in one composition or administered in the form of two or more separate compositions. In some embodiments, the anti-CD166 antibody, conjugated anti-CD166 antibody, activated anti-CD166 antibody and/or conjugated activated anti-CD166 antibody and additional agent(s) are administered sequentially, or anti-CD166 antibody, conjugated anti-CD166 antibody, activated anti-CD166 antibody and/ or a conjugated anti-CD166 activated antibody and an additional agent are administered at different times during the course of the treatment regimen.

In some embodiments, the anti-CD166 antibody, conjugated anti-CD166 antibody, activated anti-CD166 antibody, and/or conjugated activated anti-CD166 antibody is administered during and/or after treatment in combination with one or more additional agents such as, but not limited to, a chemotherapeutic agent. an agent, an anti-inflammatory agent, and/or an immunosuppressive agent such as an alkylating agent, an antimetabolite, an anti-microtubule agent, a topoisomerase inhibitor, a cytotoxic antibiotic, and/or any other nucleic acid damaging agent. In some embodiments, the additional agent is a taxane, such as paclitaxel (eg, abraxane®). In some embodiments, the additional agent is an antimetabolite such as gemcitabine. In some embodiments, the additional agent is an alkylating agent such as a platinum-based chemotherapeutic agent such as carboplatin or cisplatin. In some embodiments, the additional agent is a targeting agent such as a kinase inhibitor such as sorafenib or erlotinib. In some embodiments, the additional agent is a targeting agent, such as another antibody, such as a monoclonal antibody (eg, bevacizumab), a bispecific antibody, or a multispecific antibody. In some embodiments, the additional agent is a proteasome inhibitor such as bortezomib or carfilzomib. In some embodiments, the additional agent is an immunomodulatory agent such as lenolidomide or IL-2. In some embodiments, the additional agent is radiation exposure. In some embodiments, the additional agent is an agent considered by those skilled in the art as a standard of care. In some embodiments, the additional agent is a chemotherapeutic agent well known to those skilled in the art.

In some embodiments, the additional agent is another antibody or antigen-binding fragment thereof, another conjugated antibody or antigen-binding fragment thereof, another activated antibody or antigen-binding fragment thereof, and/or another conjugated activated antibody or antigen-binding fragment thereof. In some embodiments, the additional agent is another antibody or antigen binding fragment thereof, another conjugated antibody or antigen binding fragment thereof, another activated antibody or antigen binding fragment thereof, and/or another conjugated activated antibody or antigen binding fragment thereof against the same target as the first antibody. or an antigennegative fragment thereof, a first conjugated antibody or antigennegative fragment thereof, an activated antibody or antigennegative fragment thereof, and/or a conjugated activated antibody or antigennegative fragment thereof, for example against CD166. In some embodiments, the additional agent is another antibody or antigen binding fragment thereof, another conjugated antibody or antigen binding fragment thereof, another activated antibody or antigen binding fragment thereof, and/or another conjugated activated antibody or antigen binding fragment thereof against a target other than that of the first antibody, or its antigennegative fragment, the first conjugated antibody or its antigennegative fragment, the activated antibody or its antigennegative fragment and/or the conjugated activated antibody or its antigennegative fragment.

As a non-limiting example, an antibody or antigen-binding fragment and/or an AB-activated antibody is a binding partner for any of the targets listed in Table 1. 1.

Table 1

Illustrative targets

1-92-LFA-3 CD52 DL44 HVEM LIF-R STEAP1 Integrin alpha-4 CD56 DLK1 Hyaluronidase Lewis X STEAP2 Integrin alpha-V CD64 DLL4 IGOS LIGHT TAG-72 Integrin alpha4beta! CD70 DPP-4 TRIALFA LRP4 TAPA1 Integrin alpha4beta7 CD71 DSG1 IFN6eTa LRRC26 TGF6eTa AGR2 CD74 EGFR IFNgamma MCSP TIGIT Anti-LewisY EGFRviii IgE mesothelium TIM-3 Apelin J receptor CD80 Endothelin receptor (ETBR) IgE B receptor (FceRI) MRP 4 TLR2 APRIL CD81 ENPP3 IGF MUGI TLR4 B7-H4 CD86 Yer SAM IGF1R Mucin-16 (MUGI6, CA125) TLR6 BAFF CD95 ERHA2 IL1B Na/K ATPase TLR7 BT LA CD117 ERNV2 ILIR Neutrophil elastase TLR8 Complement component C5 CD125 ERBB3 IL2 NGF TLR9 S-242 CD132 (IL-2RG) F protein of RSV IL11 Nikastrin TMEM31 SA9 CD133 FAP IL12 Notch receptors TIEalpha SA19-9 (Lewis a) CD137 FGF-2 IL12p40 Notch 1 TNFR Carbonic anhydrase 9 CD138 FGF8 IL-12R, IL12R6eTal Notch 2 TNFRS12A CD2 CD166 FGFR1 IL13 Notch 3 TRAIL-RI CD3 CD172A FGFR2 IL13R Notch 4 TRAIL-R2 CD6 CD248 FGFR3 IL15 NOV Transferrin CD9 CDH6 FGFR4 IL17 OSM-R Transferrin a receptor

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CDlla SEACAM5 (CEA) folate receptor IL18 OX-4 0 TRK-A CD19 CEACAM6 (NCA90) GAL3ST1 IL21 PAR2 TRK-B CD2 0 CLAUDIA-3 G-CSF IL23 PDGF-AA uPAR CD22 CLAUDIA-4 G-CSFR IL23R PDGF-BB VAP1 CD24 cmet GD2 IL27/IL27R (wsxl) PDGFRalφa VCAM-1 CD25 Collagen GITR IL29 PDGFR6eTa VEGF CD27 Crypto GLUT1 IL-31R PD-1 VEGF-A CD2 8 CSFR GLUT4 IL31/IL31R PD-L1 VEGF-B CD30 CSFR-1 GM-CSF IL2R PD-L2 VEGF-C CD33 CT LA-4 GM-CSFR IL4 Phosphatidylserine VEGF-D CD38 CTGF GP receptors Ilb/IIIa IL4R P1GF VEGFR1 CD40 CXCL10 Gpl30 IL6, IL6R PSCA VEGFR2 CD40L CXCL13 GPIIB/IIIA insulin receptor PSMA VEGFR3 CD41 CXCR1 GPNMB Jagged ligands RAAG12 VISTA CD44 CXCR2 GRP7 8 Jagged 1 RAGE WISP-1 CD44v6 HER2/neu Jagged 2 SLC44A4 WISP-2 CD47 CXCR4 HGF LAG-3 Sphingosine-1phosphate WISP-3 CD51 CYR61 hGH

As a non-limiting example, the antibody or antigen-binding fragment and/or AB of an activated antibody is or is derived from an antibody listed in Table 1. 2.

table 2

Illustrative Ab sources

Trade name of the antibody (antibody name) Target Avastin™ (bevacizumab) VEGF Lucentis™ (ranibizumab) VEGF Erbitux™ (cetuximab) EGFR Vectibix™ (panitumumab) EGFR Remicade™ (infliximab) TNFa Humira™ (adalimumab) TNFa Tysabri™ (natalizumab) Integrin4 Simulect™ (basiliximab) IL2R Soliris™ (eculizumab) Complement component C5 Raptiva™ (efalizumab) CDlla Bexar™ (tositumomab) CD2 0 Zevalin™ (tiuxetan ibritutomaba) CD2 0 Rituxan™ (rituximab) CD2 0 Ocrelizumab CD2 0 Arzerra™ (ofatumumab) CD2 0 Gaziva™ (obinutuzumab) CD2 0 Zenapax™ (daclizumab) CD25 Adcetris™ (vedotin brentuximab) CD30 Myelotarg™ (gemtuzumab) CD33 Mylotarg™ (ozogamicin gemtuzumab) CD33 Campat™ (alemtuzumab) CD52 ReoPro™ (abciximab) Ilb/IIIa glycoprotein receptor Xolair™ (omalizumab) IgE Herceptin™ (trastuzumab) Her2

- 54 041958

Kadcyla™ (emtansine trastuzumab) Neg2 Synagis™ (palivizumab) Protein F RSV (ipilimumab) CTLA-4 (tremelimumab) CTLA-4 Ni5s8 CD40L (pertuzumab) Her2-neu (ertumaxomab) CD3/Her2-neu Orencia™ (abatacept) CTLA-4 (tanezumab) NGF (bavituximab) Phosphatidylserine (zalutumumab) EGFR (mapatumumab) EGFR (matuzumab) EGFR (nimotuzumab) EGFR ICR62 EGFR mAb 528 EGFR CH806 EGFR MDX-447 EGFR/CD64 (edrecolomab) Epcam RAV12 RAAG12 huJ591 PSMA Enbrel™ (etanercept) TNF-R Ameviv™ (alefacept) 1-92-LFA-3 Antril™, Kinneret™ (anakinra) IL-IRa GC1008 Tbebeta Notch, e.g. Notch 1 Jagged 1 or Jagged 2 (adecatumumab) Epcam (figitumumab) IGF1R (tocilizumab) IL-6 receptor Stelara™ (ustekinumab) IL-12/IL-23 Prolia™ (denosumab) RANKL

In some embodiments, the additional antibody or antigen-binding fragment thereof, the conjugated antibody or antigen-binding fragment thereof, the activated antibody or antigen-binding fragment thereof, and/or the conjugated activated antibody or antigen-binding fragment thereof is a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, a fragment F(ab')2, scFv, scAb, dAb, heavy chain single domain antibody, or light chain single domain antibody. In some embodiments, the additional antibody or antigen binding fragment thereof, the conjugated antibody or antigen binding fragment thereof, the activated antibody or antigen binding fragment thereof, and/or the conjugated activated antibody or antigen binding fragment thereof is a mouse, other rodent, chimeric, humanized, or fully human monoclonal antibody. antibody.

The description also relates to methods for producing an anti-CD166 antibody polypeptide and/or an activated anti-CD166 antibody by culturing a cell under conditions allowing expression of the polypeptide, wherein the cell contains an isolated nucleic acid molecule encoding an antibody and/or an activated antibody described herein, and/ or vectors containing these isolated nucleic acid sequences. The description relates to methods for producing an antibody and/or an activated antibody by culturing a cell under conditions that allow expression of the antibody and/or an activated antibody, wherein the cell contains an isolated nucleic acid molecule encoding an antibody and/or an activated antibody described herein and/or vectors containing these isolated nucleic acid sequences.

The invention also relates to a method for making activated antibodies that bind CD166 in an activated state by (a) culturing a cell containing a nucleic acid construct encoding an activated antibody under conditions that allow expression of an activated antibody, wherein the activated antibody contains a masking group (MM), a cleavable group (CM) and an antibody or antigen-binding fragment (AB) thereof specifically binding CD 166, (i) wherein CM is a polypeptide that functions as a substrate for a protease; and (ii) where the SM is located in the activated antibody such that when the activated antibody is in an uncleaved state, the MM interferes with the specific binding of AB to CD166, and in the cleaved state, the MM does not interfere with or compete with the specific binding of AB to CD166; and (b) isolating the activated antibody. Suitable AB, MM, and/or CM include any of

- 55 041958

AB, MM and/or CM as described herein.

In some embodiments, the activated antibody in the uncleaved state has the following N-terminal to C-terminal structural arrangement: MM-CM-AB or AB-CM-MM. In some embodiments, the implementation of the activated antibody contains a binding peptide between MM and SM. In some embodiments, the activated antibody comprises a binding peptide between the CM and AB. In some embodiments, the activated antibody comprises a first binding peptide (LP1) and a second binding peptide (LP2), and wherein the activated antibody in an uncleaved state has the following N-terminal to C-terminal structural arrangement: MM-LP1-CMLP2-AB or AB -LP2-CM-LP1-MM. In some embodiments, the two binding peptides need not be identical to each other. In some embodiments, the activated antibody in the uncleaved state has the following N-terminal to C-terminal structural arrangement: spacer-MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM-spacer.

In some embodiments, at least one of LP1 or LP2 contains an amino acid sequence selected from the group consisting of (GS) _n , (GGS) _n , (GSGGS) _n (SEQ ID NO: 1) and (GGGS) _n (SEQ ID NO: 2), where n is an integer, at least one.

In some embodiments, at least one of LP1 or LP2 contains an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7) and GSSSG (SEQ ID NO: 8).

In some embodiments, LP1 contains the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10), GSSGGSGGSGGS (SEQ ID NO: 11), GSSGGSGGSGGSGGGS (SEQ ID NO: 12), GSSGGSGGSG (SEQ ID NO: 12), 13) or GSSGGSGGSGS (SEQ ID NO: 14).

In some embodiments, LP2 contains the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 15), GSSGT (SEQ ID NO: 16), or GSSG (SEQ ID NO: 17).

The invention relates to methods for preventing, delaying the progression, treating, reducing a symptom, or otherwise alleviating a CD166-mediated disease in a subject by administering a therapeutically effective amount of an anti-CD166 antibody, an anti-CD166 conjugated antibody, an anti-CD166 anti-antibody and/or an anti-CD166 conjugated anti-CD166 anti-activated antibody described in this document to a subject in need.

The invention also relates to methods for preventing, delaying the progression, treating, reducing a symptom, or otherwise alleviating a cancer in a subject by administering a therapeutically effective amount of an anti-CD166 antibody, an anti-CD166 conjugated antibody, an anti-CD166 anti-antibody, and/or an anti-CD166 conjugated anti-CD166 antibody described in this document to a subject in need. CD166 is known to be expressed in a variety of cancers, such as, by way of non-limiting example, any epithelial or squamous cell cancer, any carcinoid and/or neuroendocrine cancer. Examples of cancers include, but are not limited to, adenocarcinoma, bile duct (biliary) cancer, bladder cancer, breast cancer, e.g. triple negative breast cancer, Her2 negative breast cancer, estrogen receptor positive breast cancer , carcinoid malignant tumor; cervical cancer; cholangiocarcinoma; colorectal; endometrial; glioma; head and neck cancer, such as squamous cell carcinoma of the head and neck; leukemia; liver cancer; lung cancer, eg NSCLC, SCLC; lymphoma; melanoma; oropharyngeal cancer; ovarian cancer; pancreas cancer; prostate cancer, eg, metastatic castration-resistant carcinoma of the prostate; kidney cancer; skin cancer; squamous cell carcinoma, stomach cancer; testicular cancer; thyroid cancer; and urothelial cancer.

In some embodiments, the cancer is any epithelial or squamous cell cancer. In some embodiments, the cancer is prostate cancer, breast cancer, lung cancer, cervical cancer, oropharyngeal cancer, and/or head and neck cancer.

In some embodiments, the cancer is bladder cancer, bone cancer, breast cancer, carcinoid cancer, cervical cancer, colorectal cancer, colon cancer, endometrial cancer, epithelial cancer, glioma, head and neck cancer, liver cancer. , lung cancer, melanoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, genitourinary cancer, and/or urothelial cancer.

In some embodiments, the cancer is selected from the group consisting of triple negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), Ras-mutated colorectal carcinoma, rare types of epithelial cancers, oropharyngeal cancer, cervical cancer, head and neck squamous cell carcinoma (HNSCC), and/or prostate cancer. In some embodiments, the malignant tumor is associated with a CD166-expressing tumor. In some embodiments, the cancer is due to a CD166-expressing tumor.

The anti-CD166 antibody, anti-CD166 conjugated antibody, activated anti-CD166 antibody, and/or conjugated anti-CD166 activated antibody used in any of the embodiments of these methods and uses can be administered at any stage of the disease. For example, such an anti-CD166 antibody, a conjugated anti-CD166 antibody, an activated anti-CD166 antibody, and/or a conjugated activated anti-CD166 antibody may be administered to a patient suffering from any stage of cancer from early to metastatic. The terms subject and patient are used interchangeably herein.

In some embodiments, the subject is a mammal, such as a human, non-human primate, companion animal (eg, cat, dog, horse), farm animal, working animal, or zoo animal. In some embodiments, the subject is a human. In some embodiments, the subject is a companion animal. In some embodiments, the subject is an animal under the supervision of a veterinarian.

An anti-CD166 antibody, a conjugated anti-CD166 antibody, an activated anti-CD166 antibody, and/or a conjugated activated anti-CD166 antibody and their therapeutic formulations are administered to a subject suffering from a disease or disorder or susceptible to a disease or disorder associated with altered expression and/or activity of CD166. A subject suffering from or susceptible to a disease or disorder associated with altered CD166 expression and/or activity is identified using any of a variety of methods known in the art. For example, subjects suffering from cancer or other neoplastic conditions are identified using any of a variety of clinical and/or laboratory tests, such as a physical examination and blood, urine, and/or stool testing to assess health status. For example, subjects suffering from inflammation and/or an inflammatory disorder are identified using any of a variety of clinical and/or laboratory tests, such as a physical examination and/or body fluid analysis, such as a blood, urine, and/or stool test, to assess the condition. health.

Administration of an anti-CD166 antibody, an anti-CD166 conjugated antibody, an anti-CD166 conjugated antibody, and/or a conjugated anti-CD166 antibody to a patient suffering from a disease or disorder associated with altered expression and/or activity of CD166 is considered successful if any of a variety of laboratory or clinical findings are achieved. goals. For example, administration of an anti-CD166 antibody, an anti-CD166 conjugated antibody, an anti-CD166 conjugated antibody, and/or a conjugated anti-CD166 antibody to a patient suffering from a disease or disorder associated with altered CD166 expression and/or activity is considered successful if one or more of the symptoms, associated with a disease or disorder are alleviated, reduced, inhibited, or do not progress to the next, i.e. worst, condition. Administration of an anti-CD166 antibody, an anti-CD166 conjugated antibody, an anti-CD166 conjugated antibody, and/or a conjugated anti-CD166 antibody to a patient suffering from a disease or disorder associated with altered expression and/or activity of CD166 is considered successful if the disease or disorder achieves remission or does not progresses to the next, i.e. worst, condition.

In some embodiments, an anti-CD166 antibody, an anti-CD166 conjugated antibody, an anti-CD166 conjugated antibody, and/or an anti-CD166 conjugated activated antibody and their therapeutic formulations are administered to a subject suffering from or susceptible to the disease or disorder, such as subjects suffering from cancer. or other neoplastic condition, where diseased cells of the subject express CD166. In some embodiments, diseased cells are associated with altered CD166 expression and/or activity. In some embodiments, diseased cells are associated with normal CD166 expression and/or activity. A subject suffering from a disease or disorder, or susceptible to the disease or disorder, wherein diseased cells of the subject expressing CD166 are identified using any of a variety of methods known in the art. For example, subjects suffering from cancer or other neoplastic conditions are identified using any of a variety of clinical and/or laboratory tests, such as a physical examination and blood, urine, and/or stool testing to assess health status. For example, subjects suffering from inflammation and/or an inflammatory disorder are identified using any of a variety of clinical and/or laboratory tests, such as a physical examination and/or body fluid analysis, such as a blood, urine, and/or stool test, to assess the condition. health.

In some embodiments, an anti-CD166 antibody, a conjugated anti-CD166 antibody, an anti-CD166 conjugated antibody, and/or a conjugated anti-CD166 antibody.

- 57 041958

CD166 and their therapeutic formulations are administered to a subject suffering from or susceptible to a disease or disorder associated with cells expressing CD166 or the presence, growth, proliferation, metastasis and/or activity of such cells, such as subjects suffering from a malignant tumor or other neoplastic condition. In some embodiments, cells are associated with altered expression and/or activity of CD166. In some embodiments, the cells are associated with normal CD166 expression and/or activity. A subject suffering from or susceptible to a disease or disorder associated with cells expressing CD166 is identified using any of a variety of methods known in the art. For example, subjects suffering from cancer or other neoplastic conditions are identified using any of a variety of clinical and/or laboratory tests, such as a physical examination and blood, urine, and/or stool testing to assess health status. For example, subjects suffering from inflammation and/or an inflammatory disorder are identified using any of a variety of clinical and/or laboratory tests, such as a physical examination and/or body fluid analysis, such as a blood, urine, and/or stool test, to assess health status. .

Administration of an anti-CD166 antibody, an anti-CD166 conjugated antibody, an activated anti-CD166 antibody, and/or a conjugated anti-CD166 antibody to a patient suffering from a disease or disorder associated with CD166 expressing cells is considered successful if any of a variety of laboratory or clinical goals are achieved. For example, administration of an anti-CD166 antibody, an anti-CD166 conjugated antibody, an anti-CD166 conjugated antibody, and/or a conjugated anti-CD166 antibody to a patient suffering from a disease or disorder associated with CD166-expressing cells is considered successful if one or more of the symptoms associated with the disease or impaired, alleviated, reduced, inhibited, or not progressing to the next, i.e. worst, condition. Administration of an anti-CD166 antibody, an anti-CD166 conjugated antibody, an anti-CD166 conjugated antibody, and/or a conjugated anti-CD166 antibody to a patient suffering from a disease or disorder associated with CD166-expressing cells is considered successful if the disease or disorder achieves remission or does not progress to the next , i.e. worst, condition.

In some embodiments, the CD166 antibody, CD166 conjugated antibody, CD166-activated antibody, and/or CD166-conjugated activated antibody is administered during and/or after treatment in combination with one or more additional agents, such as, for example, a chemotherapeutic agent, an anti-inflammatory agent. agent, and/or immunosuppressive agent. In some embodiments, the anti-CD166 antibody, conjugated anti-CD166 antibody, activated anti-CD166 antibody, and/or conjugated activated anti-CD166 antibody, and additional agent(s) are administered simultaneously. For example, an anti-CD166 antibody, a conjugated anti-CD166 antibody, an activated anti-CD166 antibody and/or a conjugated activated anti-CD166 antibody, and additional agent(s) can be formulated in one composition or administered in the form of two or more separate compositions. In some embodiments, the anti-CD166 antibody, conjugated anti-CD166 antibody, activated anti-CD166 antibody, and/or conjugated activated anti-CD166 antibody, and additional agent(s) are administered sequentially.

The invention also relates to methods and kits for the use of activated anti-CD166 antibodies and/or conjugated activated anti-CD166 antibodies in a variety of diagnostic and/or prophylactic indications. For example, the invention relates to methods and kits for detecting the presence or absence of a cleavage agent and a target of interest in a subject or sample by (i) contacting the subject or sample with an activated anti-CD166 antibody, wherein the activated anti-CD166 antibody contains a masking group (MM ), a cleavable group (CM) cleavable by a cleaving agent, and an antigen-binding domain or fragment thereof (AB) specifically binding to a target of interest, wherein the activated anti-CD166 antibody in the uncleaved, non-activated state has the following structural arrangement from the N-terminus to the C- end: MM-CM-AB or AB-CM-MM;

(a) where MM is a peptide that inhibits AB binding to CD166, and where MM does not have the amino acid sequence of a natural AB binding partner and is not a modified form of a natural AB binding partner; and (b) where, when the AV is in a non-cleaved, non-activated state, the MM interferes with the specific binding of the AB to CD166, and when the AV is in the cleaved, activated state, the MM does not interfere with or compete with the specific binding of the AV to CD166; and (ii) measuring the level of activated anti-CD166 antibody in the subject or sample, wherein the detectable level of activated anti-CD166 antibody in

- 58 041958 subject or sample indicates that a cleavage agent and CD166 are present in the subject or sample, and where an undetectable level of activated anti-

CD166 in the subject or in the sample indicates that the cleaving agent, CD166, or both the cleaving agent and CD166 are not present in the subject or in the sample.

In some embodiments, the activated anti-CD166 antibody is an activated anti-CD166 antibody conjugated to a drug. In some embodiments, the activated anti-CD166 antibody is not conjugated to the agent. In some embodiments, the implementation of the activated antibody against CD166 contains a detectable label. In some embodiments, a detectable label is located on the AB. In some embodiments, the level of activated CD166 antibody in a subject or sample is measured using a secondary reagent that specifically binds the activated antibody, wherein the reagent contains a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

In some embodiments of these methods and kits, the activated anti-CD166 antibody contains a detectable label. In some embodiments of these methods and kits, the detectable label includes an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments of these methods and kits, the imaging agent contains a radioactive isotope. In some embodiments of these methods and kits, the radioactive isotope is indium or technetium. In some embodiments of these methods and kits, the contrast agent contains iodine, gadolinium, or iron oxide. In some embodiments of these methods and kits, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments of these methods and kits, the fluorescent label comprises yellow fluorescent protein (YFP), blue fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein (RFP) t-dimer-2 tdimer2), HCRED or europium derivative. In some embodiments of these methods and kits, the luminescent label contains an N-methylacridinium derivative. In some embodiments of these methods, the label comprises an Alexa Fluor® label, such as Alexa Fluor® 680 or Alexa Fluor® 750. In some embodiments of these methods and kits, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.

In some embodiments of these methods and kits, the subject is a mammal. In some embodiments of these methods, the subject is a human. In some embodiments, the subject is a non-human mammal, such as a non-human primate, companion animal (eg, cat, dog, horse), farm animal, working animal, or zoo animal. In some embodiments, the subject is a rodent.

In some embodiments of these methods and kits, the method is an in vivo method. In some embodiments of these methods, the method is an in situ method. In some embodiments of these methods, the method is an ex vivo method. In some embodiments of these methods, the method is an in vitro method.

In some embodiments of the methods and kits, the method is used to identify or otherwise refine a population of patients suitable for treatment with an activated CD166 antibody as described herein, followed by treatment with the administration of that activated CD166 antibody and/or a conjugated, activated CD166 antibody. subject in need. For example, patients who test positive for both the target (eg, CD166) and the substrate-cleaving protease in the cleavable group (CM) of an activated anti-CD166 antibody when tested by these methods are identified as suitable candidates for treatment with that activated anti-CD166 antibody. containing such SM, and then the patient is administered a therapeutically effective amount of an activated anti-CD166 antibody and/or a conjugated activated anti-CD166 antibody that has been tested. Similarly, patients who test negative for either or both of the target (eg, CD166) and the CM substrate-cleaving protease in the activated antibody tested using these methods can be identified as suitable candidates for another form of therapy. In some embodiments, such patients can be tested with other CD166 activated antibodies until a suitable CD166 activated antibody for treatment is identified (eg, an activated CD166 antibody containing CM cleaved by the patient at the disease site). In some embodiments, the patient is then administered a therapeutically effective amount of an activated and/or conjugated anti-CD166 antibody for which the patient has tested positive. Suitable AB, MM and/or CM include

- 59 041958 any of the AB, MM and/or CM described in this document.

Pharmaceutical compositions of the invention may contain an antibody of the invention and a carrier. These pharmaceutical compositions can be included in kits such as diagnostic kits.

Brief description of the drawings

Fig. 1 is a graph showing the binding of various anti-CD166 antibodies in the present to human CD166 protein.

Fig. 2 is a schematic representation of a masking peptide selection scheme for anti-CD166 antibodies of the present disclosure. The boxes show the populations with the sort options specified between the boxes. All FACS sorts were performed in 0.5% BSA with excess anti-mouse isotype antibodies to block non-specific binding to labeled Alexa Fluor 488 Mab M9 (VH of SEQ ID NO: 119, VL of SEQ ID NO: 120).

Fig. 3A and 3B are a series of graphs showing the ability of an anti-CD166 CD166 M9 vK1/HcB antibody (VH of SEQ ID NO: 121, VL of SEQ ID NO: 123) as described herein and various activated anti-CD166 antibodies as described herein to bind human CD166. .

Fig. 4 is a graph showing the ability of various activated anti-CD166 antibodies of this description to bind human CD166 upon proteolytic activation.

Fig. 5 is a graph showing the ability of various conjugated anti-CD166 activated antibodies of this description to bind human CD166 upon proteolytic activation.

Fig. 6A-6D are series of images showing that activated anti-CD166 antibody is activated (ie, cleaved) in colon cancer tissue samples and activated anti-CD166 antibody is not activated in healthy tissue samples. In FIG. 6A and 6C depict the results of IHC analysis of tumor and healthy tissue samples, and FIG. 6B and 6D depict the results of in situ imaging analysis of tumor and healthy tissue samples.

Fig. 7A-7D are series of images showing that activated anti-CD166 antibody is activated (ie, cleaved) in lung cancer tissue samples and activated anti-CD166 antibody is not activated in healthy tissue samples. In FIG. 7A and 7C depict the results of IHC analysis of tumor and healthy tissue samples, and FIG. 7B and 7D depict the results of in situ imaging analysis of tumor and healthy tissue samples.

Fig. 8A-8D are a series of images showing that the activated anti-CD166 antibody is not activated in healthy tissue samples. In FIG. 8A and 8C depict the results of IHC analysis of healthy tissue samples, and FIG. 8B and 8D depict the results of an in situ imaging analysis of healthy tissue samples.

Fig. 9 is a series of graphs showing the activity of an anti-CD166 conjugated antibody of this description against a breast cancer cell line, a prostate cancer cell line, a pancreatic cancer cell line, a head and neck squamous cell line, and a negative control cell line without CD166. .

Fig. 10 is a graph showing the efficacy of a conjugated anti-CD166 antibody (AADC, Activatable Antibody Drug Complex) as described herein in a breast cancer model.

Fig. 11 is a graph showing the performance of the CD166 AADC DM4 conjugate as described in the H292 non-small cell lung cancer (NSCLC) model.

Fig. 12 is a graph showing the performance of the CD166 AADC DM4 conjugate as described in the H1975 non-small cell lung cancer (NSCLC) model.

Fig. 13 is a graph showing the ability of anti-CD166 antibodies of this description to bind human and cynomolgus monkey CD166 with equal affinity.

Fig. 14 is a graph showing the results of a tolerability study in cynomolgus monkeys using an activated anti-CD166 antibody of this specification.

Fig. 15 is a graph showing that an anti-CD166 conjugated activated antibody of the present disclosure is well tolerated at the intended therapeutic dose.

Fig. 16A, 16B and 16C are a series of graphs showing the ability of various conjugated activated anti-CD166 antibodies as described herein to bind human CD166 when such conjugated activated antibodies are proteolytically activated.

Fig. 17 is a graph showing the ability of anti-CD166 antibodies of the present disclosure to bind human H292 cells and cynomolgus monkey primary kidney epithelial cells with comparable affinity.

Fig. 18 is a graph showing the ability of anti-CD166 antibodies of the present disclosure to inhibit binding of human HuT-78 cells to the CD6 receptor.

Fig. 19A-19C are graphs showing the ability of the activated anti-CD166 antibody drug conjugates of the present disclosure to bind CD166 and human cells in protease-activated (cleaved) and non-activated (non-cleaved) forms.

Fig. 20A and 20B are graphs showing an exemplary cytotoxicity assay of activated anti-CD166 antibody drug conjugates of the present disclosure in human H292 and HCC1806 cells.

Fig. 21A-21D are graphs showing the ability of the activated anti-CD166 antibody drug conjugates of the present disclosure to induce an immunological response in cells.

Fig. 22 is a graph showing the ability of activated anti-CD166 antibody drug conjugates of the present disclosure to induce antibody-dependent cellular cytotoxicity in cells.

Fig. 23 is a graph showing the efficacy of activated anti-CD166 antibody drug conjugates of the present disclosure against a variety of cell-derived and patient-derived tumor xenograft models.

Fig. 24A-24D are graphs showing the cytotoxicity of activated anti-CD166 antibody drug conjugates of the present disclosure against multiple endometrial cancer-derived cell lines and analysis showing CD166 expression levels in endometrial cancer-derived cells.

Fig. 25A and 25B depict exemplary in situ binding studies of anti-CD166 antibodies of the present disclosure in a lung cancer xenograft model.

Fig. 26 is a graph showing the efficacy of activated anti-CD166 antibody drug conjugates of the present disclosure against a mouse lung cancer xenograft model.

Detailed description of the invention

The present invention relates to monoclonal antibodies (mAb) and activated monoclonal antibodies specifically binding to CD166, also known as activated leukocyte cell adhesion molecule (ALCAM). In some embodiments, the implementation of monoclonal antibodies and activated monoclonal antibodies are internalized containing CD166 cells. CD166 is a cell adhesion molecule that binds CD6, a cell surface receptor belonging to the superfamily (SRCRSF) of cysteine-rich scavenger receptor proteins (SRCR). CD166 is known to be associated with cell-cell and cell-matrix interactions, cell adhesion, cell migration, and T-cell activation and proliferation. Altered expression and/or activity of CD166 and CD166-associated signaling have been implicated in the pathogenesis of many diseases and disorders such as cancer, inflammation, and autoimmunity. For example, CD166 is highly expressed in a variety of cancer types, such as, for example, prostate cancer, breast cancer, lung cancer such as NSCLC and/or SCLC, oropharyngeal cancer, cervical cancer, and head and neck cancer such as like HNSCC.

This disclosure relates to anti-CD166 antibodies, conjugated anti-CD166 antibodies, activated anti-CD166 antibodies and/or conjugated activated anti-CD166 antibodies that can be used in methods of treating, preventing, delaying the progression, alleviating and/or reducing the symptom of a disease or disorder associated with altered expression and/or activity of CD166. For example, activated anti-CD166 antibodies are used in methods of treating, preventing, delaying the progression, alleviating, and/or reducing a symptom of cancer or other neoplastic condition.

This disclosure relates to anti-CD166 antibodies, conjugated anti-CD166 antibodies, activated anti-CD166 antibodies and/or conjugated activated anti-CD166 antibodies that can be used in methods of treating, preventing, delaying the progression, alleviating and/or reducing the symptom of a disease or disorder associated with cells expressing CD166. In some embodiments, cells are associated with altered expression and/or activity of CD166. In some embodiments, the cells are associated with normal CD166 expression and/or activity. For example, activated anti-CD166 antibodies are used in methods of treating, preventing, delaying the progression, alleviating, and/or reducing a symptom of cancer or other neoplastic condition.

This disclosure relates to anti-CD166 antibodies, conjugated anti-CD166 antibodies, activated anti-CD166 antibodies, and/or conjugated activated anti-CD166 antibodies that can be used in methods of treating, preventing, delaying the progression, alleviating and/or reducing the symptom of a disease or disorder in which diseased cells express CD166. In some embodiments, diseased cells are associated with altered CD166 expression and/or activity. In some embodiments, diseased cells are associated with normal CD166 expression and/or activity. For example, activated anti-CD166 antibodies are used in treatments

- 61 041958 preventing, delaying the progression, alleviating and/or reducing a symptom of a malignant tumor or other neoplastic condition.

Activated anti-CD166 antibodies and/or conjugated activated anti-CD166 antibodies comprise an antibody or antigen-binding fragment thereof that specifically binds CD166 attached to a masking group (MM) such that the addition of MM reduces the ability of the antibody or antigen-binding fragment to bind CD166. In some embodiments, the MM is attached via a sequence containing a substrate for a protease, eg, a protease, co-localized with CD166 at a treatment site in a subject.

Exemplary activated anti-CD166 antibodies of the invention include, for example, activated antibodies comprising a heavy chain and a light chain that are or are derived from the heavy chain variable region and light chain variable region sequences shown below (CDR sequences defined according to the definition AbM presented on Dr. Andrew C.R. Martin's website, available at www bioinf org uk/abs/, are shown in bold and underlined).

VH muM9:

OVTLKESGPGILOPSOTLSLTCSFSGFSLSTYGMGVGWIROPSGKGLEWLANIWWSEDK

HYNSALKSRLTISKDTSNNQVFLKIS SVDTADTATYYCVQIDYGNDYAFTYWGOGTLVTVSA (SEQ ID NO: 119)

VL muM9:

DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLOKPGOSPQLLIYQMSNL

ASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPYTFGGGTKLEIKR (SEQ ID

NO: 120)

VH huM9b:

OITLKESGPTLVKPTOTLTLTCTFSGFSLSTYGMGVGWIROPPGKALEWLANIWWSEDK

HYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCVQIDYGNDYAFTYWGQGTLVTVSS (SEQ ID NO: 121)

VH huM9c:

QITLKESGPTLVKPTQTLLTLTTFSGFSLSTYGMGVGWIRQPPGKALEWLANIWWSEDK

HYSPSLKSRLTITKDTSKNQWLTITNVDPVDTATYYCVQIDYGNDYAFTYWGQGTLVTVSS (SEQ ID NO: 122)

V L hM9vK-l:

DIVMTQSPLSLPPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNL

ASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK (SEQ ID

NO:123)

V L hM9vK-2:

DIVMTQSPLSLPPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNL

ASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK (SEQ ID

NO: 124)

V L hM9vK-3a:

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNR

ASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK (SEQ ID

NO: 125)

V L hM9vK-3b:

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNR

ASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK (SEQ ID

NO:126)

Exemplary activated anti-CD166 antibodies of the invention include, for example, activated antibodies comprising a heavy chain and a light chain that are or are derived from the heavy chain variable region and light chain variable region sequences shown below:

- 62 041958

HuCD166_HcC

Amino acid sequence.

QITLKESGPTLVKPTQTLTLTCTFSGFSLSTYGMGVGWIRQPPGKALEWLANIWWSEDK

HYSPSLKSRLTITKDTSKNQVVLTITNVDPVDTATYYCVQIDYGNDYAFTYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 239)

HuCD166_Lcl

DIVMTQSPLSLPPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNL

ASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 240)

Exemplary activated CD166 antibodies of the invention include, for example, activated antibodies comprising a combination of a heavy chain variable region complementarity determining region 1 (CDR1 VH, also referred to herein as CDRH1) sequence, a heavy chain variable region complementarity determining region 2 sequence ( CDR2 VH, also referred to herein as CDRH2), the complementarity determining sequence of variable heavy chain region 3 (CDR3 VH, also referred to herein as CDRH3), the complementarity determining sequence of variable light chain region 1 (CDR1 VL, also referred to in herein as CDRL1), the light chain variable region complementarity determining region 2 (CDR2 VL, also referred to herein as CDRL2), and the variable region complementarity determining region 3 sequence and a light chain (VL CDR3, also referred to herein as CDRL3), wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence containing the amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127);

a VH CDR2 sequence containing the amino acid sequence NIWWSEDKH (SEQ ID NO: 128);

a VH CDR3 sequence containing the amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129);

a VL CDR1 sequence containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131);

a VL CDR2 sequence containing the amino acid sequence QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133); and a VL CDR3 sequence containing the amino acid sequence AQNLELPYT (SEQ ID NO: 134).

In some embodiments, the activated anti-CD166 antibody comprises a heavy chain comprising or derived from the heavy chain amino acid sequence shown in US Patent Application Publication Nos.

In some embodiments, an activated anti-CD166 antibody comprises a heavy chain comprising a CDR1 VH, CDR2 VH, CDR3 VH, CDR1 VL, CDR2 VL, and/or a CDR3 VL sequence comprising or derived from the CDR amino acid sequence shown in US Patent Application Publication No. 20150071937 , 20090070890 and/or 20090203538, the entire contents of each of which are hereby incorporated by reference.

In some embodiments, an activated anti-CD166 antibody comprises a heavy chain containing or derived from the heavy chain amino acid sequence shown in Table 1. 12. In some embodiments, the implementation of the activated antibody against CD166 contains a light chain containing or derived from the amino acid sequence of the light chain shown in table. 12. In some embodiments, the implementation of the activated antibody against CD166 contains a heavy chain containing or derived from the amino acid sequence of the heavy chain shown in table. 12, and a light chain containing or derived from the amino acid sequence of the light chain shown in table. 12. In some embodiments, an activated anti-CD166 antibody comprises a combination of heavy chain variable region sequences

- 63 041958 pi and the variable region of the light chain from the combinations shown in group A in table. 12. In some embodiments, an activated CD166 antibody comprises a combination of heavy chain variable region and light chain variable region sequences from the sequences shown in Group B of Table 12. 12. In some embodiments, an activated anti-CD166 antibody comprises a combination of heavy chain variable region and light chain variable region sequences from the sequences shown in group C of Table 12. 12. In some embodiments, an activated anti-CD166 antibody comprises a combination of heavy chain variable region and light chain variable region sequences from the sequences shown in group D of Table 12. 12.

In some embodiments, an activated anti-CD166 antibody comprises a combination of complementarity determining region (CDR) sequences from a heavy chain sequence from the heavy chain sequences shown in Group A of Table 1. 12. In some embodiments, an activated CD166 antibody comprises a combination of CDRs from a light chain sequence from the light chain sequences shown in Group A of Table 12. 12. In some embodiments, the implementation of the activated antibody against CD166 contains a combination of CDRs from the sequence of the heavy chain of the sequences of the heavy chain shown in group a table. 12 and a CDR from a light chain sequence from the light chain sequences shown in Group A of Table 12. 12.

In some embodiments, an activated anti-CD166 antibody comprises a combination of CDRs from a heavy chain sequence from the heavy chain sequences shown in Group B of Table 1. 12. In some embodiments, an activated CD166 antibody comprises a combination of CDRs from a light chain sequence from the light chain sequences shown in Group B of Table 12. 12. In some embodiments, an activated anti-CD166 antibody comprises a combination of CDRs from a heavy chain sequence from the heavy chain sequences shown in Group B of Table 12. 12 and a CDR from a light chain sequence from the light chain sequences shown in Group B of Table 12. 12.

In some embodiments, an activated anti-CD166 antibody comprises a combination of CDRs from a heavy chain sequence from the heavy chain sequences shown in Group C of Table 1. 12. In some embodiments, an activated anti-CD166 antibody comprises a combination of CDRs from a light chain sequence from the light chain sequences shown in Group C of Table 12. 12. In some embodiments, an activated CD166 antibody comprises a combination of CDRs from a heavy chain sequence from the heavy chain sequences shown in Group C of Table 12. 12 and CDRs from the light chain sequence from the light chain sequences shown in Group C of Table 12. 12.

In some embodiments, the activated anti-CD166 antibody comprises a combination of CDRs from a heavy chain sequence from the heavy chain sequences shown in Group D of Table 1. 12. In some embodiments, the implementation of the activated antibody against CD166 contains a combination of CDRs from the light chain sequence of the light chain sequences shown in group D of table. 12. In some embodiments, an activated anti-CD166 antibody comprises a combination of CDRs from a heavy chain sequence from the heavy chain sequences shown in Group D of Table 12. 12 and CDRs from the light chain sequence from the light chain sequences shown in group D of Table. 12.

Table 12

Heavy chain variable region (VH) and light chain variable region (VL) sequences for activated CD166-binding antibodies

Group A vh QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR DNSKDTLYLQMNSLRAEDTAVYYCASRSLLDYWGQGTLVTVSS (SEQ ID NO: 249) vh QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR DNSKDTLYLQMNSLRAEDTAVYYCASRSLLDYWGQGTLVTVSS (SEQ ID NO: 250) VL NFMLTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPLLVIYGKNNRPSGIPDRFSGSSSGNTASL TITGAQAEDEADYYCNSRDSSGNPVFGGGTKVTVL (SEQ ID NO: 251) Group B vh EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGGWEFWGQGTLVTVSS (SEQ ID NO: 252)

- 64 041958

VL DIRMTOSPSFLSASVGDRVTITCRASQDISSYFAWYOOKPGKAPKLLIYAASTLRSGVPSRFSGSGSGTDFT LTISSLOPEDFATYYCQQSYSTPRITFGOGTRLEIK (SEQ ID NO: 253) Group C vh EVOLVESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSTISGSGGSTYYADSVKGRFTISR DNSKNTLYLOMNSLRAEDTAVYYCARGIVATSWGOGTLVTVSR (SEQ ID NO: 254) vh EVOLVESGGGWOPGGSLRLSCAASGFNFDVYGMNWVROVPGKGLEWVSLINGDGGLRYYADSVKGRFTVSR DNSRNSLYLOMNSLRSEDTALYYCVKGNFOQWGOGTLVTVSR (SEQ ID NO: 255) vh EVQLVES GGGLVQ PGGS LRLS CAAS GFT FDDYAMHWVRQAPGKGLEWVSLISGDGGSTYYADSVKDRFTIS R DNSKNSLYLOMNSLRAEDTAVYYCARGNYFDYWGOGTLVTVSR (SEQ ID NO: 256) vh EVQLVES GGGLVQ PGGS LRLS CAAS GFT FSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTIS R DNAKNSLYLQMNSLRDEDTAVYYCARVMPSYYYYYGMDVWGQGTTVTVSR (SEQ ID NO: 257) vh EVOLVESGGGWOPGRSLRLSCAASGFTFSSYGMHWVROAPGKGLEWVAVISYDGSNKYYADSVKGRFTIS R DNSKNTLYLOMNSLRAEDTAVYYCADYGMDVWGOGTTVTVSR (SEQ ID NO: 258) VL SYELTOPPSVSVAPGOTARITCGGNKIGSKSVHWYOOKOGOAPVLVIYLDRDRPSGIPERFSGSNSGNTATL TITRVEAEDEADYYCHLWDSGSDQVFGGGTKLTVLG (SEQ ID NO: 259) VL SYVLTOPPSVSVAPGKTARITCGGNNIGSKSVHWYOOKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATL TISRVEAGDEADYYCQVWDSSSDHWFGGGTKLTVLG (SEQ ID NO: 260) VL SYELTOPLSVSVALGOTARITCGGNNIGSKNVHWYOOKPGOAPVLVIYRDSNRPSGIPERFSGSNSGNTATL TISRAOAGDEADYYCQVWDSSWFGGGTKLTVLG (SEQ ID NO: 261) VL NFMLTOPHSVSESPGKTVTISCTGSSGSIASNYVQWYOORPGSAPTTVIYEDSERPSGVPDRFSGSIDSSSN SASLTISGLKTODEADYYCQSYDGVNWVFGGGTKLTVLG (SEQ ID NO: 262) VL DIOMTOSPSSLSASVGDRVTITCRASOSISSYLNWYOOKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQSYSTPVTFGQGTKVEIK (SEQ ID NO: 263) Group D VH/VL QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR DNSKDTLYLQMNSLRAEDTAVYYCASRSLLDYWGQGTLVTVSSGGGGSGGGGSGGGGSNFMLTQDPAVSVAL GQTVRITCQGDSLRSYYASWYQQKPGQAPLLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYY CNSRDSSGNPVFGGGTKVTVL (SEQ ID NO: 475) VH/VL QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR DNSKDTLYLQMNSLRAEDTAVYYCASRSLLDYWGQGTLVTVSSGGGGSGGGGSGGGGSNFMLTQDPAVSVAL GQTVRITCQGDSLRSYYASWYQQKPGQAPLLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYY CNSRDSSGNPVFGGGTKVTVL (SEQ ID NO: 476)

In some embodiments, the implementation of the activated antibody against CD166 contains the CDR sequence shown in table. 13, a combination of CDR VL sequences (CDR1 VL, CDR2 VL, CDR3 VL) selected from the group consisting of these combinations shown in one row of Table. 13, a combination of CDR VH sequences (CDR1 VH, CDR2 VH, CDR3 VH) selected from the group consisting of these combinations shown in table. 13, or a combination of CDR VL and CDR VH sequences (CDR1 VL, CDR2 VL, CDR3 VL, CDR1 VH, CDR2 VH, CDR3 VH) selected from the group consisting of these combinations shown in Table. 13.

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Table 13

CDR sequences for antibodies and activated antibodies that bind CD166

VL CDR1 (SEQID NO) CDR2 (SEQID NO) CDR3 (SEQ ID NO) QGDSLRSYYAS YGKNNRPS NSRDSSGNPV (264) (265) (266) QGDSLRSYYAS YGKNNRPS NSRDSSGNPV (264) (265) (266) RASQDISSYFA AASTLRS QQSYSTPRIT (270) (271) (272) GGNKIGSKSVH LDRDRPS HLWDSGSD (274) (275) (276) GGNNIGSKSVH YDSDRPS QVWDSSSDH (279) (280) (281) GGNNIGSKNVH (285) RDSNRPs (286) QVWDSS (287) TGSSGSIASNY EDSERPS QSYDGVN VQ (291) (292) (293) RASQSISSYLN AASSLQS QQSYSTP (297) (298) (299)

vh CDR1 (SEQID NO) CDR2 (SEQID NO) CDR3 (SEQ ID NO) SYAMS (267) AISGSGGSTYYADSVKG (268) RSLLDY (269) SYAMG(477) AISGSGGSTYYADSVKG( 268) RSLLDY (269) SYAMS (267) AISGSGGSTYYADSVKG (268) GGGWEF (273) SYAMS (267) TISGSGGSTYYADSVKG (277) GIVATS (278) VYGMN (282) LINGDGGLRYYADSVKG (283) GNFQQ (284) DYAMH (288) LISGDGGSTYYADSVKD (289) GNYFDY (290) SYSMN(294) YISSSSTIYYADSVKG (295) VMPSYYYYYGMDV (296) SYGMH(300) VISYDGSNKKYYADSVKG (301) YGMDV(302)

In some embodiments, an activated CD166 antibody comprises or is derived from an antibody made, secreted, or otherwise produced by a hybridoma, such as, for example, the hybridoma(s) described in U.S. Patent Application Publication No. 20040048319 and deposited with the American Type Culture Collection ( АТСС) under the deposit number РТА-4478. In some embodiments, the activated CD166 antibody comprises or is derived from an antibody made, secreted, or otherwise produced by a hybridoma, such as, for example, the hybridoma(s) described in U.S. Patent No. 6,022,540 and deposited with ATCC under deposit number HB 11789.

In some embodiments, the activated anti-CD166 antibody comprises or is derived from an antibody made, secreted, or otherwise produced by a hybridoma, such as, for example, the hybridoma(s) described in US Pat. HB 12137, HB 12138, HB12139, HB 12140 and/or HB 12141.

Anti-CD166 and AB antibodies in the activated antibodies of this disclosure specifically bind to a CD166 target, such as, for example, mammalian CD166 and/or human CD166. Also included in this description are anti-CD166 antibodies and ABs that bind the same CD166 epitope as an antibody of this specification and/or an activated activable antibody described herein. Also included in this specification are anti-CD166 antibodies and ABs that compete with an anti-CD166 antibody and/or an activated, activated anti-CD166 antibody described herein for binding to a CD166 target, eg, human CD166. Also included in this specification are anti-CD166 antibodies and ABs that cross-compete with an anti-CD166 antibody and/or an activated anti-CD166 antibody described herein for binding to a CD166 target, eg human CD166.

The activated anti-CD166 antibodies provided herein contain a masking group. In some embodiments, the masking group is an amino acid sequence attached to or otherwise attached to an anti-CD166 antibody and located within an activated anti-CD166 antibody construct such that the masking group reduces the ability of the anti-CD166 antibody to specifically bind CD166. Suitable masking groups are identified using any of a variety of known methods. For example, peptide masking groups are identified using the methods described in PCT Publication No. WO 2009/025846 of Daugherty et al., the entire contents of which are incorporated herein by reference.

The activated anti-CD166 antibodies provided herein contain a cleavable group. In some embodiments, the cleavable group contains an amino acid sequence that is a substrate for a protease, typically an extracellular protease. Suitable substrates are identified using any of a variety of known methods. For example, peptide substrates are identified using the methods described in US Pat. No. 7,666,817 to Daugherty et al.; in US patent No. 8563269 from Stagliano et al.; and in PCT Publication No. WO 2014/026136 by La Porte et al., the full contents of each of which are hereby incorporated by reference (see also Boulware et al., Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics , Biotechnol Bioeng., 106.3(2010):339-46).

Illustrative substrates include, without limitation, substrates cleavable by one or more of the following enzymes or proteases listed in Table. 4.

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Table 4

Illustrative proteases and/or enzymes

ADAMS, ADAMTS, for example ADAM 8 ADAM 9 ADAMI0 ADAM12 ADAM15 ADAMI7/TACE ADAMDEC1 ADAMTSI ADAMTS4 ADAMTS5 Cysteine proteases, such as Cruzipaine Legumain Otubain-2 Serine proteases such as Activated protein C Cathepsin A Cathepsin G Chymase protease - coagulation factors (eg, FVIIa, FIXa, FXa, FXIa, FXIIa) KLK, for example, KLK4 KLK5 KLK6 KLK7 KLK8 KLK10 KLK11 KLK13 KLK14 Elastase Granzym B Guanidinobenzoatase HtrAl Aspartate proteases, such as BACE ranin Human neutrophil elastase lactoferrin Marapsin NS3/4A PACE 4 Plasmin PSA tPA Thrombin Tryptase and RA Type II transmembrane serine proteases (TTSP), e.g. DESC1 DPP-4 FAP Hepsin Matriptase-2 MT-SPl/Matryptase TMPRSS2 TMPRSS3 TMPRSS4 Aspartate cathepsins, e.g. Cathepsin D Cathepsin E Metalloproteases, for example, Meprin Neprilysin PSMA VMP-1 Caspases, for example, Caspase 1 Caspase 2 Caspase 3 Caspase 4 Caspase 5 Caspase 6 Caspase 7 Caspase 8 Caspase 9 Caspase 10 Caspaza 14 MMR, for example, MMP1 MMP2 MMRP MMP7 MMR 8 MMP 9 MMP10 MMP11 MMP12 MMP13 MMP14 MMP15 MMP16 MMP17 MMP19 MMP2 0 MMP23 MMP24 MMP2 6 MMP27 Cysteine cathepsins, e.g. Cathepsin B Cathepsin C Cathepsin K Cathepsin L Cathepsin S Cathepsin V/L2 Cathepsin X/Z/P

The activated anti-CD166 antibodies described herein overcome the limitation of antibody drugs, in particular antibody drugs known to be toxic in vivo, at least to some extent. Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. The activated anti-CD166 antibodies provided herein are designed to combat toxicity associated with target inhibition in normal tissues by conventional therapeutic antibodies. These activated anti-CD166 antibodies remain masked until proteolytic activation at the disease site. Starting with an anti-CD166 antibody as the initial therapeutic antibody, the activated anti-CD166 antibodies of the invention were constructed by attaching the antibody to an inhibitory mask via a linker containing a protease substrate.

When an AV is modified with MM and is in the presence of a target, the specific binding of the AV to its target is reduced or inhibited, compared to the specific binding of the AV not modified with MM or the specific binding of the original AV to the target.

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K _d AB modified with MM relative to the target at least 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000, 1000000 , 50000000 or more or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1000000, 10-10000000, 100-1000, 100-10000, 100-100000, 100000, 100-10000, 100-100000 1000000, 100-10000000, 1000-10000, 1000-100000, 1000-1000000, 1000-10000000, 10000-100000, 1000-1000000, 10000-10000000, 100000-1000000 or 100000-10000,000 times higher than K _D AB, not modified from using MM, or the original AB in relation to the target. In contrast, the binding affinity of AB modified with MM for the target is at least 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, 50000, 100000 , 500000, 1000000, 5000000, 10000000, 50000000 or more or between 5-10, 10-100, 10-1000, 10-10000, 10-100000, 10-1000000, 10-10000000, 10.00 10000, 100100000, 100-1000000, 100-10000000, 1000-10000, 1000-100000, 1000-1000000, 1000-10000000, 1000100000, 1000-1000000, 1000-10000000, 10000 0-1000000 or 10000 0-10000000 times lower, lower, lower than the binding affinity of the AV not modified with MM or the original AV for the target.

The dissociation constant (K _d ) MM with respect to AB, as a rule, exceeds K _d AB with respect to the target. K _d MM with respect to AB can be at least 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, 100000, 1000000 or even 10000000 greater than K _d for AB with respect to the target . In contrast, the binding affinity of MM for AB is generally lower than the binding affinity of AB for the target. The binding affinity of MM for AB can be at least 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, 100000, 1000000 or even 10,000,000 times lower than the binding affinity of AV for in relation to the target.

In some embodiments, the dissociation constant (K _d ) MM with respect to AB is approximately equal to Kd AB with respect to the target. In some embodiments, the dissociation constant (Kd) of MM with respect to AB does not exceed the dissociation constant of AB with respect to the target.

In some embodiments, the dissociation constant (K _d ) of MM with respect to AB is less than the dissociation constant of AB with respect to the target.

In some embodiments, the dissociation constant (K _d ) of MM with respect to AB is greater than the dissociation constant of AB with respect to the target.

In some embodiments, the implementation of the MM has a K _d for binding to AB, not exceeding the K _d for binding AB to the target.

In some embodiments, the MM has a Kd for binding to an AB that is not less than a Kd for binding an AB to a target.

In some embodiments, the implementation of the MM has a Kd for binding to AB, approximately equal to the Kd for binding AB to the target.

In some embodiments, the implementation of the MM has a Kd for binding to AB, less than the Kd for binding AB to the target.

In some embodiments, the MM has a K _d for binding to an AB that is greater than the K _d for binding an AB to a target.

In some embodiments, the MM has a K _d for binding to AB no greater than 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1000 times the K _d for binding AB to a target. In some embodiments, the implementation of the MM has a K _d for binding to AB, in the range between 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10- 1,000, 20-100, 20-1000 or 100-1000 times the K _d for AB binding to the target.

In some embodiments, the implementation of the MM has an affinity for binding to AB, less than the affinity of binding of AB to the target.

In some embodiments, the implementation of the MM has an affinity for binding to AB, not exceeding the affinity of binding to the target of AB.

In some embodiments, the implementation of the MM has an affinity for binding to AB, approximately equal to the binding affinity of AB to the target.

In some embodiments, the MM has an affinity for binding to an AB that is not less than the binding affinity of the AB to a target.

In some embodiments, the MM has an affinity for binding to AB that is greater than the binding affinity of AB for the target.

In some embodiments, the MM has an affinity for binding to AB that is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1000 less than the binding affinity of AB to the target. In some embodiments, MM has an affinity for binding to AB between 1-5, 25, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1000, 20 -100, 20-1000, or 100-1000 times less than the binding affinity of AB to the target. In some embodiments, the implementation of the MM has an affinity for binding to AB, 2-20 times less than the affinity of binding to the target of AB. In some embodiments, the implementation of the MM is not covalently associated with AB and at equimolar concentration with AB does not inhibit the binding of AB to the target.

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When an AV is modified with MM and is in the presence of a target, the specific binding of the AV to its target is reduced or inhibited compared to the specific binding of the AV not modified with MM or the specific binding of the parent AV to the target. Compared to binding of AB not modified with MM or binding of the original AB to the target, the ability of AB to bind to the target when modified with MM can be reduced by at least 50, 60, 70, 80, 90, 92, 93, 94, 95, 96, 97, 98, 99 and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84 or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more when measured in vivo or in an in vitro assay.

MM inhibits AV binding to the target. MM binds the antigen-binding domain of AB and inhibits AB binding to the target. MM can sterically inhibit AV binding to the target. MM can allosterically inhibit the binding of AV to its target. In these embodiments, when the AV is modified or bound to the MM and is in the presence of a target, there is no or substantially no binding of the AB to the target, or no more than 0.001, 0.01, 0.1, 1, 2, 3 is present, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or 50% binding of AB to the target, compared with the binding of AB not modified with MM, original AB or AB, unbound to MM, to the target, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30 , 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or longer when measured in vivo or assayed in vitro .

When the AV is associated with or modified by the MM, the MM masks or reduces or otherwise inhibits the specific binding of the AV to the target. When an AV is associated with or modified by MM, such binding or modification may cause a structural change that reduces or inhibits the ability of the AV to specifically bind to its target.

AB associated with MM or modified with MM can be represented by the following formulas (in order from amino(M)-terminal region to carboxy(C)-terminal region):

(MM)-(AB), (AB)-(MM), (MM)-L-(AB), (AB)-L-(MM), where MM is a masking group, AB is an antibody or fragment thereof antibodies, and L is a linker. In many embodiments, it may be desirable to include one or more linkers, such as flexible linkers, in a formulation to provide flexibility.

In specific embodiments, MM is not a natural binding partner for AB. In some embodiments, the MM has no or substantially no homology with any natural AB binding partner. In some embodiments, the MW is no more than 55%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner. AB. In some embodiments, the MW is no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% identical to any natural AB binding partner . In some embodiments, the MM is no more than 25% identical to any natural AB binding partner. In some embodiments, the MM is no more than 50% identical to any natural AB binding partner. In some embodiments, the MM is no more than 20% identical to any natural AB binding partner. In some embodiments, MM is no more than 10% identical to any natural AB binding partner.

In some embodiments, the activated antibodies contain AB modified with MM and also contain one or more cleavable groups (CMs). Such activated antibodies have activated/switchable binding to the AB target. Activated antibodies typically contain an antibody or antibody fragment (AB) modified by or linked to a masking group (MM) and a modifiable or cleavable group (CM). In some embodiments, the implementation of the CM contains an amino acid sequence that serves as a substrate for at least one protease.

The elements of the activated antibodies are arranged so that the MM and SM are arranged in such a way that in the cleaved (or relatively active) state and in the presence of the target, the AB binds the target, while the activated antibody is in the uncleaved (or relatively inactive) state in in the presence of a target, the specific binding of AB to its target is reduced or inhibited. The specific binding of an AB to its target can be reduced by inhibiting or masking by MM the ability of the AB to specifically bind to its target.

Kd AB modified using MM and SM, in relation to the target at least 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000, 10000000, 50000000 or more or between 5-10, 10-100, 10-1000, 10-10000, 10-100000,

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10-1000000, 10-10000000, 100-1000, 100-10000, 100-100000, 100-1000000, 100-10000000, 1000-10000, 1000-100000, 1000-1000000, 1000-10000000, 10000-100000, 10000- 1000000, 10000-10000000, 100000-1000000 or 100000-10000000 times the Kd of the AB not modified with MM and SM or the original AB relative to the target. In contrast, the binding affinity of AB modified with MM and SM for the target is at least 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, 50000, 100000, 500000, 1000000 , 5000000, 10000000, 50000000 or more or between 5-10, 10-100, 10-1000, 10-10000, 10-100000, 10-1000000, 10-10000000, 100-1000, 100-1000 100000, 100-1000000, 100-10000000, 1000-10,000, 1000-100000, 1000-1000000, 1000-10000000, 10000-100000, 1000-1000000, 10000-10000000, 10000 0-1000000 or 10000 0-10000000 times lower, lower, lower than the binding affinity of AB not modified with MM and SM or the original AB for the target.

When an AV is modified with MM and SM, and is in the presence of a target but not in the presence of a modifying agent (e.g., at least one protease), the specific binding of the AV to its target is reduced or inhibited, compared to the specific binding of the AB, not modified using MM and SM, or the original AB with a target. Compared to the binding of the original AB or the binding of an AB not modified with MM and SM to its target, the ability of AB to bind the target when modified with MM and SM can be reduced by at least 50, 60, 70, 80, 90, 92, 93, 94, 95, 96, 97, 98, 99 and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84 or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer when measured in vivo or in an in vitro assay.

As used herein, the term cleaved state refers to the state of activated antibodies after modification by the CM with at least one protease. The term uncleaved state, as used herein, refers to the state of activated antibodies in the absence of CM cleavage by the protease. As discussed above, the term activated antibodies is used herein to refer to an activated antibody in its uncleaved (natural) state, as well as in its cleaved state. One skilled in the art will appreciate that in some embodiments, the cleaved, activated antibody may lack MM due to cleavage of the CM by a protease resulting in the release of at least one MM (e.g., where the MM is not attached to the activated antibodies via a covalent bond (e.g., disulfide bond between cysteine residues).

By activated or switchable is meant that the activated antibody has a first level of target binding when the activated antibody is in an inhibited, masked, or uncleaved state (i.e., first conformation) and a second level of target binding when uninhibited, unmasked, and/or cleaved. state (ie the second conformation), where the second level of binding of the target exceeds the first level of binding. As a rule, access to the AB target of an activated antibody is greater in the presence of a cleaving agent capable of cleaving SM, i.e. protease than in the absence of such a degrading agent. Thus, when the activated antibody is in the uncleaved state, AB binding to the target is inhibited and AB can be masked from binding to the target (i.e., the first conformation is such that AB cannot bind to the target) and in the cleaved state, AB is not inhibited, or is unmasked for target binding.

The CM and AB of the activated antibodies are chosen such that AB is the linking group for the target and CM is the substrate for the protease. In some embodiments, the protease is colocalized with a target at a treatment site or diagnostic site in a subject. As used herein, co-localization refers to being in the same area or relatively close neighborhood. In some embodiments, the protease cleaves the CM to form an activated antibody that binds to a target located in the vicinity of the cleavage site. The activated antibodies described herein find particular use when, for example, a protease capable of cleaving a site in the CM, i.e. the protease is present at relatively higher levels in target-containing tissue at the site of treatment or diagnosis than in tissue at non-treatment sites (eg, healthy tissue). In some embodiments, the implementation of the SM according to this description is also cleaved by one or more other proteases. In some embodiments, it is one or more other proteases co-localized with the target and responsible for cleaving CM in vivo.

In some embodiments, activated antibodies provide reduced toxicity and/or adverse side effects that may otherwise result from AV binding at non-treatable sites, unless the AV is masked or its binding to the target is otherwise inhibited.

In general, an activated antibody can be designed by selecting an AB of interest and designing the remaining activated antibody such that, under conformational constraints, MM masks the AB or reduces binding of the AB to its target. Structural design criteria may be taken into account to provide this functional feature.

Activated antibodies are provided that have a switchable phenotype of the desired dynamic range of target binding in an inhibited versus uninhibited conformation. Dynamic range generally refers to the ratio of (a) the maximum detected level of a parameter under a first set of conditions to (b) the minimum detected value of that parameter under a second set of conditions. For example, in the context of an activated antibody, dynamic range refers to the ratio of (a) the maximum detected level of target protein binding to the activated antibody in the presence of at least one protease capable of cleaving the CM of the activated antibody to (b) the minimum detected level of target protein binding to the activated antibody in no protease. The dynamic range of an activated antibody can be calculated as the ratio of the dissociation constant when the activated antibody is treated with a cleavage agent (eg, enzyme) to the dissociation constant when the activated antibody is treated with a cleavage agent. The greater the dynamic range of the activated antibody, the better the switchable phenotype of the activated antibody. Activated antibodies having relatively higher dynamic range values (e.g., greater than 1) have more desirable switchable phenotypes such that the activated antibodies bind to the target protein more (e.g., predominantly) in the presence of a cleaving agent (e.g., enzyme ) capable of cleaving the CM of activated antibodies than in the absence of a cleaving agent.

Activated antibodies can be provided in a variety of structural configurations. Illustrative formulas for activated antibodies are provided below. It is specifically contemplated that the N- to C-terminal order of AB, MM, and CM can be maintained within an activated antibody. It is also specifically contemplated that CM and MM may overlap in amino acid sequence, for example such that CM is contained within MM.

For example, activated antibodies can be represented by the following formula (in order from amino(N)-terminal region to carboxy(C)-terminal region:

(MM)-(CM)-(AB), (AB)-(CM)-(MM), where MM is a masking group, CM is a cleavable group, and AB is an antibody or fragment thereof. It should be noted that while MM and CM are listed as separate components in the formula above, all exemplary embodiments (including formulas) described herein provide that the amino acid sequences of MM and CM may overlap, for example, as that the SM is wholly or partly contained within the MM. In addition, the formulas above provide additional amino acid sequences that can be located at the N-terminus or C-terminus of the elements of the activated antibodies.

In specific embodiments, MM is not a natural binding partner for AB. In some embodiments, the MM has no or substantially no homology with any natural AB binding partner. In some embodiments, the MW is no more than 55%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural AB binding partner. . In some embodiments, the MW is no more than 55%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% identical to any natural AB binding partner. In some embodiments, the MM is no more than 50% identical to any natural AB binding partner. In some embodiments, the MM is no more than 25% identical to any natural AB binding partner. In some embodiments, the MM is no more than 20% identical to any natural AB binding partner. In some embodiments, the MM is no more than 10% identical to any natural AB binding partner.

In many embodiments, it may be desirable to insert one or more linkers, such as flexible linkers, into the antibody being activated in such a manner as to provide flexibility at one or more of the MM-CM junction, the CM-AB junction, or both. For example, AB, MM, and/or CM may not contain sufficient residues (eg, Gly, Ser, Asp, Asn, especially Gly and Ser, especially Gly) to provide the desired flexibility. Therefore, the switchable phenotype of such activated antibody constructs may benefit from the introduction of one or more amino acids to provide a flexible linker. In addition, as described below, when an activated antibody is provided in the form of a conformationally constrained construct, a flexible linker can be inserted functionally to facilitate the formation and maintenance of the cyclic structure of an uncleaved, activated antibody.

For example, in specific embodiments, the activated antibody comprises one of the following formulas (wherein the formula below is an amino acid sequence in either

- 71 041958 direction from N- to C-terminus, or in the direction from C- to N-terminus):

(MM)-L1-(CM)-(AB), (MM)-(CM)-L2-(AB), (mm)-L1-(CM)-L2-(AB), where MM, CM, and AB are as defined above, wherein L1 and L2, independently and optionally present or absent, are each the same or different flexible linkers containing at least 1 flexible amino acid (eg, Gly). In addition, additional amino acid sequences are provided in the above formulas, which may be located at the N-terminus or C-terminus of the activated antibody elements. Examples include, but are not limited to, targeting groups (eg, a ligand for a receptor on a cell present in the target tissue) and serum half-life increasing groups (eg, serum protein-binding polypeptides such as immunoglobulin (eg, IgG) or serum albumin (eg, human serum albumin (HAS)).

SM specifically cleaves at least one protease at a rate of approximately 0.001-1500x104 M ^-1 s ^-1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250 or 1500x104 M ^-1 s ^-1 . In some embodiments, CM is specifically cleaved at a rate of approximately 100,000 M ^-1 s ^-1 . In some embodiments, CM is specifically cleaved at a rate of about 1x10E2 to about 1x10E6 M' ¹ s' ¹ (ie, about 1x10 ² to about 1x106 M' ¹ s' ¹ ).

For specific cleavage by an enzyme, the enzyme and SM are brought into contact. When an activated antibody containing an AB attached to the MM and SM is in the presence of a target and sufficient enzyme activity, the SM can be cleaved. Sufficient enzyme activity may refer to the ability of the enzyme to come into contact with the SM and effect degradation. It can be easily assumed that an enzyme may be in the vicinity of the CM but be unable to be degraded due to other cellular factors or protein modification of the enzyme.

Linkers suitable for use in the compositions described herein are typically linkers that provide the flexibility of the modified AB or activated antibodies to facilitate inhibition of AB binding to the target. Such linkers are generally referred to as flexible linkers. Suitable linkers can be readily selected and can be any of a variety of suitable lengths, for example, 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, from 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids and can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 in length, 16, 17, 18, 19 or 20 amino acids.

Exemplary flexible linkers include (G)n glycine polymers, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 339) and (GGGS)n (SEQ ID NO: 340) where n is an integer of at least one), glycine-alanine polymers, alanine serine polymers, and other flexible linkers known in the art. Glycine and glycineserine polymers are relatively unstructured, and thus may be capable of serving as a neutral bond between the components. Glycine spans a much larger psi space even compared to alanine and is much less restricted than longer side chain residues (see Scheraga, Rev. Computational Chem., 11173-142 (1992)). Illustrative flexible linkers include, but are not limited to, Gly-Gly-Ser-Gly (SEQ ID NO: 341), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 342), Gly-Ser-Gly-Ser -Gly (SEQ ID NO: 343), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 344), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 345), Gly-Ser-Ser- Ser-Gly (SEQ ID NO: 346) and the like. One skilled in the art will appreciate that the design of activated antibodies may include linkers that are fully or partially flexible such that the linker may contain a flexible linker as well as one or more less flexible structure portions to provide the desired structure of the activated antibodies.

The description also relates to compositions and methods, including an activated anti-CD166 antibody, containing an antibody or antibody fragment (AB) that specifically binds CD166, where the AB is associated with a masking group (MM) that reduces the ability of the AB to bind its target. In some embodiments, the activated CD166 antibody further comprises a cleavable group (CM) that is a substrate for the protease. The compositions and methods provided herein allow the attachment of one or more agents to one or more cysteine residues in an AB without interfering with the activity (eg, masking, activating, or binding activity) of an activated anti-CD166 antibody. In some embodiments, the compositions and methods provided herein allow the attachment of one or more agents to one or more cysteine residues in the AB without reducing or otherwise breaking one or more disulfide bonds within the MM. Using the compositions and methods provided herein, an activated anti-CD166 antibody is prepared that is conjugated to one or more agents, e.g.

- 72 041958 any of a variety of therapeutic, diagnostic and/or prophylactic agents, for example, In some embodiments, where none of the agent(s) is conjugated to an MM of an activated anti-CD166 antibody. Using the compositions and methods provided herein, conjugated anti-CD166 activated antibodies are prepared in which MM retains the ability to efficiently and effectively mask the AV activated antibody in an uncleaved state. Using the compositions and methods provided herein, conjugated activated anti-CD166 antibodies are prepared in which the activated antibody is still activated, i. cleaved, in the presence of a protease that can cleave SM.

Activated anti-CD166 antibodies have at least one agent conjugation site, but in the methods and compositions provided herein, fewer than all possible agent conjugation sites are available for agent conjugation. In some embodiments, one or more conjugation points are sulfur atoms involved in disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms involved in interchain disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms involved in interchain sulfide bonds, but not sulfur atoms involved in intrachain disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms from cysteine or other amino acid residues containing a sulfur atom. Such residues may occur naturally in the structure of the antibody, or they may be introduced into the antibody by site-directed mutagenesis, chemical transformation, or mismatched incorporation of unnatural amino acids.

Also provided are methods for preparing an activated anti-CD166 antibody conjugate having one or more interchain disulfide bonds in AB and one or more intrachain disulfide bonds in MM, and a drug reactive with free thiols is provided. The method generally includes partial reduction of inter-chain disulfide bonds in the activated antibody with a reducing agent such as TCEP; and conjugating the free thiol-reactive drug with the partially reduced, activated antibody. As used herein, the term partial reduction refers to situations where an activated anti-CD166 antibody is contacted with a reducing agent and less than all disulfide bonds, eg, less than all possible conjugation sites, are reduced. In some embodiments, and less than 99, 98, 97, 96, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10 or less than 5% of all possible conjugation sites are reduced.

In other embodiments, a method is provided for reconstituting and conjugating an agent, eg, a drug, with an activated anti-CD166 antibody to selectively target the agent. The method generally includes partially reducing an activated anti-CD166 antibody using a reducing agent such that any conjugation sites in the masking group or other non-AB portion of the activated antibody are not reduced, and conjugating the agent with interchain thiols in the AB. The conjugation site(s) is chosen to allow the desired location of the agent to allow conjugation to occur at the desired site. The restoring agent is, for example, TCEP. Conditions of the reduction reaction, such as the ratio of reducing agent to activated antibody, duration of incubation, temperature during incubation, pH of the reducing reaction solution, etc., are determined by identifying the conditions under which a conjugated activated antibody is prepared in which MM retains the ability to effectively and efficiently mask the AV-activated antibody in the uncleaved state. The ratio of reducing agent to activated anti-CD166 antibody can be varied depending on the activated antibody. In some embodiments, the ratio of reducing agent to activated anti-CD166 antibody may range from about 20:1 to 1:1, from about 10:1 to 1:1, from about 9:1 to 1:1, from about 8: 1 to 1:1, about 7:1 to 1:1, about 6:1 to 1:1, about 5:1 to 1:1, about 4:1 to 1:1, about 3: 1 to 1:1, about 2:1 to 1:1, about 20:1 to 1:1.5, about 10:1 to 1:1.5, about 9:1 to 1:1, 5, about 8:1 to 1:1.5, about 7:1 to 1:1.5, about 6:1 to 1:1.5, about 5:1 to 1:1.5, about 4:1 to 1:1.5, about 3:1 to 1:1.5, about 2:1 to 1:1.5, about 1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In some embodiments, the ratio is in the range of about 5:1 to 1:1. In some embodiments, the ratio is in the range of about 5:1 to 1.5:1. In some embodiments, the ratio is in the range of about 4:1 to 1:1. In some embodiments, the ratio is in the range of about 4:1 to 1.5:1. In some embodiments, the ratio is in the range of about 8:1 to about 1:1. In some embodiments, the ratio is in the range of about 2.5:1 to 1:1.

In some embodiments, a method is provided for reducing inter-chain disulfide bonds in an AV of an activated anti-CD166 antibody and conjugating an agent, eg, a thiol-containing agent, such as a drug, with the resulting inter-chain thiols to selectively locate the agent(s) on the AB. The method generally includes partial reduction of AB using a reducing agent to form at least two interchain thiols without generating all possible interchain thiols in the activated antibody; and conjugation of the agent with interchain thiols of the partially reduced AB. For example, the AV of an activated antibody is partially reduced over about 1 hour at about 37° C. at the desired ratio of reducing agent: activated antibody. In some embodiments, the ratio of reducing agent to activated antibody may range from about 20:1 to 1:1, from about 10:1 to 1:1, from about 9:1 to 1:1, from about 8:1 to 1:1, about 7:1 to 1:1, about 6:1 to 1:1, about 5:1 to 1:1, about 4:1 to 1:1, about 3:1 to 1:1, about 2:1 to 1:1, about 20:1 to 1:1.5, about 10:1 to 1:1.5, about 9:1 to 1:1.5, about 8:1 to 1:1.5, about 7:1 to 1:1.5, about 6:1 to 1:1.5, about 5:1 to 1:1.5, about 4:1 to 1:1.5, about 3:1 to 1:1.5, about 2:1 to 1:1.5, about 1.5:1 to 1:1.5, or approximately 1:1 to 1:1.5. In some embodiments, the ratio is in the range of about 5:1 to 1:1. In some embodiments, the ratio is in the range of about 5:1 to 1.5:1. In some embodiments, the ratio is in the range of about 4:1 to 1:1. In some embodiments, the ratio is in the range of about 4:1 to 1.5:1. In some embodiments, the ratio is in the range of about 8:1 to about 1:1. In some embodiments, the ratio is in the range of about 2.5:1 to 1:1.

The thiol-containing reagent may be, for example, cysteine or N-acetylcysteine. The restorative agent may be, for example, TCEP. In some embodiments, the reconstituted activated antibody may be purified prior to conjugation, using, for example, column chromatography, dialysis, or diafiltration. Alternatively, the reconstituted antibody is not purified after partial reconstitution and before conjugation.

The invention also relates to partially reduced activated antibodies against CD166, in which at least one inter-chain disulfide bond in the activated antibody is restored using a reducing agent without affecting any intra-chain disulfide bonds in the activated antibody, where the activated antibody contains the antibody or its antigen-binding fragment ( AB) specifically binding to CD166, a masking group (MM) inhibiting binding of an AB activated antibody in an uncleaved state to a CD166 target, and a cleavage group (CM) attached to AB, where CM is a polypeptide that functions as a substrate for a protease. In some embodiments, the implementation of the MM is attached to the AB through the SM. In some embodiments, one or more intrachain disulfide bond(s) of the activated antibody is unaffected by the reducing agent. In some embodiments, one or more of the intrachain MM disulfide bond(s) within the activated antibody is unaffected by the reducing agent. In some embodiments, the activated antibody in the uncleaved state has the following N-terminal to C-terminal structural arrangement: MM-CM-AB or AB-CM-MM. In some embodiments, the restoring agent is TCEP.

The description also relates to partially reduced activated antibodies, in which at least one inter-chain disulfide bond in the activated antibody is restored using a reducing agent without affecting any intra-chain disulfide bonds in the activated antibody, where the activated antibody contains an antibody or an antigen-binding fragment (AB), specifically binding a target, such as CD166, a masking group (MM) that inhibits binding of an AB activated antibody in an uncleaved state to the target, and a cleavable group (CM) attached to the AB, where CM is a polypeptide that functions as a substrate for at least one protease. In some embodiments, the implementation of the MM is attached to the AB through the SM. In some embodiments, one or more intrachain disulfide bond(s) of the activated antibody is unaffected by the reducing agent. In some embodiments, one or more of the intrachain MM disulfide bond(s) within the activated antibody is unaffected by the reducing agent. In some embodiments, the activated antibody in the uncleaved state has the following N-terminal to C-terminal structural arrangement: MM-CM-AB or AB-CM-MM. In some embodiments, the restoring agent is TCEP.

In other embodiments, using a method of reconstitution and conjugation of an agent, e.g. a drug, with an activated anti-CD166 antibody, site selectivity of the agent is obtained by providing an activated anti-CD166 antibody with a certain amount and location of lysine and/or cysteine residues. In some embodiments, the implementation of a certain number of lysine and/or cysteine residues above or below the number of corresponding residues in the amino acid sequence of the original antibody or activated antibodies. In some embodiments, a certain number of lysine and/or cysteine residues may result in a certain number of equivalents of an agent that can be conjugated to an anti-CD166 antibody or an activated anti-CD166 antibody. In some embodiments, a certain number of lysine and/or cysteine residues may result in a certain number of equivalents of an agent that can be conjugated to an anti-CD166 antibody or an activated anti-CD166 antibody in a site-specific manner. In some embodiments, the modified activated antibody is modified with one or more non-natural amino acids in a site-specific manner, thereby limiting the conjugation of agents to only non-natural amino acid regions in some embodiments. In some embodiments, an anti-CD166 antibody or an activated anti-CD166 antibody with a certain number and positions of lysine and/or cysteine residues can be partially reduced using a reducing agent, as discussed herein, such that any conjugation sites in a masking group or other unrelated The AV portions of the activated antibody are not reconstituted, and conjugation of the agent with interchain thiols in the AB.

In some embodiments, the activated antibodies described herein also contain an agent conjugated to the activated antibody. In some embodiments, the conjugate agent is a drug, such as an anti-inflammatory and/or antineoplastic agent. In such embodiments, the agent is conjugated to the carbohydrate group of the activated antibody, for example, In some embodiments, where the carbohydrate group is located outside the antigen-binding region of the antibody or an antigen-binding fragment in the activated antibody. In some embodiments, the agent is conjugated to a sulfhydryl group of an antibody or antigen-binding fragment in an activated antibody.

In some embodiments, the agent is a cytotoxic agent, such as a toxin (eg, an enzymatically active bacterial, fungal, plant, or animal toxin or fragments thereof) or a radioactive isotope (ie, a radioconjugate).

In some embodiments, the agent is a detectable group, such as a label or other marker, for example. For example, the agent is or contains a radiolabeled amino acid, one or more biotinylated groups that can be detected by labeled avidin (for example, streptavidin having a fluorescent marker or an enzymatic activity that can be detected by optical or calorimetric methods), one or more radioactive isotopes or radioactive nuclei, one or more fluorescent labels, one or more fluorescent labels, and/or one or more chemiluminescent agents. In some embodiments, detectable agents are attached via spacer molecules.

The description also relates to immunoconjugates containing an antibody conjugated to a cytotoxic agent, such as a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). Suitable cytotoxic agents include, for example, dolastatins and their derivatives (eg, auristatin E, AFP, MMAF, MMAE, MMAD, DMAF, DMAE). For example, the agent is monomethylauristatin E (MMAE) or monomethylauristatin D (MMAD). In some embodiments, the agent is an agent selected from the groups listed in Table. 5. In some embodiments, the agent is dolastatin. In some embodiments, the agent is auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is pyrrolobenzodiazepine. In some embodiments, the agent is a dimer of pyrrolobenzodiazepine.

In some embodiments, the agent is linked to AB using a maleimide-caproyl-valine-citrulline linker or a maleimide-PEG-valine-citrulline linker. In some embodiments, the agent is linked to an AB using a maleimide-caproyl-valine-citrulline linker. In some embodiments, the agent is linked to AB using a maleimide-PEG-valine-citrulline linker. In some embodiments,

- 75 041958 the agent is monomethylauristatin D (MMAD) linked to AB using a maleimide-PEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, and the construct loaded with this linker is referred to herein as vc-MMAD. In some embodiments, the agent is monomethylauristatin E (MMAE) linked to AB using a maleimide-PEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, and the construct loaded with this linker is referred to herein as vc-MMAE. In some embodiments, the agent is linked to AB using a maleimide-PEG-valinecitrulline linker. In some embodiments, the agent is monomethylauristatin D (MMAD) linked to AB using a maleimide-bis-PEG-valine-citrulline-p-aminobenzyloxycarbonyl linker, and the construct loaded with this linker is referred to herein as PEG2-VC-MMAD. The vc-MMAD, vc-MMAE, and PEG2-VC-MMAD structures are shown below.

Load for S-26

The description also refers to conjugated activated antibodies containing an activated antibody associated with a load of monomethylauristatin D (MMAD), where the activated antibody contains an antibody or an antigen-binding fragment (AB) that specifically binds a target, a masking group (MM) that inhibits binding of the AB activated antibody to uncleaved state with the target, and a cleavable group (CM) attached to AB, and CM is a polypeptide that functions as a substrate for at least one MMP protease.

In some embodiments, the MMAD-conjugated, activated antibody can be conjugated using any of several methods for attaching agents to the AB:

(a) attachment to AB carbohydrate groups, or (b) attachment to AB sulfhydryl groups, or (c) attachment to AB amino groups, or (d) attachment to AB carboxylate groups.

In some embodiments, the MMAD load is conjugated to AB via a linker. In some embodiments, the MMAD load is cysteine-conjugated in the AB via a linker. In some embodiments, the MMAD load is lysine-conjugated in the AB via a linker. In some embodiments, the MMAD load is conjugated to another AB residue via a linker, such as the residues described herein. In some embodiments, the linker is a thiol-containing linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the implementation of the linker is selected from the group consisting of the linkers shown in table. 6 and 7. In some embodiments, the activated antibody and the MMAD loading are linked via a maleimide-caproyl-valine-citrulline linker. In some embodiments

- 76 041958 the activated antibody and the MMAD load are linked via a maleimide-PEG-valinecitrulline linker. In some embodiments, the activated antibody and the MMAD loading are linked via a maleimide-caproyl-valine-citrulline-para-aminobenzyloxycarbonyl linker. In some embodiments, the activated antibody and the MMAD loading are linked via a maleimide-PEG-valine-citrulline-para-aminobenzyloxycarbonyl linker. In some embodiments, the MMAD load is conjugated to AB using the partial reduction and conjugation technology described herein.

In some embodiments, the polyethylene glycol (PEG) component of the linker of the present disclosure is formed from 2 ethylene glycol monomers, 3 ethylene glycol monomers, 4 ethylene glycol monomers, 5 ethylene glycol monomers, 6 ethylene glycol monomers, 7 ethylene glycol monomers, 8 ethylene glycol monomers, 9 ethylene glycol monomers, or at least 10 ethylene glycol monomers. In some embodiments as described herein, the PEG component is a branched polymer. In some embodiments, as described herein, the PEG component is a straight polymer. In some embodiments, the PEG polymer component is functionalized with an amino group or a derivative thereof, a carboxyl group or a derivative thereof, or both an amino group or a derivative thereof and a carboxyl group or a derivative thereof.

In some embodiments, the implementation of the PEG component of the linker according to the present description is an amino-tetraethylene glycol-carboxyl group or its derivative. In some embodiments, the implementation of the PEG component of the linker according to the present description is an amino triethylene glycol carboxyl group or a derivative thereof. In some embodiments, the implementation of the PEG component of the linker according to the present description is an amino-diethylene glycol carboxyl group or its derivative. In some embodiments, the amino derivative is the formation of an amide bond between the amino group and the carboxyl group to which it is conjugated. In some embodiments, the carboxyl derivative is the formation of an amide bond between the carboxyl group and the amino group to which it is conjugated. In some embodiments, the a carboxyl derivative is the formation of an ester bond between the carboxyl group and the hydroxyl group to which it is conjugated.

Enzymatically active toxins and fragments thereof that can be used include diphtheria toxin A chain, diphtheria toxin non-binding active fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modecin A chain, alpha-sarcin, Aleurites fordii proteins, diantine proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, fenomycin, enomycin and tricothecenes. Many radioactive isotopes are available for the production of radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re.

Antibody-cytotoxic agent conjugates are prepared using a variety of bifunctional protein crosslinkers such as K-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate HCL), active compounds esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene -2,6 diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, the ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating a radioactive isotope to an antibody. (See WO94/11026).

In table. 5 lists some of the illustrative drugs that can be used in the description described herein, but this is by no means considered as an exhaustive list.

- 77 041958

Table 5

Exemplary Drugs for Conjugation

CYTOTOXIC AGENTS

Auristatins Auristatin E Monomethylauristatin D (MMAD) Monomethylauristatin E (MMAE) Desmethylauristatin E (DMAE) Auristatin F

Monomethylauristatin F (MMAF) Desmethylauristatin F (DMAF) Derivatives of auristatin, e.g. its amides Auristatintyramine Auristatinquinoline Dolastatins

Dolastatin derivatives Dolastatin 16 DmJ

Dolastatin 16 Dpv

Maytansinoids, eg DM-1; DM-4

Maytansinoid derivatives

Duocarmycin

Duocarmycin derivatives Alpha-amanitin Anthracyclines Doxorubicin

Daunorubicin Briostatins Camptothecin Derivatives of camptothecin 7-substituted camptothecin

10, 11 Difluoromethylenedioxycamptothecin Combretastatins

Debromaplysiatoxin

Kagalalid-F

Discodermolide Ecteinascidins

ANTIVIRALS

Acyclovir

Vira A

Symmetrel

ANTIFUNGAL AGENTS

Nystatin

ADDITIONAL

ANTINEOPLASTIC AGENTS

Adriamycin

Cerubidin Bleomycin Alkeran Velban Oncovin Fluorouracil Methotrexate Thiope Bisantren Novantron Thioguanine Procarbizine

Turbostatin

Fenstatins

Hydroxifenstatin

Spongistatin 5

Spongistatin 7

Galistatin 1

Galistatin 2

Galistatin 3

Modified bryostatins

halocomstatins

Pyrrolobenzimidazoles (PBI)

Cybrostatin 6

Doxaliform

Anthracycline analogues

Sematodin analog (CemCH2-SH) Pseudomonas toxin A variant (PE38)

Pseudomonas toxin variant A (ZZ-PE38)

ZJ-101

OSW-1

4-nitrobenzyloxycarbonyl 06-benzylguanine derivatives Topoisomerase inhibitors Hemiasterlin

Cephalotaxin Homoharringtonin Pyrrolobenzodiazepine dimers (PBD)

Pyrrolobenzodiazepines

Functionalized Pyrrolobenzodiazepines Functionalized Dimers of Pyrrolobenzodiazepines Potassium e amycins

Podophyllotoxins Taxanes

Vinca alkaloids

CONJUGABLE DETECTION REAGENTS

Fluorescein and its derivatives Fluorescein isothiocyanate (FITC)

RADIOACTIVE DRUGS

125J 131!

89 _Zr

1111η

1231

1311 mTc 2°1 _T1

133 _Xe ^Y C ⁶² C

18r ⁶⁸ Ga ⁱ³ N i5 ₀ 38 _K

- 78 041958

Cytarabine ANTIBACTERIALS ⁸² Rb mTc (Technetium) Amino GLYCO ZIDES HEAVY METALS Streptomycin Barium Neomycin Gold Kanamycin Amikacin Platinum Gentamicin MEDICINES AGAINST MYCOPLASMA Tobramycin Tylosin Streptomycin B Spectinomycin Ampicillin Sulfanilamide Polymyxin Chloramphenicol Spectinomycin

One skilled in the art will recognize that a wide variety of possible groups can be attached to antibodies made according to this disclosure (see, for example, Conjugate Vaccines, Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr (eds), Carger Press, New York, (1989), the contents of which are incorporated herein by reference).

Attachment can be carried out by any chemical reaction that links two molecules, provided that the antibody and other group retain their respective activity. This bonding may include a variety of chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordination bonding, and complexation. In some embodiments, the implementation of the link, however, is a covalent link. Covalent bonding can be done either by direct condensation of existing side chains or by incorporation of external bridge molecules. Many divalent or polyvalent binding agents can be used to connect protein molecules, such as antibodies according to the present description, with other molecules. For example, representative binders may include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylenediamines. This listing is not intended to be exhaustive of the various classes of binders known in the art, but is instead illustrative of the most common binders (see Killen and Lindstrom, Jour. Immun., 133:1335-2549 (1984); Jansen et al., Immunological Reviews, 62:185-216 (1982) and Vitetta et al., Science, 238:1098 (1987).

In some embodiments, in addition to the compositions and methods provided herein, the conjugated activated antibody can also be modified for site-specific conjugation by modified amino acid sequences inserted or otherwise incorporated into the sequence of the activated antibody. These modified amino acid sequences are designed to allow controlled placement and/or dosing of the conjugate agent within the conjugated, activated antibody. For example, an activated antibody can be designed to include cysteine substitutions at light and heavy chain positions that provide reactive thiol groups and neither adversely affect protein folding and assembly nor alter antigen binding. In some embodiments, an activated antibody can be engineered to incorporate or otherwise introduce one or more non-natural amino acid residues into the activated antibody to provide suitable sites for conjugation. In some embodiments, an activated antibody can be designed to incorporate or otherwise introduce enzymatically activated peptide sequences into the sequence of the activated antibody.

Suitable linkers are described in the literature (see, for example, Ramakrishnan S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, US Pat. No. 5,030,719, which describes the use of a halogenated acetylhydrazide derivative attached to an antibody via an oligopeptide linker In some embodiments, suitable linkers include (i) EDC ((1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride);

(ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)toluene (Pierce Chem. Co., cat. (21558G);

(iii) SPDP (succinimidyl-6-[3-(2-pyridyldithio)propionamido]hexanoate) (Pierce Chem. Co., cat. #21651G);

(iv) Sulfo-LC-SPDP (sulfosuccinimidyl-6-[3-(2-pyridyldithio)propionamide]hexanoate (Pierce Chem. Co. cat #2165-G) and (v) sulfo-NHS (N-hydroxysulfosuccinimide) : Pierce Chem. Co., Cat. #24510), conjugated

- 79 041958 with EDC.

Additional linkers include, but are not limited to, SMCC ((succinimidyl 4-(Nmaleimidomethyl)cyclohexane-1-carboxylate), sulfo-SMCC (sulfosuccinimidyl-4-(Nmaleimidomethyl)cyclohexane-1-carboxylate), SPDB (N-succinimidyl- 4-(2-pyridyldithio)butanoate), or sulfo-SPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate).

The linkers described above contain components having different characteristics, resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters with alkyl carboxylates are more stable than sulfo-NHS esters with aromatic carboxylates. Linkers containing NHS esters are less soluble than sulfo-NHS esters. In addition, the SMPT linker contains a sterically hindered disulfide bond and can form conjugates with increased stability. Disulfide bonds tend to be less stable than other bonds because the disulfide bond is cleaved in vitro, reducing the amount of conjugate available. Sulfo-NHS, in particular, can increase the stability of carbodiimide coupling agents. Carbodiimide coupling agents (such as EDC) when used in combination with sulfo-NHS form esters that are more resistant to hydrolysis than compounds made by carbodiimide coupling alone.

In some embodiments, the linkers are cleavable. In some embodiments, the linkers are non-cleavable. In some embodiments, two or more linkers are present. Two or more linkers are all the same, ie. cleavable or non-cleavable, or two or more linkers are different, ie. at least one cleavable and at least one non-cleavable.

According to the present description, several methods of attaching funds to the AB are used:

(a) attachment to AB carbohydrate groups, or (b) attachment to AB sulfhydryl groups, or (c) attachment to AB amino groups, or (d) attachment to AB carboxylate groups.

As described, the AB can be covalently attached to the agent via an intermediate linker having at least two reactive groups, one for reaction with the AB and one for reaction with the agent. The linker, which may include any compatible organic compound, may be chosen such that reaction with the AB (or agent) does not adversely affect the reactivity and selectivity of the AB. In addition, attaching a linker to an agent may not interfere with the activity of the agent. Suitable linkers for reaction with oxidized antibodies or oxidized antibody fragments include linkers containing an amine selected from the group consisting of primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide, and thiosemicarbazide groups. Such reactive functional groups may exist as part of the linker structure, or they may be introduced by appropriate chemical modification of linkers free of such groups.

As used herein, suitable linkers for attaching reduced ABs include linkers containing reactive groups capable of reacting with the sulfhydryl group of the reduced antibody or fragment. Such reactive groups include, but are not limited to, reactive haloalkyl groups (including, for example, haloacetyl groups), p-mercury benzoate groups, and groups capable of Michael addition reactions (including, for example, maleimides and groups of the type described in Mitra and Lawton , 1979, J. Amer. Chem. Soc., 101:3097-3110).

As used herein, suitable linkers for attachment to neither oxidized nor reduced Abs include linkers having defined functional groups capable of reacting with the primary amino groups present on unmodified lysine residues in the Ab. Such reactive groups include, but are not limited to, NHS carboxylic or carboxylic esters, sulfo-NHS carboxylic or carboxylic esters, 4-nitrophenyl carboxylic or carboxylic esters, pentafluorophenyl carboxylic or carboxylic esters, acylimidazoles, isocyanates, and isothiocyanates.

As used herein, suitable linkers for attachment to neither oxidized nor reduced Abs include those having specific functional groups capable of reacting with carboxylic acid groups present on aspartate or glutamate residues in Ab activated using suitable reagents. . Suitable activating agents include EDC, with or without the addition of NHS or sulfo-NHS, and other dehydrating agents used to form carboxamide. In these instances, functional groups present on suitable linkers may include primary and secondary amines, hydrazines, hydroxylamines, and hydrazides.

The agent can be attached to the linker before or after the linker is attached to the AB. At

- 80 041958 specific applications may be desirable to first obtain an intermediate connection

An AV linker wherein the linker is free of the associated agent. Depending on the particular application, the particular agent may be covalently attached to the linker. In some embodiments, the AB is first attached to the MM, SM, and associated linkers and then attached to the linker for conjugation purposes.

branched linkers.

In specific embodiments, branched linkers are used having multiple sites for attachment of agents. For multi-site linkers, a single covalent attachment to AB can result in an AB-linker intermediate capable of binding the agent at multiple sites. The sites may be aldehyde or sulfhydryl groups, or any chemical site to which agents can be attached.

In some embodiments, higher specific activity (or higher agent to AV ratio) can be achieved by attaching a single site linker at multiple sites on the AB. This plurality of regions can be introduced into the AB in either of two ways. First, you can get a lot of aldehyde groups and/or sulfhydryl groups in the same AB. Secondly, a branched linker with multiple functional sites can be attached to the aldehyde or sulfhydryl AB for subsequent attachment to the linkers. The functional sites of a branched or multi-site linker may be aldehyde or sulfhydryl groups, or may be any chemical site to which linkers can be attached. Even higher specific activity can be obtained by a combination of these two methods, ie. attaching multi-site linkers at multiple sites on the AB.

Cleavable Linkers.

Peptide linkers that are cleavage-sensitive enzymes of the complement system, such as, but not limited to, γ-plasminogen activator, tissue plasminogen activator, trypsin, plasmin, or other enzyme having proteolytic activity, can be used in one embodiment of the present disclosure. In accordance with one method according to the present description, the agent is attached via a linker sensitive to cleavage by complement. The antibody is selected from a class that can activate complement. The antibody-drug conjugate thus activates the complement cascade and releases the agent at the target site. According to another method of the present disclosure, the agent is attached via a linker sensitive to cleavage by enzymes having proteolytic activity, such as y-plasminogen activator, tissue plasminogen activator, plasmin or trypsin. These cleavable linkers can be used in conjugated activated antibodies containing an extracellular toxin, for example, as a non-limiting example, any of the extracellular toxins shown in table. 5.

Non-limiting examples of cleavable linker sequences are shown in Table 1. 6.

- 81 041958

Table 6 Exemplary linker sequences for conjugation

Amino Acid Sequence Cleavable Sequence Types

Plasmin-cleavable sequences

Prourokinase

TGFP

Plasminogen

Stafilskinase

Xa-cleavable sequences

PRFKIIGG (SEQ ID NO: 89) PRFRIIGG (SEQ ID NO: 90) SSRHRRALD (SEQ ID NO: 91) RKSSIIIRMRDVVL (SEQ ID NO: 92) SSSFDKGKYKKGDDA (SEQ ID NO: 93)

SSSFDKGKYKRGDDA (SEQ ID NO: 94)

IEGR (SEQ ID NO: 95)

IDGR (SEQ ID NO: 96)

GGSIDGR (SEQ ID NO: 97)

Cleavable MMP sequences

Gelatinase A

PLGLWA (SEQ ID NO: 98)

Collagenase cleavable sequences

Calf skin collagen (al(I) chain) Calf skin collagen (α2(Ι) chain) Bovine cartilage collagen (al(II) chain) Human liver collagen (al(III) chain) α ₂ Μ human ΡΖΡ human α _χ Μ rat α ₂ Μ rats

0-13(2 J) rats (scTs-13(2 J) rats Human fibroblast collagenase (autolytic cleavage)

GPQGIAGQ (SEQ ID NO: 99) GPQGLLGA (SEQ ID NO: 100) GIAGQ (SEQ ID) NO:101) GPLGIAGI (SEQ ID NO: 102) GPEGLRVG (SEQ ID NO: 103) YGAGLGVV (SEQ ID NO: 104) AGLGVVER (SEQ ID NO: 105) AGLGISST (SEQ ID NO: 106) EPQALAMS (SEQ ID NO: 107) QALAMSAI (SEQ ID NO: 108) AAYHLVSQ (SEQ ID NO: 109) MDAFLESS (SEQ ID NO: 110) ESLPVVAV (SEQ ID NO: 111) SAPAVESE (SEQ ID NO: 112) DVAQFVLT (SEQ ID NO: 113) VAQFVLTE (SEQ ID NO: 114) AQFVLTEG (SEQ ID NO: 115) PVQPIGPQ (SEQ ID NO: 116)

In addition, agents can be attached via disulfide bonds (eg, disulfide bonds on a cysteine molecule) to the AB. Because many tumors naturally release high levels of glutathione (the reducing agent), this can repair disulfide bonds, followed by release of the agent at the delivery site. In some embodiments, a reducing agent that can modify CM can also modify the linker of the conjugated, activated antibody.

Spacer and split elements.

In some embodiments, it may be necessary to design the linker in such a way as to optimize the distance between the agent and the AV of the activated antibody. This can be done by using a linker of the general structure

W-(CH2)nQ, where W is either -NH--CH2- or -CH ₂ -,

Q is an amino acid, a peptide, and n is an integer from 0 to 20.

In some embodiments, the linker may comprise a spacer element and a cleavable element. The spacer element serves to position the cleavable element away from the AB core so that the cleavable element is more accessible to the enzyme responsible for cleavage. The specific branched linkers described above can serve as spacer elements.

Throughout this description, it should be understood that the attachment of a linker to a tool (or a spacer element to a cleavable element, or a cleavable element to a tool) need not be a specific method of attachment or reaction. Any reaction

- 82 041958 providing a product of suitable stability and biocompatibility is acceptable.

Serum complement and linker selection.

According to one method of the present disclosure, when release of the agent is desired, an AB is used, which is an antibody of the class that can activate complement. The resulting conjugate retains the ability to both bind the antigen and activate the cascade of complement reactions. Thus, according to this embodiment of the present disclosure, the agent is attached to one end of a cleavable linker or cleavable element, and the other end of the linker group is attached to a specific site on the AB. For example, if the agent has a hydroxy or amino group, it can be attached to the carboxy terminus of a peptide, amino acid, or other suitably chosen linker via an ester or amide bond, respectively. For example, such agents can be attached to the linker peptide via a carbodimide reaction. If the agent contains functional groups that may interfere with attachment to the linker, these interfering functional groups can be blocked prior to attachment and unblocked after the product conjugate or intermediate is prepared. The opposite, or amino, end of the linker is then used, either directly or after further modification, to bind to an AB capable of activating complement.

Linkers (or spacer elements of linkers) can be of any desired length, one end can be covalently attached to specific sites on the AB of the activated antibody. The other end of the linker or spacer element can be attached to the amino acid or peptide linker.

Thus, when these conjugates bind an antigen in the presence of complement, the amide or ester bond that attaches the agent to the linker is cleaved, releasing the agent in its active form. These conjugates, when administered to a subject, can deliver and release an agent at a target site and are particularly effective for in vivo delivery of drugs, antibiotics, antimetabolites, antiproliferative agents, and the like, as shown in, but not limited to, Table 1. 5.

Linkers for release without complement activation: In another application of targeted delivery, it is desirable to release the agent without complement activation because activation of the complement cascade may eventually lyse the target cell. Thus, this method can be used when the delivery and release of the agent is to be accomplished without destroying the target cell. This is the goal if it is desired to deliver cellular mediators such as hormones, enzymes, corticosteroids, neurotransmitters, genes or enzymes to target cells. These conjugates can be prepared by attaching the agent to an AV that is not capable of activating complement via a linker that is weakly susceptible to cleavage by serum proteases. When this conjugate is administered to an individual, antigen-antibody complexes are rapidly formed while cleavage of the agent is slow, thus resulting in release at the target site.

Biochemical cross-linking agents.

In some embodiments, an activated antibody can be conjugated to one or more drugs using specific biochemical crosslinkers. Cross-linking reagents form molecular bridges linking the functional groups of two different molecules. To link two different proteins in a stepwise manner, hetero-bifunctional crosslinkers can be used to avoid the formation of an undesirable homopolymer.

You can also use peptidyl linkers that are cleaved by lysosomal proteases, such as Val-Cit, Val-Ala or other dipeptides. In addition, acid-labile linkers cleavable in the low pH environment of the lysosome, such as bis-sialyl ester, can be used. Other useful linkers include cathepsin-labile substrates, in particular those having optimal functions at acidic pH.

Illustrative hetero-bifunctional cross-linking agents are shown in table. 7.

- 83 041958

Table 7

Exemplary Hetero-Bifunctional Crosslinkers

HETERO-BIFUNCTIONAL CROSS-LINKING FACILITIES Linker Reactivity Benefits and Applications Spacer arm length after cross stitching (Angstrom)

SMPT Primary amines Sulfhydryls Greater stability 11.2 A SPDP Primary amines Sulfhydryls Thiolation fissile cross-linking 6, 8 A LC-SPDP Primary amines Sulfhydryls Extended spacer arm 15.6 A Sulfo-LC-SPDPPrimary Amines Sulfhydryls Extended spacer arm 15.6 A SMCC Primary amines Sulfhydryls stable maleimide reactive group Enzyme-antibody conjugation Hapten-carrier protein conjugation 11.6 A Sulfo-SMCC Primary amines Sulfhydryls Stable maleimide reactive group Water soluble Enzyme-antibody conjugation 11.6 A MBS Primary amines Sulfhydryls Enzyme-antibody conjugation Hapten-carrier protein conjugation 9.9 A Sulfo-MBS Primary amines Sulfhydryls water soluble 9.9 A SIAB Primary amines Sulfhydryls Enzyme-antibody conjugation 10.6 A Sulfo-SIAB Primary amines Sulfhydryls water soluble 10.6 A SMPB Primary amines Sulfhydryls Extended spacer arm Enzyme-antibody conjugation 14.5 A Sulfo-SMPB Primary amines Sulfhydryls Extended spacer arm 14.5 A EDE/Sulfo-] MHS Primary Amines Hapten-conjugation 0

carrier Carboxyl groups

ABH Carbohydrates Reaction with 11.9 A sugar groups Non-selective

Non-cleavable linkers or direct attachment.

In some embodiments of this disclosure, the conjugate may be conjugated to deliver the agent to the target but not release. This can be done by attaching the agent to the AB, either directly or via a non-cleavable linker.

These non-cleavable linkers may include amino acids, peptides, D-amino acids, or other organic compounds that can be modified to include functional groups that can then be used to attach to AB by the methods described herein. The general formula for such an organic linker may be

W-(CH2) _n -Q, where W is either -NH--CH2- or -CH ₂ -,

Q is an amino acid, a peptide, and

- 84 041958 n is an integer from 0 to 20.

non-cleavable conjugates.

In some embodiments, a compound can be attached to ABs that do not activate complement. When using AVs incapable of complement activation, this attachment can be performed using linkers sensitive to cleavage by activated complement or using linkers not sensitive to cleavage by activated complement.

The antibodies described herein can also be formulated into immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as those 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 US Pat. Nos. 4,485,045 and 4,544,545. Liposomes with extended circulation time are described in US Pat.

Particularly useful liposomes can be obtained by reverse phase evaporation from a lipid composition containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through filters with a defined pore size to obtain liposomes of the desired diameter. Fab' fragments of an antibody of the present disclosure can be conjugated to liposomes as described in Martin et al., J. Biol. Chem., 257:286-288 (1982), via a disulfide exchange reaction.

Definitions.

Unless otherwise defined, scientific and technical terms used in connection with the present description should have the meanings that are generally accepted by a person skilled in the art. The term object refers to one or more such objects, eg connection refers to one or more connections, so the terms one or more and at least one can be used interchangeably. In addition, unless the context otherwise requires, singular terms must include the plural and plural terms must include the singular. In general, the nomenclature used in connection with cell and tissue culture, molecular biology and chemistry, and the hybridization of proteins and oligo-or polynucleotides, and in their methods, as described herein, is well known and generally accepted in the art. Standard methods are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection). Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's instructions, as is generally accepted in the art or as described herein. The above methods and procedures are usually carried out in accordance with conventional methods, as is well known in this field, and as described in various and more specialized references mentioned and discussed throughout the present description. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry and their laboratory procedures and methods as described herein is well known and generally accepted in the art. Standard methods are used for chemical synthesis, chemical assays, pharmaceuticals, formulations, and patient delivery and treatment.

As used in accordance with the present description, the following terms, unless otherwise indicated, should be understood as having the following meanings.

As used herein, the term antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i. molecules containing an antigen-binding site that specifically binds an antigen (immunoreactive with respect to the antigen). By specifically binds or immunoreactive with respect to or immunospecifically binds means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds to them with a much lower affinity (K _d >10'6 ). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, domain antibody, single chain, Fab and F(ab') ₂ fragments, scFvs, and Fab expression library.

It is known that the basic structural unit of an antibody contains a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, where each pair contains one light (approximately 25 kDa) and one heavy chain (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Typically, human-derived antibody molecules belong to any of the IgG, IgM, IgA, IgE, and IgD classes, which differ from one another in the nature of the heavy chain present in the molecule. Specific classes also have subclasses such as IgG1, IgG ₂ and others. Moreover, in humans, the light chain can be a kappa chain or a lambda chain.

- 85 041958

The term monoclonal antibody (mAb) or monoclonal antibody composition as used herein refers to a population of antibody molecules containing only one molecular species of antibody molecules, consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of a monoclonal antibody are identical across all molecules in the population. MAb contain antigennegative site capable of immune response with a specific epitope of the antigen, characterized by a unique binding affinity for it.

The term antigen-binding site or binding portion refers to the portion of an immunoglobulin molecule that is involved in antigen binding. The antigen-binding site is formed by the amino acid residues of the N-terminal variable (V) regions of the heavy (H) and light (L) chains. Three highly diverse fragments within the V regions of the heavy and light chains, designated hypervariable regions, are located between more conserved flanking fragments known as framework regions or FRs. Thus, the term FR refers to amino acid sequences that are found in nature between and adjacent to the hypervariable regions of immunoglobulins. In an antibody molecule, the three light chain hypervariable regions and the three heavy chain hypervariable regions are arranged with respect to each other in three-dimensional space so as to form an antigen-binding surface.

The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are designated complementarity-determining regions or CDRs. The assignment of amino acids to each of the domains is in accordance with the definitions of Rabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987), Chothia et al. Nature, 342:878-883 (1989).

As used herein, the term epitope includes any protein determinant capable of specifically binding to an immunoglobulin, scFv, or T cell receptor. The term epitope includes any protein determinant capable of specifically binding to an immunoglobulin or T cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, antibodies can be generated against the N-terminal or C-terminal peptides of the polypeptide. An antibody is said to specifically bind an antigen when the dissociation constant is <1 μM. In some embodiments, <100 nM. In some embodiments, <10 nM.

As used herein, the terms specific binding, immunological binding, and immunological binding properties refer to non-covalent interactions of the type that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity, of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, where a smaller Kd represents a greater affinity. The immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method involves measuring the rates of formation and dissociation of the antigen-binding site/antigen complex, where these rates depend on the concentrations of partners in the complex, the affinity of the interaction and geometric parameters that equally affect the rate in both directions. Thus, both the binding rate constant (K _on ) and the dissociation rate constant (K _off ) can be determined by calculating concentrations and actual binding and dissociation rates (see Nature, 361:186-87 (1993)). The K _o ff/K _on ratio eliminates all non-affinity parameters and is equal to the dissociation constant K _d (see generally Davies et al. (1990), Annual. Rev. Biochem., 59:439-473). An antibody of the present disclosure is said to specifically bind to a target when the binding constant (K _d ) is <1 μM, in some embodiments, <100 nM, in some embodiments, <10 nM, and in some embodiments, <100 pM to about 1 pM as measured by assays such as radioactive ligand binding assays or similar assays known to those skilled in the art.

The term isolated polynucleotide, as used herein, shall mean a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, where, by virtue of its origin, the isolated polynucleotide (1) is not associated with all or part of the polynucleotide in which the isolated polynucleotide is found in nature, (2) is operably linked to a polynucleotide to which it is not naturally linked, or (3) does not occur naturally as part of a larger sequence. Polynucleotides as described herein include nucleic acid molecules encoding the heavy chain of the immunoglobulin molecules shown herein and nucleic acid molecules encoding the light chain of the immunoglobulin molecules shown herein.

- 86 041958

The term isolated protein as used herein means a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof, where by virtue of its origin or source of preparation, the isolated protein (1) is not associated with proteins found in nature, (2 ) is free from other proteins from the same source, such as free from mouse proteins, (3) is expressed by cells from another species, or (4) is not naturally occurring.

The term polypeptide is used herein as a general term to refer to native protein, fragments or analogs of a polypeptide sequence. Thus, native protein fragments and analogs are species of the polypeptide genus. The polypeptides as described comprise the heavy chain of the immunoglobulin molecules shown herein and the light chain of the immunoglobulin molecules shown herein, as well as antibody molecules formed by combinations containing the heavy chain of immunoglobulin molecules with the light chain of immunoglobulin molecules, such as light kappa chain of immunoglobulin molecules, and vice versa, as well as their fragments and analogues.

The term natural, as used herein with respect to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) that can be isolated from a natural source and that has not been purposefully modified by humans in a laboratory or otherwise is naturally occurring.

The term operably linked, as used herein, refers to the arrangement of components thus described in such a relationship that allows them to function in the manner intended for them. A control sequence operably linked to a coding sequence is ligated such that expression of the coding sequence occurs under conditions compatible with the control sequences.

The term control sequence, as used herein, refers to polynucleotide sequences necessary for the expression and processing of the coding sequences to which they are ligated. The nature of such control sequences varies depending on the host organism: in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, such control sequences typically include promoters and a transcription termination sequence. The term control sequences is intended to include, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence provides an advantage, such as leader sequences and fusion partner sequences. The term polynucleotide as used herein means nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides, or a modified form of any type of nucleotide. The term includes single- and double-stranded forms of DNA.

The term oligonucleotide as used herein includes natural and modified nucleotides linked to each other by natural and non-natural oligonucleotide linkages. Oligonucleotides are a subset of polynucleotides typically having a length of 200 bases or less. In some embodiments, the oligonucleotides are 10-60 bases in length, and in some embodiments, 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 bases in length. Oligonucleotides are typically single stranded, eg for probes, although oligonucleotides may be double stranded, eg for use in the construction of a gene mutant. Oligonucleotides as used herein are sense or antisense oligonucleotides.

The term natural nucleotides as used herein includes deoxyribonucleotides and ribonucleotides. The term modified nucleotides as used herein includes nucleotides with modified or substituted sugar groups and the like. The term oligonucleotide linkages, as used herein, includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphorimidate, and the like. See, for example, LaPlanche et al., Nucl. Acids Res. 14:9081 (1986); Stec et al., J. Am. Chem. Soc., 106:6077 (1984), Stein et al., Nucl. Acids Res., 16:3209 (1988), Zon et al., Anticancer Drug Design, 6:539 (1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, p. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al., US patent No. 5151510; Uhlmann and Peyman Chemical Reviews, 90:543 (1990). The oligonucleotide may include a detection label, if desired.

As used herein, the twenty common amino acids and their abbreviations correspond to common use. See Immunology - A Synthesis (2nd edition, E.S. Golub and D.R. Green, Eds., Sinauer Associates, Sunderland, Mass. (1991)). Stereoisomers (e.g. D-amino acids) Twenty common amino acids, non-natural amino acids,

- 87 041958 such as α-,α-disubstituted amino acids, N-alkylamino acids, lactic acid, and other unusual amino acids may also be useful components of the polypeptides of the present description. Examples of unusual amino acids include 4 hydroxyproline, γ-carboxyglutamate, ε-N,N,Ntrimethyllysine, ε-N-acetylysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N -methylarginine and other similar amino acids and imino acids (eg 4-hydroxyproline). In the polypeptide annotation used herein, the left direction is the direction towards the amino terminus and the direction to the right is the direction towards the carboxy terminus, in accordance with standard usage and conventions.

Similarly, unless otherwise indicated, the left end of a single stranded polynucleotide sequence is the 5' end, the left direction of a double stranded polynucleotide sequence is indicated by the 5' direction. The direction of attachment from 5' to 3' of growing RNA transcripts is designated as the direction of transcription. Regions of the DNA chain sequence that have the same sequence as RNA and are located 5' from the 5' end of the RNA transcript are designated as upstream sequences, regions of the DNA chain sequence that have the same sequence as RNA and are located 3 ' from the 3' end of the RNA transcript are designated as downstream sequences.

In the context of polypeptides, the term significant identity means that two peptide sequences, when optimally aligned, such as through the GAP or BESTFIT program using default gap penalties, share at least 80 percent sequence identity, in some embodiments at least 90 percent sequence identity, in some embodiments at least 95 percent sequence identity, and in some embodiments at least 99 percent sequence identity.

In some embodiments, residues at non-identical positions differ by conservative amino acid substitutions.

As discussed herein, minor changes in the amino acid sequences of antibody or immunoglobulin molecules are contemplated as being within the scope of this disclosure, provided that the amino acid sequence variants retain at least 75%, in some embodiments at least 80%, 90%, 95%, and in some embodiments 99% implementation. In particular, conservative amino acid substitutions are contemplated.

Conservative substitutions are those that occur within a family of amino acids related in their side chains. Genetically encoded amino acids are generally divided into the following families:

(1) acidic amino acids are aspartate, glutamate;

(2) the basic amino acids are lysine, arginine, histidine;

(3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine.

Hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include (i) serine and threonine, which form an aliphatic hydroxyl family;

(ii) asparagine and glutamine, which form an amide-containing family;

(iii) alanine, valine, leucine and isoleucine, which form an aliphatic family; and (iv) phenylalanine, tryptophan and tyrosine, which form an aromatic family.

For example, it is reasonable to expect that an isolated substitution of a leucine for an isoleucine or valine, an aspartate for a glutamate, a threonine for a serine, or a similar substitution of an amino acid for a structurally related amino acid will not have a significant effect on the binding or properties of the resulting molecule, especially if the substitution does not affect the amino acid in the backbone. plot. Whether an amino acid substitution results in a functional peptide can be readily determined by analyzing the specific activity of the polypeptide derivative. The analyzes are described in detail in this document. Fragments or analogs of antibody or immunoglobulin molecules can easily be obtained by one skilled in the art. Suitable amino and carboxy ends of the fragments or analogues are located near the boundaries of the functional domains. Structural and functional domains can be identified by comparing nucleotide and/or amino acid sequence data with public or privately owned sequence databases. In some embodiments, computerized comparison methods are used to identify sequence motifs or predict protein conformational domains present in other proteins of known structure and/or function. Known methods for identifying proteins

- 88 041958 sequences that fit into a known three-dimensional structure. Bowie et al., Science,

253:164 (1991). Thus, the above examples show that one skilled in the art can recognize sequence motifs and structural conformations that can be used to determine structural and functional domains as described.

Suitable amino acid substitutions are those that (1) decrease the sensitivity to proteolysis, (2) decrease the sensitivity to oxidation, (3) change the binding affinity for the formation of protein complexes, (4) change the binding affinity, and (5) confer or modify other physical properties. -chemical or functional properties of such analogues.

Analogues may include different mutein sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (eg, conservative amino acid substitutions) can be made in natural sequence (eg, on the portion of the polypeptide outside of the domain(s) forming intermolecular contacts.

A conservative amino acid substitution should not significantly change the structural characteristics of the original sequence (eg, an amino acid substitution should not tend to break the helix existing in the original sequence or destroy other types of secondary structure that characterize the original sequence). Examples of secondary and tertiary structures of polypeptides known in the art are described in Proteins, Structures and Molecular Principles (Creighton, ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et at. Nature, 354:105 (1991).

The term polypeptide fragment, as used herein, refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion and/or one or more internal deletion(s), but in which the remaining amino acid sequence is identical to the corresponding positions in a natural sequence derived, for example, from a full-length cDNA sequence. Fragments are typically at least 5, 6, 8 or 10 amino acids long, in some embodiments at least 14 amino acids long, in some embodiments at least 20 amino acids long, typically at least 50 amino acids long, and in some embodiments, the implementation is at least 70 amino acids long. The term analog, as used herein, refers to polypeptides consisting of a fragment of at least 25 amino acids, having significant identity with a portion of the deduced amino acid sequence, and having specific binding to a target, under suitable binding conditions. Typically, polypeptide analogs contain a conservative amino acid substitution (or addition or deletion) relative to the naturally occurring sequence. Analogues are typically at least 20 amino acids in length, in some embodiments at least 50 amino acids or more in length, and can often be as long as a full-length naturally occurring polypeptide.

The term agent is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract derived from biological materials.

As used herein, the terms labeled or labeled refer to the incorporation of a detectable marker, for example, by incorporating a radiolabeled amino acid or by attaching biotinylated groups to a polypeptide that can be detected by labeled avidin (for example, streptavidin having a fluorescent marker or an enzymatic activity that can be detected by optical or calorimetric methods). In specific situations, the label or marker may also be therapeutic. Various methods for labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioactive isotopes or radionuclides (e.g., ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹ⁿ In, ¹²⁵ I, ¹³¹ I), fluorescent labels (eg, FITC, rhodamine, phosphorus lanthanides), enzymatic labels (eg, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinylated groups, predetermined polypeptide epitopes recognized by the secondary reporter (eg, leucine zipper pair sequences, secondary antibody binding sites, metal-binding domains, epitope tags). In some embodiments, labels are attached using spacer arms of various lengths to reduce potential steric hindrances. The term pharmaceutical agent or drug, as used herein, refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

Other chemical terms in this document are used in accordance with the generally accepted

- 89 041958 in this field of application, as illustrated in The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

As used herein, substantially pure means that the target species are the predominant species present (i.e., molarly, they are more numerous than any other individual species in the composition), and In some embodiments, the substantially purified fraction is is a composition, where the target objects are at least about 50% (in molar terms) of all present macromolecular objects.

Typically, a substantially pure composition contains greater than about 80% of all macromolecular entities present in the composition, in some embodiments greater than about 85%, 90%, 95%, and 99%. In some embodiments, the target objects are purified to a substantially homogeneous state (contaminating objects cannot be detected in the composition by conventional detection methods), where the composition consists mainly of one kind of macromolecular objects.

The term patient includes human and veterinary subjects.

Antibodies and/or activated antibodies as described herein specifically bind a given target, for example a human target protein such as human CD166. Also included in this description are antibodies and/or activated antibodies that bind the same epitope as the antibodies and/or activated antibodies described herein. Also included in this specification are antibodies and/or activated antibodies that compete with an anti-CD166 antibody and/or an activated anti-CD166 antibody described herein for binding to CD166, eg, human CD166. Also included in this specification are antibodies and/or activated antibodies that cross-compete with an anti-CD166 antibody and/or an activated anti-CD166 antibody described herein for binding to CD166, eg, human CD166.

One skilled in the art will appreciate that it is possible to determine, without undue experimentation, whether a monoclonal antibody (e.g., a mouse monoclonal or humanized antibody) has the same specificity as the monoclonal antibody used in the methods described herein by checking whether it prevents the first binding of the latter to the target. If the tested monoclonal antibody competes with the monoclonal antibody of this description, as shown by the reduced binding of the monoclonal antibody of this description, then the two monoclonal antibodies bind the same or a closely related epitope. An alternative way to determine if a monoclonal antibody has the specificity of a monoclonal antibody of this description is to preincubate the monoclonal antibody of this description with a target and then add the test monoclonal antibody to determine if the ability of the test monoclonal antibody to bind to the target is inhibited. If the test monoclonal antibody is inhibited, then it is likely to have the same or functionally equivalent epitope specificity as the monoclonal antibody of this specification.

Multispecific activated antibodies.

The description also applies to multispecific activated antibodies against CD166. The multispecific activated antibodies provided herein are multispecific antibodies that recognize CD166 and at least one or more other antigens or epitopes and that contain at least one masking group (MM) associated with at least one antigen-binding or epitope-binding domain of a multispecific antibody, so that the attachment of MM reduces the ability of the antigen-binding or epitope-binding domain to bind its target. In some embodiments, the MM is attached to the antigen-binding or epitope-binding domain of the multispecific antibody via a cleavable group (CM) that functions as a substrate for at least one protease. The activated multispecific antibodies provided herein are stable in the circulation, activated in the areas targeted for therapy and/or diagnostics, but not in the normal, i.e. healthy tissue, and upon activation exhibit target binding at least comparable to that of the corresponding, unmodified, multispecific antibody.

In some embodiments, the multispecifically activated antibodies are designed to attract immunoeffector cells and are also referred to herein as immunoeffector cell-attracting multispecifically activated antibodies. In some embodiments, the multispecifically activated antibodies are designed to attract leukocytes and are also referred to herein as leukocyte-attracting multispecifically activated antibodies. In some embodiments, multispecifically activated antibodies designed to attract T cells are also referred to herein as T cell-attracting multispecifically activated antibodies. In some embodiments, the implementation of multispecific activated antibodies attract surface antigen on

- 90 041958 leukocyte, for example on a T cell, on a natural killer (NK) cell, on a myeloid mononuclear cell, on a macrophage and/or on another immunoeffector cell. In some embodiments, the implementation of the immunoeffector cell is a leukocyte. In some embodiments, the implementation of the immunoeffector cell is a T cell. In some embodiments, the immunoeffector cell is an NK cell. In some embodiments, the immunoeffector cell is a mononuclear cell, such as a myeloid mononuclear cell. In some embodiments, multispecific activated antibodies are designed to bind or otherwise interact with more than one target and/or more than one epitope, and are also referred to herein as multi-antigen-targeted activated antibodies. As used herein, the terms target and antigen are used interchangeably.

In some embodiments, the immunoeffector cell-attracting multispecific activated antibodies of this description comprise a targeting antibody or an antigen-binding fragment thereof that binds CD166 and an immunoeffector cell-attracting antibody or an antigen-binding portion thereof, wherein at least one of the targeting antibody or antigen-binding fragment thereof and/or the attracting immune effector cells of the antibody or its antigen-binding portion is masked. In some embodiments, the immunoeffector cell-attracting antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to the first immunoeffector cell-attracting target, wherein the AB1 is attached to a masking group (MM1) in such a way that the addition of MM1 reduces the ability of AB1 to bind first target. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, including a second antibody or antigen-binding fragment (AB2) that binds CD166, where AB2 is attached to a masking group (MM2) such that attachment of MM2 reduces the ability AB2 bind CD166. In some embodiments, the immunoeffector cell-attracting antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to the first immunoeffector cell-attracting target, wherein the AB1 is attached to a masking group (MM1) in such a way that the addition of MM1 reduces the ability of AB1 to bind the first target, and the targeting antibody or antigen-binding fragment thereof contains a second antibody or fragment thereof, including a second antibody or antigen-binding fragment (AB2) that binds CD166, where AB2 is attached to a masking group (MM2) in such a way that the addition of MM2 reduces the ability AB2 bind CD166. In some embodiments, the non-immunoeffector cell-attracting antibody is a cancer-targeting antibody. In some embodiments, the non-immunocyte effector antibody is an IgG. In some embodiments, the immunoeffector cell-attracting antibody is scFv. In some embodiments, the CD166-targeting antibody (eg, a non-immunocyte effector antibody) is IgG and the immunoeffector cell-attracting antibody is scFv. In some embodiments, the implementation of the immunoeffector cell is a leukocyte. In some embodiments, the immunoeffector cell is a T cell. In some embodiments, the immunoeffector cell is an NK cell. In some embodiments, the implementation of the immunoeffector cell is a myeloid mononuclear cell.

In some embodiments, the T cell-attracting multispecific activated antibodies of this disclosure comprise a CD166-targeted antibody or antigen-binding fragment thereof and a T-cell-attracting antibody or antigen-binding portion thereof, wherein at least one of the CD166-targeted antibody or antigen-binding fragment thereof, and /or the T-cell-attracting antibody or antigen-binding portion thereof is masked. In some embodiments, the T cell-attracting antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment (AB1) that binds the first T-cell-attracting target, wherein the AB1 is attached to a masking group (MM1) such that attachment of MM1 reduces the ability AB1 bind the first target. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, including a second antibody or antigen-binding fragment (AB2) that binds CD166, where AB2 is attached to a masking group (MM2) such that attachment of MM2 reduces the ability AB2 bind CD166. In some embodiments, the T cell recruiting antibody or antigen binding fragment thereof comprises a first antibody or antigen binding fragment (AB1) that binds the first T cell recruiting target, wherein the AB1 is attached to a masking group (MM1) such that attachment of MM1 reduces the ability of AB1 bind to the first target, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, including a second antibody or antigen-binding fragment (AB2) that binds CD166, where AB2 is attached to a masking group (MM2) and

- 91 041958 that the addition of MM2 reduces the ability of AB2 to bind CD166.

In some embodiments of an immunoeffector cell-attracting multispecific activated antibody, one antigen is CD166 and the other antigen is generally a stimulatory or inhibitory receptor present on the surface of a T cell, a natural killer (NK) cell, a myeloid mononuclear cell, a macrophage and/or another immune effector cell such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3 or VISTA. In some embodiments, the antigen is a stimulatory receptor present on the surface of a T cell or NK cell; examples of such stimulatory receptors include, but are not limited to, CD3, CD27, CD28, CD137 (also referred to as 4-1BB), GITR, HVEM, ICOS, NKG2D, and OX40. In some embodiments, the antigen is an inhibitory receptor present on the surface of a T cell; examples of such inhibitory receptors include, but are not limited to, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and expressed NK KIRs. An antibody domain conferring specificity for a T cell surface antigen may also be substituted for a ligand or ligand domain binding a T cell receptor, an NK cell receptor, a macrophage receptor, and/or another immune effector cell receptor, such as, but not limited to, B7-1 , B7-2, B7H3, PDL1, PDL2 or TNFSF9.

In some embodiments, the T-cell-attracting multispecific activated antibody comprises an anti-CD3 epsilon (CD3ε, also referred to herein as CD3e and CD3) scFv and a targeting antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and/or targeting the antibody or its antigen-binding portion is masked. In some embodiments, the anti-CD3ε scFv comprises a first antibody or antigen-binding fragment (AB1) thereof that binds CD3ε, wherein AB1 is attached to a masking group (MM1) such that the addition of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, including a second antibody or antigen-binding fragment (AB2) that binds CD166, where AB2 is attached to a masking group (MM2) such that attachment of MM2 reduces the ability AB2 bind CD166. In some embodiments, the anti-CD3ε scFv comprises a first antibody or antigen-binding fragment (AB1) thereof that binds CD3ε, where AB1 is attached to a masking group (MM1) such that attachment of MM1 reduces the ability of AB1 to bind CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2) that binds CD166, wherein AB2 is attached to a masking group (MM2) such that the addition of MM2 reduces the ability of AB2 to bind CD166.

In some embodiments, the multi-antigen-targeted antibodies and/or multi-antigen-targeted, activated antibodies comprise at least a first antibody or antigen-binding fragment thereof that binds a first target and/or a first epitope and a second antibody or antigen-binding fragment thereof that binds a second target and /or the second epitope. In some embodiments, multi-antigen-targeted antibodies and/or multi-antigen-targeted activated antibodies bind two or more different targets. In some embodiments, multi-antigen-targeted antibodies and/or multi-antigen-targeted activated antibodies bind two or more different epitopes on the same target. In some embodiments, multi-antigen-targeted antibodies and/or multi-antigen-targeted, activated antibodies bind a combination of two or more different targets and two or more different epitopes on the same target.

In some embodiments, an IgG-containing multispecifically activated antibody has masked IgG variable domains. In some embodiments, the scFv-containing multispecific activation antibody has masked scFv domains. In some embodiments, the multispecific activation antibody has both IgG variable domains and scFv domains, wherein at least one of the IgG variable domains is attached to a masking group. In some embodiments, the multispecific activation antibody has both IgG variable domains and scFv domains, wherein at least one of the scFv domains is attached to a masking group. In some embodiments, the multispecific activated antibody has both IgG variable domains and scFv domains, wherein at least one of the IgG variable domains is attached to a masking group and at least one of the scFv domains is attached to a masking group. In some embodiments, the multispecific activation antibody has both IgG variable domains and scFv domains, where each of the IgG variable domains and scFv domains is attached to its own masking group. In some embodiments, a single antibody domain from a multispecific activated antibody has specificity for a target antigen.

- 92 041958 and another domain of the antibody has specificity for the surface antigen of the T cell. In some embodiments, one antibody domain of a multispecific activated antibody has specificity for a target antigen and another antibody domain has specificity for a different target antigen. In some embodiments, one antibody domain of a multispecific activated antibody has specificity for a target antigen epitope and another antibody domain has specificity for a different target antigen epitope.

In a multispecific activated antibody, scFv can be fused to the carboxy-terminus of the heavy chain of an activated IgG antibody, to the carboxy-terminus of the light chain of an activated IgG antibody, or to the carboxy-terminus of both the heavy and light chains of an activated IgG antibody. In a multispecific activated antibody, the scFv can be fused to the amino terminus of the heavy chain of an activated IgG antibody, to the amino terminus of the light chain of an activated IgG antibody, or to the amino ends of both the heavy and light chains of an activated IgG antibody. In a multispecific activated antibody, the scFv can be fused to any combination of one or more carboxy-termini and one or more amino-termini of an IgG activated antibody. In some embodiments, a masking group (MM) associated with a cleavable group (CM) is attached to and masks an IgG antigen-binding domain. In some embodiments, a masking group (MM) associated with a cleavable group (CM) is attached to and masks the antigen-binding domain of at least one scFv. In some embodiments, a masking group (MM) associated with a cleavable group (CM) is attached to and masks the antigen-binding domain of an IgG, and a masking group (MM) associated with a cleavable group (CM) is attached to the antigen-binding domain of at least one scFv and masks it.

This description relates to examples of multispecific activated antibody structures, which include, but are not limited to, the following:

(VL-CL)2: (VH-CH1-CH2-CH3-L4-VH*-L3-VL*-L2-CM-L1-MM)2;

(VL-Cl) ₂ : (vH-CH1-CH2-CH3-L4-VL*-L3-VH*-L2-CM-L1-Mm) ₂ ;

(mM-L1-CM-L2-VL-CL) ₂ : (VH-CH1-CH2-CH3-L4-VH*-L3-VL*)2;

(mM-L1-CM-L2-VL-Cl) ₂ : (vH-CH1-CH2-CH3-L4-VL*-L3-VH*) ₂ ;

(VL-CL) ₂ : (MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2;

(VL-Cl) ₂ : (mM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3) ₂ ;

(mM-L1-CM-L2-VL-CL) ₂ : (VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2;

(mM-L1-CM-L2-VL-Cl) ₂ : (vH*-L3-VL*-L4-VH-CH1-CH2-CH3) ₂ ;

(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)2: (VH-CH1-CH2-CH3)2;

(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-Mm) ₂ : (vH-CH1-CH2-CH3) ₂ ;

(mM-L1-CM-L2-VL*-L3-VH*-L4-VL-Cl) ₂ : (VH-CH1-CH2-CH3)2;

(MM-L1-CM-L2-VH*-L3-VL*-L4-VL-CL) ₂ : (vH-CH1-CH2-CH3) ₂ ;

(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-Mm) ₂ : (mM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2- CH3) ₂ ;

(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-Mm) ₂ : (mM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2- CH3) ₂ ;

(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-Mm) ₂ : (mM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2- CH3) ₂ ;

(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)2 : (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2- CH3)2;

(VL-CL-L4-VH*-L3-VL*) ₂ : (MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2;

(VL-CL-L4-VH*-L3-VL*) ₂ : (mM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3) ₂ ;

(VL-CL-L4-VL*-L3-VH*) ₂ : (mM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3) ₂ ;

(VL-CL-L4-VL*-L3-VH*) ₂ : (mM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3) ₂ ;

(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)2: (VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2;

(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM) ₂ : (vH*-L3-VL*-L4-VH-CH1-CH2-CH3) ₂ ;

(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM) ₂ : (VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; or (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM) ₂ : (vH*-L3-VL*-L4-VH-CH1-CH2-CH3) ₂ , where VL and VH are light and heavy chain variable domains for the first specificity contained in IgG;

VL* and VH* are second specificity variable domains contained in scFv;

L1 is a linker peptide connecting a masking group (MM) and a cleavable group (CM);

L2 is a linker peptide connecting a cleavable group (CM) and an antibody;

L3 is a linker peptide that connects scFv variable domains;

L4 is a linker peptide connecting an antibody of the first specificity with an antibody of the second specificity;

CL is a light chain constant domain; And

CH1, CH2, CH3 are heavy chain constant domains.

The first and second specificities can target any antigen or epitope.

In some embodiments of a T-cell-attracting multispecific activated antibody, one antigen is CD166 and the other antigen is typically

- 93 041958 is a stimulatory (also referred to herein as an activating) or inhibitory receptor present on the surface of a T cell, natural killer (NK) cell, myeloid mononuclear cell, macrophage, and/or other immunoeffector cell, such as, but not limited to , B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137 (also referred to as TNFRSF9), CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD -1, TIGIT, TIM3 or VISTA. An antibody domain conferring specificity for a T cell surface antigen can also be replaced with a ligand or ligand domain binding a T cell receptor, an NK cell receptor, a macrophage receptor, and/or another immunoeffector cell receptor.

In some embodiments, the targeting antibody is an anti-CD166 antibody described herein. In some embodiments, the targeting antibody may be in the form of an activated antibody. In some embodiments, scFvs (multiple scFvs) may be in pro-scFv form (see, for example, WO 2009/025846, WO 2010/081173).

In some embodiments, the scFv is specific for CD3ε binding and contains or is derived from an antibody or fragment thereof that binds CD3e, such as CH2527, FN18, H2C, OKT3, 2C11, UCHT1, or V9. In some embodiments, the scFv is specific for binding CTLA-4 (also referred to herein as CTLA and CTLA4).

In some embodiments, the anti-CTLA-4 scFv contains the amino acid sequence

GGGSGGGGSGSGGGSGGGGSGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAW

YQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFG GGTKVEIKRSGGSTITSYNVYYTKLSSSGTQVQLVQTGGWQPGRSLRLSCAASGTFSSYAM SWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYNODLSS1 NSLYWYFAS1

In some embodiments, the anti-CTLA-4 scFv contains an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 117.

In some embodiments, the anti-CD3s scFv comprises the amino acid sequence

GGGSGGGGSGSGGGSGGGGSGGGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHW

VKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYY DDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVS YMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSGTSLTISGMEAQEDAA NOTYYCQQWSSNP8

In some embodiments, the anti-CD3s scFv contains an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 118.

In some embodiments, the scFv is specific for binding one or more T cells, one or more NK cells, and/or one or more macrophages. In some embodiments, the scFv is specific to bind to a target selected from the group consisting of B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM , ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3 or VISTA.

In some embodiments, the implementation of the multispecific activated antibody also contains an agent conjugated to AB. In some embodiments, the agent is a drug. In some embodiments, the agent is an antineoplastic agent. In some embodiments, the agent is a toxin or a fragment thereof. In some embodiments, the agent is conjugated to a multispecifically activated antibody via a linker. In some embodiments, the agent is AB-conjugated via a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalating agent, or other DNA damaging agent. In some embodiments, the linker is a cleavable linker. In some embodiments, the implementation of the tool is a tool selected from the group listed in table. 5. In some embodiments, the agent is dolastatin. In some embodiments, the agent is auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some

- 94 041958 embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is pyrrolobenzodiazepine. In some embodiments, the agent is a dimer of pyrrolobenzodiazepine.

In some embodiments, the implementation of the multispecific activated antibody also contains a detectable group. In some embodiments, the detectable group is a diagnostic tool.

In some embodiments, the implementation of the multispecifically activated antibody naturally contains one or more disulfide bonds. In some embodiments, a multispecific activated antibody can be designed to include one or more disulfide bonds.

The description also refers to an isolated nucleic acid molecule encoding the multispecifically activated antibody described herein, as well as vectors containing these isolated nucleic acid sequences. The description relates to methods for producing a multispecifically activated antibody by culturing a cell under conditions that allow expression of the activated antibody, wherein the cell contains such a nucleic acid molecule. In some embodiments, the implementation of the cell contains such a vector.

The description also relates to a method of manufacturing multispecifically activated antibodies according to this description by (a) culturing a cell containing a nucleic acid construct encoding a multispecifically activated antibody under conditions that allow expression of the multispecifically activated antibody; and (b) isolating the multispecific activated antibody.

Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM described herein.

The description also relates to multispecifically activated antibodies and/or compositions of a multispecifically activated antibody containing at least a first antibody or antigen-binding fragment (AB1) that specifically binds a first target or a first epitope, and a second antibody or antigen-binding fragment (AB2) that binds a second target or a second epitope, wherein at least AB1 is associated or otherwise connected to a masking group (MM1) such that the addition of MM1 reduces the ability of AB1 to bind to its target. In some embodiments, MM1 is fused to AB1 via a cleavable group (CM1) sequence containing a substrate for a protease, e.g., a protease, co-localized with an AB1 target at a treatment or diagnostic site in a subject. The multispecific activated antibodies provided herein are circulating stable, activated at sites targeted for therapy and/or diagnosis, but not in normal, i. healthy, tissue and when activated have binding to the AB1 target at least comparable to the corresponding, unmodified multispecific antibody. Suitable AB, MM and/or CM include any of the AB, MM and/or CM described herein.

The description also relates to compositions and methods, including a multispecific activated antibody, containing at least a first antibody or antibody fragment (AB1) that specifically binds the target, and a second antibody or antibody fragment (AB2), where at least the first AB in the multispecific the activated antibody is attached to a masking group (MM1) that reduces the ability of AB1 to bind its target. In some embodiments, each AV is attached to an MM that reduces the ability of its corresponding AV to bind to each target. For example, in embodiments of a bispecific activated antibody, AB 1 is attached to a first masking group (MM1) that reduces the ability of AB1 to bind to its target, and AB2 is attached to a second masking group (MM2) that reduces the ability of AB2 to bind to its target. In some embodiments, the multispecifically activated antibody comprises more than two AB regions; in such embodiments, AB1 is attached to a first masking group (MM1) that reduces the ability of AB1 to bind its target, AB2 is attached to a second masking group (MM2) that reduces the ability of AB2 to bind to its target, AB3 is attached to a third masking group (MM3) that reduces the ability of AB3 to bind its target, and so on for each AB in the multispecific activated antibody. Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM described herein.

In some embodiments, the implementation of the multispecific activated antibody further contains at least one cleavable group (CM), which is a substrate for the protease, where the CM binds MM to AB. For example, in some embodiments, the multispecific activated antibody comprises at least a first antibody or antibody fragment (AB1) that specifically binds to a target and a second antibody or antibody fragment (AB2) where at least

- 95 041958 the first AB in the multispecific activated antibody is attached via a first cleavable group (CM1) to a masking group (MM1) that reduces the ability of AB1 to bind to its target. In some embodiments of the bispecific activated antibody, AB1 is CM1 attached to MM1 and AB2 is fused via a second cleavable group (CM2) to a second masking group (MM2) that reduces the ability of AB2 to bind to its target. In some embodiments, the multispecifically activated antibody comprises more than two AB regions; in some of these embodiments, AB1 is attached via CM1 to MM1, AB2 is attached via CM2 to MM2, and AB3 is attached via a third cleavable group (CM3) to a third masking group (MM3) reducing the ability of AB3 to bind to its target, and so on for each AB in a multispecific activated antibody. Suitable AB, MM and/or CM include any of the AB, MM and/or CM described herein.

Activated antibodies having non-binding steric groups, or binding partners for non-binding steric groups.

The description also applies to activated antibodies containing non-binding steric groups (NB) or binding partners (BP) for non-binding steric groups, where the BP recruits or otherwise attracts the NB to the activated antibody. The activated antibodies provided herein include, for example, an activated antibody containing a non-binding steric group (NB), a cleavable linker (CL), and an antibody or antibody fragment (AB) that binds a target;

an activated antibody containing a binding partner for a non-binding steric group (BP), CL and AB; and an activated antibody containing BP to which NB, CL and AB are recruited to bind the target.

Activated antibodies in which NB is covalently attached to CL and AB of an activated antibody, or associated through interaction with a BP covalently attached to CL and AB of an activated antibody, are, as mentioned herein, containing NB activated antibodies. By activated or switchable is meant that the activated antibody has a first level of target binding when the activated antibody is in an inhibited, masked or uncleaved state (i.e., the first conformation), and a second level of target binding when the activated antibody is in an uninhibited state, unmasked and/or cleaved state (ie second conformation, ie activated antibody), where the second level of target binding is greater than the first level of target binding. Activated antibody compositions may have increased bioavailability and more favorable biodistribution compared to conventional antibody-based drugs.

In some embodiments, activated antibodies provide reduced toxicity and/or adverse side effects that may otherwise result from binding at non-treatable and/or non-diagnostic sites if the AV is not masked or its binding to such site is otherwise way is not inhibited.

Activated anti-CD166 antibodies containing a non-binding steric group (NB) can be prepared using the methods outlined in PCT Publication No. WO 2013/192546, the contents of which are incorporated herein by reference.

The use of antibodies, conjugated antibodies, activated antibodies and conjugated activated antibodies.

It will be appreciated that when therapeutic molecules are administered as described, they are administered with suitable carriers, excipients, and other agents that are included in formulations to provide improved transfer, delivery, tolerability, and the like. A variety of suitable formulations can be found in the handbook known to all those skilled in the art of pharmaceutical chemistry: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), in particular section 87 from Blaug, Seymour, in this document. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorbent pastes, oil-in-water emulsions and water-in-oil emulsions, Carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing Carbowax. Any of the above mixtures may be suitable for treatment and therapy in accordance with the present description, provided that the active ingredient in the composition is not inactivated during the preparation of the composition and the composition is physiologically compatible and tolerated by this route of administration. See also Baldrick P., Pharmaceutical excipient development: the need for preclinical guidance, Regul. Toxicol Pharmacol., 32(2):210-8 (2000); Wang W., Lyophilization and development of solid protein Pharmaceuticals, Int. J. Pharm., 203(1-2):1-60 (2000); Charman W.N., Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts, J. Pharm. Sci., 89(8):967-78 (2000), Powell et al., Compendium of excipients for parenteral formulations, PDA, J. Pharm. sci. Technol., 52:238-311 (1998) and references cited therein for additional information regarding formulations, excipients and carriers well known to those skilled in the art.

- 96 041958 pharmaceutical chemistry.

Therapeutic formulations as described herein, comprising an anti-CD166 antibody and/or an anti-CD166 activated antibody, such as, but not limited to, an antibody, a conjugated antibody, an activated antibody, and/or a conjugated activated antibody, are used to prevent, treat, or otherwise alleviate a disease or disorder. associated with altered expression and/or activity of the target. For example, therapeutic compositions as described herein, comprising an antibody, a conjugated antibody, an activated antibody, and/or a conjugated activated antibody, are used to treat or otherwise alleviate a cancer or other neoplastic condition, inflammation, inflammatory disorder, and/or an autoimmune disease. In some embodiments, the cancer is a solid tumor or hematologic cancer where the target is expressed. In some embodiments, the cancer is a solid tumor where the target is expressed. In some embodiments, the cancer is a hematologic cancer where the target is expressed. In some embodiments, the target is expressed on the parenchyma (eg, in a cancer, in a part of an organ or tissue that often performs the function(s) of an organ or tissue). In some embodiments, the target is expressed on a cell, tissue, or organ. In some embodiments, the target is expressed on the stroma (ie, the connective support scaffold of a cell, tissue, or organ). In some embodiments, the target is expressed on an osteoblast. In some embodiments, the target is expressed on the endothelium (in the vasculature). In some embodiments, the target is expressed on cancer stem cells. In some embodiments, the agent to which the antibody and/or the activated antibody is conjugated is a microtubule inhibitor. In some embodiments, the agent to which the antibody and/or the activated antibody is conjugated is a nucleic acid damaging agent.

The effectiveness of prevention, alleviation or treatment is determined in accordance with any known method for diagnosing or treating a disease or disorder associated with the expression and/or activity of the target, such as altered expression and/or activity of the target. Prolonging a subject's survival or otherwise delaying the progression of a disease or disorder associated with target expression and/or activity, e.g., altered target expression and/or activity, in a subject indicates that the antibody, conjugated antibody, activated antibody, and/or conjugated activated antibody provides clinical benefit.

The antibody, conjugated antibody, activated antibody, and/or conjugated activated antibody may be administered in the form of pharmaceutical compositions. The principles and considerations involved in making such compositions, as well as guidance on the selection of ingredients, are presented, for example, in Remington: The Science And Practice Of Pharmacy, 19th ed. (Alfonso R. Gennaro et al., editors) Mack Pub. Co., Easton, Pa., 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, vol. 4), 1991, M. Dekker, New York.

In some embodiments where antibody fragments are used, the smallest fragment that specifically binds to the binding domain of the target protein is selected. For example, based on the sequences of the variable region of an antibody, peptide molecules can be designed that retain the ability to bind a target protein sequence. Such peptides can be synthesized chemically and/or obtained by recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci., USA, 90:7889-7893 (1993)). The formulation may also contain more than one active compound as needed for the particular indication being treated, eg, in some embodiments, compounds with complementary activities that do not adversely affect each other. In some embodiments, or additionally, the composition may contain an agent that improves its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

The active ingredients can also be encapsulated in microcapsules, for example, by coacervation processes or by intersurface polymerization, for example hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions.

Formulations for use for in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes.

Sustained release formulations can be obtained. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic

- 97 041958 antibody-containing polymers, where the matrices are in the form of molded articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g. poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides US Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-O-(-)-3-hydroxybutyric acid. While polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow molecules to be released for over 100 days, specific hydrogels release proteins for shorter periods of time.

In some embodiments, the antibody, conjugated antibody, activated antibody, and/or conjugated activated antibody contains a detectable label. An intact antibody or fragment thereof is used (eg Fab, scFv, or F(ab) ₂ ). The term labeled with respect to a probe or antibody is intended to include direct labeling of a probe or antibody by attaching (i.e. physically binding) a detectable substance to the probe or antibody, as well as indirectly labeling a probe or antibody by reactivity with another reagent. , which is labeled directly. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end labeling of a DNA probe using biotin so that it can be detected using a fluorescently labeled streptavidin. The term biological sample is intended to include tissues, cells, and body fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject. Thus, the use of the term biological sample includes blood and a fraction or component of blood, including blood serum, blood plasma or lymph. Those. The detection method of this disclosure can be used to detect an mRNA, protein or genomic DNA analyte in a biological sample in vitro as well as in vivo. For example, in vitro methods for detecting an mRNA analyte include Northern hybridization and in situ hybridization methods. In vitro methods for detecting a protein analyte include enzyme-linked immunosorbent assays (ELISA), Western blotting, immunoprecipitations, immunochemical staining, and immunofluorescence. In vitro methods for detecting a genomic DNA analyte include Southern hybridization methods. Methods for conducting immunoassays are described, for example, in ELISA: Theory and Practice: Methods in Molecular Biology, vol. 42, JR Crowther (ed.), Human Press, Totowa, NJ, 1995; Immunoassay, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and Practice and Theory of Enzyme Immunoassays, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo methods for detecting a protein analyte include administering a labeled antibody against the protein analyte to the subject. For example, an antibody can be labeled with a radioactive marker whose presence and location can be detected by standard imaging techniques.

Antibodies, conjugated antibodies, activated antibodies, and/or conjugated activated antibodies as described herein may also be used in a variety of diagnostic and prophylactic formulations. In one embodiment, the antibody, conjugated antibody, activated antibody, and/or conjugated activated antibody is administered to patients at risk for developing one or more of the aforementioned disorders. The predisposition of a patient or organ to one or more of the aforementioned disorders can be determined using genotypic, serological, or biochemical markers.

In some embodiments as described herein, the antibody, conjugated antibody, activated antibody, and/or conjugated activated antibody is administered to human individuals diagnosed with a clinical indicator associated with one or more of the aforementioned disorders. After diagnosis, the antibody, conjugated antibody, activated antibody, and/or conjugated activated antibody is administered to mitigate or reverse the effects of the clinical indicator.

An antibody, a conjugated antibody, an activated antibody, and/or a conjugated activated antibody as described herein may also be used to detect a target in patient samples and, accordingly, may be used as diagnostics. For example, antibodies and/or activated antibodies and conjugated variants thereof as described herein are used in in vitro assays, such as ELISA, to detect target levels in a patient sample.

In one embodiment, an antibody, a conjugated antibody, an activated antibody, and/or a conjugated activated antibody as described herein is immobilized on a solid support (eg, well(s) of a microtiter plate). The immobilized antibody, conjugated antibody, activated antibody, and/or conjugated activated antibody serves as a binding antibody for any target that may be present in the test sample. Before bringing the immobilized antibody and/or the activated antibody and/or their conjugated variants into contact with a patient sample, the solid support is washed and treated with a blocking agent such as milk protein or albumin to prevent non-specific adsorption analyte.

The wells are then treated with a test sample suspected to contain an antigen or a solution containing a standard amount of antigen. Such a sample is, for example, a serum sample from a subject suspected of having levels of circulating antigen considered diagnostic of pathology. After washing the test sample or standard, the solid support is treated with a secondary antibody labeled with a detectable label. The labeled secondary antibody serves as the detection antibody. The level of detectable label is measured and the concentration of the target antigen in the test sample is determined by comparison with a standard curve obtained for standard samples.

It should be understood that based on the results obtained using antibodies and activated antibodies according to this description, and their conjugated variants in an in vitro diagnostic assay, it is possible to determine the stage of the disease in a subject based on the expression levels of the target antigen. For a given disease, blood samples are taken from subjects diagnosed as being at various stages of disease progression and/or at various points in the therapeutic treatment of the disease. Using a sample population that provides statistically significant results for each stage of progression or therapy, a range of antigen concentrations is developed that can be considered representative of each stage.

The antibody, conjugated antibody, activated antibody, and/or conjugated activated antibody may also be used in diagnostic and/or imaging methods. In some embodiments, such methods are in vitro methods. In some embodiments, such methods are in vivo methods. In some embodiments, such methods are in situ methods. In some embodiments, such methods are ex vivo methods. For example, activated antibodies having enzymatically cleavable CMs can be used to detect the presence or absence of an enzyme capable of cleaving CMs. Such activated antibodies can be used in diagnostics, which can include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or in some embodiments, an environment with increased reducing potential, such as one that can allow for disulfide bond reduction) via measuring the accumulation of activated antibodies (ie, antibodies resulting from cleavage of an activated antibody) in a given cell or tissue of a given host organism. This accumulation of activated antibodies not only indicates that the tissue has enzymatic activity (or increased reducing potential, depending on the nature of the SM), but also that the tissue expresses a target to which the activated antibody binds.

For example, SM can be chosen to be a substrate for at least one protease found at a tumor site, at a viral or bacterial infection site at a biologically restricted site (e.g., in an abscess, in an organ, etc.), etc. . The AB may be an AB that binds a target antigen. Using the methods described herein, or when appropriate, methods known to one of skill in the art, a detectable label (e.g., a fluorescent label or a radioactive label or a radiotracer) can be conjugated to an AB or other region of an antibody and/or an activated antibody. . Suitable detectable labels are discussed in the context of the above screening methods, and additional specific examples are provided below. By using an AB specific for a protein or peptide in a disease state, together with at least one protease whose activity is increased in the diseased tissue of interest, the activated antibodies have an increased rate of binding to the diseased tissue compared to tissues where the CM-specific the enzyme is not present at a detectable level, or is present at a lower level than in diseased tissue, or is inactive (eg, in the form of a zymogen or in combination with an inhibitor). Because small proteins and peptides are rapidly cleared from the blood by the filtration system of the kidneys, and because the enzyme specific for SM is not present at detectable levels (or present at lower levels in non-diseased tissues, or present in an inactive conformation), the accumulation of activated antibodies in diseased tissue is increased compared to non-affected tissues.

In another example, activated antibodies can be used to detect the presence or absence of a disintegrating agent in a sample. For example, where the activated antibodies contain a CM that is sensitive to enzyme digestion, the activated antibodies can be used to detect (either qualitatively or quantitatively) the presence of the enzyme in the sample. In another example, where the activated antibodies contain a SM that is sensitive to cleavage by a reducing agent, the activated antibodies can be used to detect (either qualitatively or quantitatively) the presence of reducing conditions in a sample. To facilitate analysis in these methods, activated antibodies

- 99 041958 can be labeled with a detectable label, and can be associated with a substrate (eg a solid substrate such as glass or a bead). A detectable label can be placed on the part of the activated antibody that is not released after cleavage, for example, the detectable label can be a quenched fluorescent label or other label that is not detectable until cleavage occurs. The assay can be performed, for example, by contacting immobilized, detectably labeled, activated antibodies with a sample suspected of containing an enzyme and/or reducing agent for a time sufficient for cleavage to occur, then washing to remove excess sample and contaminants. impurities. The presence or absence of a cleavage agent (e.g., an enzyme or a reducing agent) in the sample is then assessed by a change in the detectable signal of activated antibodies prior to contact with the sample, such as the presence and/or an increase in a detectable signal due to cleavage of the activated antibody by the cleavage agent in the sample.

Such detection methods can be adapted to also detect the presence or absence of a target capable of binding AV-activated antibodies upon digestion. Thus, the assays can be adapted to assess the presence or absence of a dissolving agent and the presence or absence of a target of interest. The presence or absence of a cleavage agent can be detected by the presence and/or increase in detectable label from activated antibodies as described above, and the presence or absence of a target can be detected by detection of the target-AB complex, for example by using a detectably labeled anti-target antibody.

Activated antibodies can also be used for in situ imaging to confirm the activation of an activated antibody, for example by protease digestion and binding to a specific target. In situ imaging is a technique that allows localization of proteolytic activity and target in biological samples such as cell cultures or tissue sections. Using this method, both binding to a given target and proteolytic activity can be verified based on the presence of a detectable label (eg, a fluorescent label).

These methods can be used on any frozen cells or tissue obtained from a disease site (eg tumor tissue) or healthy tissue. These methods can also be used on fresh cell or tissue samples.

In these methods, the activated antibody is labeled with a detectable label. The detectable label can be a fluorescent dye (eg, fluorophore, fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (TRITC), Alexa Fluor® label), near infrared (NIR) dye (eg, Qdot® nanocrystals), colloidal metal , a hapten, a radioactive marker, biotin, and an enhancer such as streptavidin, or an enzyme (eg, horseradish peroxidase or alkaline phosphatase).

Detection of a label in a sample that has been incubated with a labeled, activated antibody indicates that the sample contains a target and contains a protease that is specific for the CM of the activated antibody. In some embodiments, the presence of a protease can be confirmed using a broad spectrum of protease inhibitors, such as those described herein, and/or using a protease-specific agent, e.g., an antibody such as A11, that is specific for the matriptase protease and inhibits proteolytic activity. matriptase; see, for example, International Publication Number WO 2010/129609, publ. Nov. 11, 2010 The same method using a broad spectrum of protease inhibitors such as those described herein and/or using a more selective inhibitory agent can be used to identify a protease specific for the CM of an activated antibody. In some embodiments, the presence of a target may be confirmed using a target-specific agent, such as another antibody, or a detectable label may compete with an unlabeled target. In some embodiments, an unlabeled, activated antibody can be used with detection by a labeled secondary antibody or a more sophisticated detection system.

Similar methods can also be used for in vivo imaging, where detection of a fluorescent signal in a subject, eg, a mammal, including a human, indicates that the disease site contains a target and contains a protease specific for the CM of the activated antibody.

These methods can also be used in kits and/or as reagents for detecting, identifying, or characterizing protease activity in a variety of cells, tissues, and organisms based on the protease-specific SM in an activated antibody.

The description relates to methods of using antibodies and/or activated antibodies in a variety of diagnostic and/or prophylactic indications. For example, the description relates to methods for detecting the presence or absence of a disintegrating agent and a target of interest in subjects.

- 100 041958 that or in the sample by (i) bringing the subject or sample into contact with an activated antibody, where the activated antibody contains a masking group (MM), a cleavable group (CM) cleavable by a cleaving agent, for example, a protease, an antigen-binding domain, or a fragment thereof (AB) specifically binding a target of interest, wherein the activated antibody in the uncleaved, non-activated state has the following structural arrangement from N-terminus to C-terminus: MM-CMAB or AB-CM-MM;

(a) where MM is a peptide that inhibits AB binding to a target, and where MM does not have the amino acid sequence of a natural AB binding partner and is not a modified form of a natural AB binding partner; and (b) where in the uncleaved, non-activated state, MM interferes with the specific binding of AB to the target and, in the cleaved, activated state, MM does not interfere with or compete with the specific binding of AB to the target; and (ii) measuring the level of activated activated antibody in the subject or sample, where the detectable level of activated activated antibody in the subject or sample indicates that the cleavage agent and target are present in the subject or sample, and where the undetectable level of activated activated antibody in the subject or in the sample indicates that the cleavage agent, the target, or both the cleavage agent and the target are absent and/or not present in sufficient quantity in the subject or in the sample.

In some embodiments, the activated antibody is an activated drug-conjugated antibody. In some embodiments, the activated antibody is not conjugated to the agent. In some embodiments, the implementation of the activated antibody contains a detectable label. In some embodiments, a detectable label is located on the AB. In some embodiments, measurement of the level of an activated antibody in a subject or sample is performed using a secondary reagent that specifically binds the activated antibody, where the reagent contains a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

The description also relates to methods for detecting the presence or absence of a cleaving agent in a subject or sample by (i) bringing the subject or sample into contact with an activated antibody in the presence of a target of interest, such as a target, where the activated antibody contains a masking group (MM), a cleavable group (CM), cleavable by a cleaving agent, such as a protease, and an antigen-binding domain or fragment (AB) specifically binding to a target of interest, where the activated antibody in an uncleaved, non-activated state has the following structural arrangement from the N-terminus to the C-terminus: MM -SM-AB or AB-SM-MM;

(a) where MM is a peptide that inhibits AB binding to a target, and where MM does not have the amino acid sequence of a natural AB binding partner and is not a modified form of a natural AB binding partner; and (b) where in the uncleaved, non-activated state, MM interferes with the specific binding of AB to the target and, in the cleaved, activated state, MM does not interfere with or compete with the specific binding of AB to the target; and (ii) measuring the level of activated activated antibody in the subject or in the sample, where the detectable level of activated activated antibody in the subject or sample indicates that the degrading agent is present in the subject or in the sample, and where the undetectable level of activated activated antibody in the subject or in the sample indicates that the disintegrating agent is absent and/or not present in sufficient quantity in the subject or in the sample. In some embodiments, the activated antibody is an activated drug-conjugated antibody.

In some embodiments, the activated antibody is not conjugated to the agent. In some embodiments, the implementation of the activated antibody contains a detectable label. In some embodiments, a detectable label is located on the AB. In some embodiments, measurement of the level of an activated antibody in a subject or sample is performed using a secondary reagent that specifically binds the activated antibody, where the reagent contains a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

The description also relates to kits for use in methods for detecting the presence or absence of a cleaving agent and a target in a subject or sample, where the kits contain at least one activated antibody containing a masking group (MM), a cleavable group (CM) cleavable by a cleaving agent , for example proteases, and an antigen-binding domain or fragment (AB) specifically binding to a target of interest, where the activated antibody in an uncleaved, non-activated state has the following structural

- 101 041958 arranged from N-terminus to C-terminus: MM-CM-AB or AB-CM-MM;

(a) where MM is a peptide that inhibits AB binding to a target, and where MM does not have the amino acid sequence of a natural AB binding partner and is not a modified form of a natural AB binding partner; and (b) where in the uncleaved, non-activated state, MM interferes with the specific binding of AB to the target and, in the cleaved, activated state, MM does not interfere with or compete with the specific binding of AB to the target; and (ii) measuring the level of activated activated antibody in the subject or in the sample, where the detectable level of activated activated antibody in the subject or sample indicates that the degrading agent is present in the subject or in the sample, and where the undetectable level of activated activated antibody in the subject or in the sample indicates that the disintegrating agent is absent and/or not present in sufficient quantity in the subject or in the sample. In some embodiments, the activated antibody is an activated drug-conjugated antibody.

In some embodiments, the activated antibody is not conjugated to the agent. In some embodiments, the implementation of the activated antibody contains a detectable label. In some embodiments, a detectable label is located on the AB. In some embodiments, measurement of the level of an activated antibody in a subject or sample is performed using a secondary reagent that specifically binds the activated antibody, where the reagent contains a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

The description also relates to methods for detecting the presence or absence of a cleaving agent in a subject or sample by (i) bringing the subject or sample into contact with an activated antibody, where the activated antibody contains a masking group (MM), a cleavable group (CM) cleavable by the cleaving agent , for example, proteases, an antigen-binding domain (AB) that specifically binds the target, and a detectable label, where the activated antibody in an uncleaved, non-activated state has the following structural arrangement from the N-terminus to the C-terminus: MMCM-AB or AB-CM-MM ;

(a) where MM is a peptide that inhibits AB binding to a target, and where MM does not have the amino acid sequence of a natural AB binding partner and is not a modified form of a natural AB binding partner;

(b) where in the uncleaved, non-activated state, MM interferes with the specific binding of AB to the target and, in the cleaved, activated state, MM does not interfere with or compete with the specific binding of AB to the target; and (c) where the detectable label is located on the part of the activated antibody that is released after cleavage of the SM; and (ii) measuring the level of a detectable label in the subject or sample, where the detectable level of detectable label in the subject or sample indicates that the cleaving agent is absent and/or not present in sufficient quantity in the subject or sample, and where not a detectable level of detectable label in the subject or sample indicates that the disintegrating agent is present in the subject or sample.

In some embodiments, the activated antibody is an activated drug-conjugated antibody. In some embodiments, the activated antibody is not conjugated to the agent. In some embodiments, the implementation of the activated antibody contains a detectable label. In some embodiments, a detectable label is located on the AB. In some embodiments, measurement of the level of an activated antibody in a subject or sample is performed using a secondary reagent that specifically binds the activated antibody, where the reagent contains a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

The description also relates to kits for use in methods for detecting the presence or absence of a cleavage agent and a target in a subject or sample, where the kits contain at least one activated antibody and/or a conjugated activated antibody (e.g., an activated drug-conjugated antibody), described herein, for use in contact with a subject or biological sample, and means for detecting the level of activated activated antibody and/or conjugated activated antibody in the subject or biological sample, where the detectable level of activated activated antibody in the subject or biological sample shows that the cleavage agent and the target are present in the subject or in the biological sample, and where an undetectable level of activated activated antibody in the subject or in the biological sample indicates that the cleavage agent, the target, or as the cleavage agent, and the target is absent and/or not present in sufficient quantity in the subject or biological sample such that target binding and/or protease cleavage of the activated antibody cannot be detected in the subject or biological sample.

The description also relates to methods for detecting the presence or absence of a disintegrating agent in a subject or sample by (i) bringing the subject or biological sample into contact with an activated antibody in the presence of a target; and (ii) measuring the level of activated activated antibody in the subject or biological sample, where the detectable level of activated activated antibody in the subject or biological sample indicates that the degrading agent is present in the subject or biological sample, and where the undetectable level of activated activated antibody antibody in the subject or biological sample indicates that the cleavage agent is absent and/or not present in sufficient quantity in the subject or biological sample at a detectable level such that protease cleavage of the activated antibody cannot be detected in the subject or biological sample.

Such an activated antibody contains a masking group (MM), a cleavable group (CM) cleavable by a cleaving agent, such as a protease, and an antigen-binding domain or fragment thereof (AB) that specifically binds to the target, where the activated antibody in an uncleaved (i.e., unactivated) state has the following structural arrangement from the N-terminus to the C-terminus: MM-CM-AB or AB-CM-MM;

(a) where MM is a peptide that inhibits AV binding to a target, and where MM does not have the amino acid sequence of a natural AB binding partner; and (b) where the MW of the activated antibody in the uncleaved state interferes with the specific binding of the AB to the target, and where the MW of the activated antibody in the cleaved (ie, activated) state does not interfere with or compete with the specific binding of the AB to the target.

In some embodiments, the activated antibody is an activated drug-conjugated antibody. In some embodiments, the activated antibody is not conjugated to the agent. In some embodiments, a detectable label is attached to a masking group. In some embodiments, a detectable label is attached to the cleavable group N-terminally from the protease cleavage site. In some embodiments, a single AB antigen-binding site is masked. In some embodiments, where the antibody of this description has at least two antigen-binding sites, where at least one antigen-binding site is masked and at least one antigen-binding site is not masked. In some embodiments, all antigen-binding sites are masked. In some embodiments, the measurement step includes the use of a secondary reagent containing a detectable label.

The description also relates to kits for use in methods for detecting the presence or absence of a cleavage agent and a target in a subject or sample, where the kits contain at least one activated antibody and/or conjugated activated antibody described herein, for use to bring into contact subject or biological sample with an activated antibody in the presence of a target, and measuring the level of activated activated antibody in the subject or in the biological sample, where the detectable level of activated activated antibody in the subject or in the biological sample indicates that the degrading agent is present in the subject or in the biological sample, and where a non-detectable level of activated activated antibody in the subject or in the biological sample indicates that the cleavage agent is not present and/or is not present in sufficient quantity in the subject or in the biological sample at a detectable level e, so that protease cleavage of the activated antibody cannot be detected in the subject or in the biological sample. Such an activated antibody contains a masking group (MM), a cleavable group (CM) cleavable by a cleaving agent, such as a protease, and an antigen-binding domain or fragment thereof (AB) that specifically binds the target, where the activated antibody in an uncleaved (i.e., unactivated ) state has the following structural arrangement from the N-terminus to the C-terminus: MM-CM-AB or AB-CM-MM;

(a) where MM is a peptide that inhibits AV binding to a target, and where MM does not have the amino acid sequence of a natural AB binding partner; and (b) where the MW of the activated antibody in the uncleaved state interferes with the specific binding of the AB to the target, and where the MW of the activated antibody in the cleaved (ie, activated) state does not interfere with or compete with the specific binding of the AB to the target.

In some embodiments, the activated antibody is an activated drug-conjugated antibody. In some embodiments, the activated antibody is not conjugated to the agent. In some embodiments, a detectable label is attached to a masking group. In some embodiments, a detectable label is attached to a cleavable group at the N-terminus of the protease cleavage site. In some embodiments, a single AB antigen-binding site is masked. In some embodiments, where an antibody of this description has at least two antigen-binding sites, at least one antigen-binding site is masked and at least one antigen-binding site is not masked. In some embodiments, all antigen-binding sites are masked. In some embodiments, the measurement step includes the use of a secondary reagent containing a detectable label.

The description also relates to kits for use in methods for detecting the presence or absence of a disintegrating agent in a subject or in a sample, where the kits contain at least one activated antibody and/or conjugated activated antibody described herein, for use in bringing into contact with the subject or biological sample, and means for detecting the level of activated activated antibody and/or conjugated activated antibody in the subject or in the biological sample, where the activated antibody contains a detectable label located on the part of the activated antibody that is released after SM cleavage, where the detectable level of activated of an activated antibody in a subject or biological sample indicates that a cleaving agent is absent and/or not present in sufficient quantity in the subject or biological sample such that target binding and/or protease cleavage of the activated antibody is undetectable in the subject or in the biological sample, and where the undetectable level of activated activated antibody in the subject or in the biological sample indicates that the degrading agent is present in the subject or in the biological sample at a detectable level.

The description relates to methods for detecting the presence or absence of a cleavage agent and a target in a subject or sample by (i) bringing the subject or biological sample with an activated antibody, where the activated antibody contains a detectable label located on a portion of the activated antibody that is released after CM cleavage, and (ii) measuring the level of activated activated antibody in the subject or biological sample, wherein the detectable level of activated activated antibody in the subject or biological sample indicates that the cleavage agent, the target, or both the cleavage agent and the target, are absent and/or not are present in a sufficient amount in the subject or in the biological sample such that target binding and/or protease cleavage of the activated antibody cannot be detected in the subject or in the biological sample, and where a decrease in the detectable level of activated activated antibody in subject or biological sample indicates that the disintegrating agent and target are present in the subject or biological sample.

The reduction in detectable label level is, for example, a decrease of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50 %, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%.

Such an activated antibody contains a masking group (MM), a cleavable group (CM) cleavable by a cleaving agent, and an antigen-binding domain or fragment thereof (AB) that specifically binds the target, where the activated antibody in the uncleaved (i.e., unactivated) state has the following structural arrangement from the N-terminus to the C-terminus: MM-CMAB or AB-CM-MM;

(a) where MM is a peptide that inhibits AV binding to a target, and where MM does not have the amino acid sequence of a natural AB binding partner; and (b) where the MW of the activated antibody in the uncleaved state interferes with the specific binding of the AB to the target, and where the MW of the activated antibody in the cleaved (ie, activated) state does not interfere with or compete with the specific binding of the AB to the target.

In some embodiments, the activated antibody is an activated drug-conjugated antibody. In some embodiments, the activated antibody is not conjugated to the agent. In some embodiments, the implementation of the activated antibody contains a detectable label. In some embodiments, a detectable label is located on the AB. In some embodiments, measurement of the level of an activated antibody in a subject or sample is performed using a secondary reagent that specifically binds the activated antibody, where the reagent contains a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

The description also relates to kits for use in methods for detecting the presence or absence of a disintegrating agent and a target in a subject or sample, where the kits contain at least

- 104 041958 at least one activated antibody and/or conjugated activated antibody described herein for use in bringing into contact with a subject or biological sample, and means for detecting the level of activated activated antibody and/or conjugated activated antibody in a subject or in a biological a sample, where a detectable level of activated activated antibody in the subject or in the biological sample indicates that the cleavage agent, the target, or both the cleavage agent and the target, is absent and/or not present in sufficient quantity in the subject or in the biological sample such that binding target and/or protease cleavage of an activated antibody cannot be detected in the subject or biological sample, and where a decrease in the detectable level of activated activated antibody in the subject or biological sample indicates that the cleavage agent and target are present in the subject or in a biological sample. The reduction in detectable label level is, for example, a decrease of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50 %, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%.

The description also relates to methods for detecting the presence or absence of a disintegrating agent in a subject or sample by (i) bringing the subject or biological sample into contact with an activated antibody, where the activated antibody contains a detectable label located on a portion of the activated antibody that is released after cleavage of the SM ; and (ii) measuring the level of a detectable label in the subject or biological sample, wherein the detectable level of detectable label in the subject or biological sample indicates that the cleavage agent is not present and/or is not present in sufficient quantity in the subject or biological sample at at a detectable level such that protease-activated antibody cleavage is undetectable in the subject or biological sample, and wherein a decrease in the detectable level of the detectable label in the subject or biological sample indicates that the cleavage agent is present in the subject or biological sample.

The reduction in detectable label level is, for example, a decrease of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50 %, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%.

Such an activated antibody contains a masking group (MM), a cleavable group (CM) cleavable by a cleaving agent, and an antigen-binding domain or fragment (AB) specifically binding to the target, where the activated antibody in the uncleaved (i.e., unactivated) state has the following structural arrangement from N-terminus to C-terminus: MMSM-AB or AB-CM-MM;

(a) where MM is a peptide that inhibits AV binding to a target, and where MM does not have the amino acid sequence of a natural AB binding partner; and (b) where the MW of the activated antibody in the uncleaved state interferes with the specific binding of the AB to the target, and where the MW of the activated antibody in the cleaved (ie, activated) state does not interfere with or compete with the specific binding of the AB to the target.

In some embodiments, the activated antibody is an activated drug-conjugated antibody. In some embodiments, the activated antibody is not conjugated to the agent. In some embodiments, the implementation of the activated antibody contains a detectable label. In some embodiments, a detectable label is located on the AB. In some embodiments, the measurement of the level of an activated antibody in a subject or sample is performed using a secondary reagent that specifically binds the activated antibody, where the reagent contains a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

The description also relates to kits for use in methods for detecting the presence or absence of a disintegrating agent of interest in a subject or sample, where the kits contain at least one activated antibody and/or conjugated activated antibody described herein, for use in bringing into contact with a subject or biological sample, and means for detecting the level of activated activated antibody and/or conjugated activated antibody in the subject or in the biological sample, where the activated antibody contains a detectable label located on the part of the activated antibody that is released after cleavage of the CM, where the detectable detectable label level

- 105 041958 in the subject or in the biological sample indicates that the cleavage agent, the target, or both the cleavage agent and the target are absent and/or not present in sufficient quantity in the subject or in the biological sample such that target binding and/or cleavage by the protease the activated antibody is undetectable in the subject or in the biological sample, and where a decrease in the detectable level of the detectable label in the subject or in the biological sample indicates that the cleavage agent and target are present in the subject or in the biological sample. The reduction in detectable label level is, for example, a decrease of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50 %, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%.

In some embodiments of these methods and kits, the activated antibody contains a detectable label. In some embodiments of these methods and kits, the detectable label includes an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments of these methods and kits, the imaging agent contains a radioactive isotope. In some embodiments of these methods and kits, the radioactive isotope is indium or technetium. In some embodiments of these methods and kits, the contrast agent contains iodine, gadolinium, or iron oxide. In some embodiments of these methods and kits, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments of these methods and kits, the fluorescent label comprises yellow fluorescent protein (YFP), blue fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein dimer-2 (RFP tdimer2) , HCRED or europium derivative. In some embodiments of these methods and kits, the luminescent label contains an N-methylacridinium derivative. In some embodiments of these methods, the label contains an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments of these methods and kits, the ligand-based label contains biotin, avidin, streptavidin, or one or more haptens.

In some embodiments of these methods and kits, the subject is a mammal. In some embodiments of these methods and kits, the subject is a human. In some embodiments, the subject is a non-human mammal, such as a non-human primate, companion animal (eg, cat, dog, horse), farm animal, working animal, or zoo animal. In some embodiments, the subject is a rodent.

In some embodiments of these methods, the method is an in vivo method. In some embodiments of these methods, the method is an in situ method. In some embodiments of these methods, the method is an ex vivo method. In some embodiments of these methods, the method is an in vitro method.

In some embodiments, in situ imaging and/or in vivo imaging can be used in methods for identifying patients to be treated. For example, in in situ imaging, activated antibodies are used to screen samples from patients to identify patients having the appropriate protease(s) and target(s) at the appropriate location, such as at a tumor site.

In some embodiments, in situ imaging is used to identify or otherwise refine a patient population suitable for treatment with an activated antibody as described herein. For example, patients who test positive for both the target (e.g., that target) and the substrate-cleaving protease in the cleavable group (CM) of the activated antibody being tested (e.g., with accumulation of the activated antibody at the disease site) are identified as suitable candidates for treatment. using such an activated antibody containing such a SM. Similarly, patients who test negative for either or both of the target (eg, this target) and the SM substrate-cleaving protease in an activated antibody tested using these methods can be identified as suitable candidates for another form of therapy. In some embodiments, such patients who test negative for the first activated antibody may be tested with other activated antibodies containing other SMs until a suitable activated antibody for treatment is identified (e.g., an activated antibody containing a SM that is cleaved by the patient at the disease site). ). In some embodiments, the patient is then administered a therapeutically effective amount of an activated antibody for which the patient has tested positive.

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In some embodiments, in vivo imaging is used to identify or otherwise refine a patient population suitable for treatment with an activated antibody as described herein. For example, patients who test positive for both the target (e.g., this target) and the substrate-cleaving protease in the cleavable group (CM) of the activated antibody being tested (e.g., with accumulation of the activated antibody at the disease site) are identified as suitable candidates for treatment with using such an activated antibody containing such a CM. Similarly, patients who test negative may be identified as suitable candidates for another form of therapy. In some embodiments, such patients who test negative for the first activated antibody may be tested with other activated antibodies containing other SMs until a suitable activated antibody for treatment is identified (e.g., an activated antibody containing a SM that is cleaved by the patient at the disease site). ). In some embodiments, the patient is then administered a therapeutically effective amount of an activated antibody for which the patient has tested positive.

In some embodiments of the methods and kits, the method or kit is used to identify or otherwise refine a population of patients suitable for treatment with an activated antibody as described herein. For example, patients who test positive for both a target (e.g., that target) and a protease that cleaves a substrate in the cleavable group (CM) of an activated antibody tested in these methods are identified as suitable candidates for treatment with that activated antibody containing such CM. Similarly, patients who test negative for both the target (eg, this target) and the substrate-cleaving protease in SM in an activated antibody tested using these methods can be identified as suitable candidates for another form of therapy. In some embodiments, such patients can be tested with other activated antibodies until a suitable activated antibody for treatment is identified (eg, an activated antibody containing CM cleaved by the patient at the disease site). In some embodiments, patients who test negative for any target (eg, that target) are identified as suitable candidates for treatment with such an activated antibody containing such a SM. In some embodiments, patients who test negative for any target (eg, that target) are identified as not being suitable candidates for treatment with such an activated antibody containing such a SM. In some embodiments, such patients can be tested with other activated antibodies until a suitable activated antibody for treatment is identified (eg, an activated antibody containing CM cleaved by the patient at the disease site). In some embodiments, the activated antibody is an activated drug-conjugated antibody. In some embodiments, the activated antibody is not conjugated to the agent. In some embodiments, the implementation of the activated antibody contains a detectable label. In some embodiments, a detectable label is located on the AB. In some embodiments, measurement of the level of an activated antibody in a subject or sample is performed using a secondary reagent that specifically binds the activated antibody, where the reagent contains a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

In some embodiments, the method or kit is used to identify or otherwise refine a patient population suitable for treatment with an activated antibody and/or a conjugated activated antibody (e.g., an activated drug-conjugated antibody) against a target as described herein, followed by treatment by administering the activated antibody and/or the conjugated activated antibody to a subject in need thereof. For example, patients who test positive for both the target (e.g., that target) and the substrate-cleaving protease in the cleavable group (CM) of the activated antibody and/or conjugated activated antibody tested in these methods are identified as suitable candidates for treatment with using such antibody and/or such conjugated activated antibody containing such SM, and then the patient is administered a therapeutically effective amount of the tested activated antibody and/or conjugated activated antibody. Similarly, patients who test negative for any or both of a target (eg, that target) and a substrate-cleaving protease in the SM in an activated antibody tested using these methods can be identified as suitable candidates for another form of therapy. In some embodiments, such patients can be tested with another antibody and/or conjugated activated antibody until a suitable antibody and/or conjugated activated antibody for treatment is identified (e.g. disease area). In some embodiments, the patient is then administered a therapeutically effective amount of an activated antibody and/or a conjugated activated antibody for which the patient is tested positive.

In some embodiments of these methods and kits, MM is a peptide having a length of approximately 4 to 40 amino acids. In some embodiments of these methods and kits, the activated antibody comprises a linker peptide, wherein the linker peptide is located between the MM and the SM. In some embodiments of these methods and kits, the activated antibody comprises a linker peptide, wherein the linker peptide is located between the AB and the CM. In some embodiments of these methods and kits, the activated antibody comprises a first linker peptide (L1) and a second linker peptide (L2), wherein the first linker peptide is located between MM and CM and the second linker peptide is located between AB and CM. In some embodiments of these methods and kits, each of L1 and L2 is a peptide of approximately 1-20 amino acids in length, and wherein each of L1 and L2 is not necessarily the same linker. In some embodiments of these methods and kits, one or both of L1 and L2 contains a glycineserine polymer. In some embodiments of these methods and kits, at least one of L1 and L2 comprises an amino acid sequence selected from the group consisting of (GS)n, (GSGGS)n (SEQ ID NO: 1) and (GGGS)n (SEQ ID NO: 2), where n is an integer, at least one. In some embodiments of these methods and kits, at least one of L1 and L2 contains an amino acid sequence having the formula (GGS) _n , where n is an integer of at least one. In some embodiments of these methods and kits, at least one of L1 and L2 contains an amino acid sequence selected from the group consisting of Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 4), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 5), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 6), Gly-Gly-Gly-Ser- Gly (SEQ ID NO: 7) and Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 8).

In some embodiments of these methods and kits, the AB contains an antibody or antibody fragment sequence selected from the cross-reactive antibody sequences provided herein. In some embodiments of these methods and kits, the AB contains a Fab fragment, a scFv, or a single chain antibody (scAb).

In some embodiments of these methods and kits, the cleaving agent is a protease co-localized with a target in a subject or sample, and the CM is a polypeptide that functions as a substrate for the protease, wherein the protease cleaves the CM in the activated antibody when the activated antibody is exposed to proteases. In some embodiments of these methods and kits, the SM is a polypeptide up to 15 amino acids in length. In some embodiments of these methods and kits, the CM is attached to the N-terminus of the AB. In some embodiments of these methods and kits, the CM is attached to the C-terminus of the AB. In some embodiments of these methods and kits, the CM is attached to the N terminus of the VL AB chain.

Antibodies, conjugated antibodies, activated antibodies and/or conjugated activated antibodies as described herein are used in diagnostic and prophylactic formulations. In one embodiment, an activated antibody is administered to patients at risk for developing one or more of the aforementioned inflammation, inflammatory disorders, cancer, or other disorders.

The predisposition of a patient or organ to one or more of the aforementioned disorders can be determined using genotypic, serological, or biochemical markers.

In some embodiments as described herein, the antibody, conjugated antibody, activated antibody, and/or conjugated activated antibody is administered to human individuals diagnosed with a clinical indicator associated with one or more of the aforementioned disorders. After diagnosis, the antibody, conjugated antibody, activated antibody, and/or conjugated activated antibody is administered to mitigate or reverse the effects of the clinical indicator.

Antibodies, conjugated antibodies, activated antibodies, and/or conjugated activated antibodies of this disclosure can also be used to detect a target in patient samples and, accordingly, can be used as diagnostics. For example, antibodies, conjugated antibodies, activated antibodies, and/or conjugated activated antibodies as described herein are used in in vitro assays, such as ELISA, to detect target levels in a patient sample.

In one embodiment, an antibody and/or an activated antibody as described herein is immobilized on a solid support (eg, well(s) of a microtiter plate). The immobilized antibody and/or the activated antibody serves as a binding antibody for any target that may be present in the test sample. Before bringing the immobilized antibody and/or activated antibody into contact with a patient sample, the solid support is washed and treated with a blocking agent such as milk protein or albumin to prevent non-specific adsorption of the analyte.

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The wells are then treated with a test sample suspected to contain an antigen or a solution containing a standard amount of antigen. Such a sample is, for example, a serum sample from a subject suspected of having levels of circulating antigen considered diagnostic of pathology. After washing the test sample or standard, the solid support is treated with a secondary antibody labeled with a detectable label. The labeled secondary antibody serves as the detection antibody. The level of a detectable label is measured and the concentration of the target antigen in the test sample is determined by comparison with a standard curve obtained from standard samples.

It should be understood that based on the results obtained using antibodies and/or activated antibodies of this description in an in vitro diagnostic assay, one can determine the stage of the disease in a subject based on the expression levels of the target antigen. For a given disease, blood samples are taken from subjects diagnosed as being at different stages of disease progression and/or at different points in the therapeutic treatment of the disease. Using a sample population that provides statistically significant results for each stage of progression or therapy, a range of antigen concentrations is developed that can be considered representative of each stage.

Antibodies, conjugated antibodies, activated antibodies, and/or conjugated activated antibodies can also be used in diagnostic and/or imaging methods. In some embodiments, such methods are in vitro methods. In some embodiments, such methods are in vivo methods. In some embodiments, such methods are in situ methods. In some embodiments, such methods are ex vivo methods. For example, activated antibodies having enzymatically cleavable SM can be used to detect the presence or absence of an enzyme capable of cleaving SM. Such activated antibodies can be used in diagnostics, which can include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or in some embodiments, an environment with increased reducing potential, such as one that can allow for disulfide bond reduction) via measuring the accumulation of activated antibodies (ie, antibodies resulting from cleavage of an activated antibody) in a given cell or tissue of a given host organism. This accumulation of activated antibodies not only indicates that the tissue has enzymatic activity (or increased reducing potential, depending on the nature of the SM), but also that the tissue expresses a target to which the activated antibody binds.

For example, SM can be chosen to be a protease substrate for a protease found at a tumor site, at a viral or bacterial infection site at a biologically restricted site (eg, in an abscess, in an organ, etc.), and the like. The AB may be an AB that binds a target antigen. Using methods known to one of skill in the art, a detectable label (eg, a fluorescent label or a radioactive label or a radiotracer) can be conjugated to the AB or other region of the antibody to be activated. Suitable detectable labels are discussed in the context of the above screening methods, and additional specific examples are provided below. By using an AV specific for a protein or peptide in a disease state, together with at least one protease whose activity is elevated in the diseased tissue of interest, the activated antibodies have an increased rate of binding to the diseased tissue compared to tissues where the CM-specific the enzyme is not present at a detectable level, or is present at a lower level than in diseased tissue, or is inactive (eg, in the form of a zymogen or in combination with an inhibitor). Because small proteins and peptides are rapidly cleared from the blood by the filtration system of the kidneys, and because the enzyme specific for SM is not present at detectable levels (or present at lower levels in non-diseased tissues, or present in an inactive conformation), the accumulation of activated antibodies in diseased tissue is increased compared to non-affected tissues.

In another example, activated antibodies can be used to detect the presence or absence of a disintegrating agent in a sample. For example, where the activated antibodies contain a SM that is sensitive to enzyme digestion, the activated antibodies can be used to detect (either qualitatively or quantitatively) the presence of the enzyme in the sample. In another example, where the activated antibodies contain a CM that is sensitive to cleavage by a reducing agent, the activated antibodies can be used to detect (either qualitatively or quantitatively) the presence of reducing conditions in a sample. To facilitate analysis in these methods, activated antibodies can be labeled with a detectable label, and can be associated with a support (eg, a solid support such as glass or bead). A detectable label can be placed on the part of the activated antibody that is not released after cleavage, for example, the detectable label can be a quenched fluorescent label or other label that is not under

- 109 041958 is given for detection until splitting occurs. The assay can be performed, for example, by bringing immobilized, detectably labeled, activated antibodies into contact with a sample suspected of containing an enzyme and/or reducing agent for a time sufficient for cleavage to occur, then washing to remove excess sample and contaminants. impurities. The presence or absence of a degrading agent (e.g., an enzyme or a reducing agent) in the sample is then assessed by a change in the detectable signal of activated antibodies prior to contact with the sample, e.g., the presence and/or an increase in a detectable signal due to cleavage of the activated antibody by the degrading agent in the sample .

Such detection methods can be adapted to also detect the presence or absence of a target capable of binding AV-activated antibodies upon digestion. Thus, the assays can be adapted to assess the presence or absence of a dissolving agent and the presence or absence of a target of interest. The presence or absence of a cleavage agent can be detected by the presence and/or increase in detectable label from activated antibodies as described above, and the presence or absence of a target can be detected by detection of the target-AB complex, for example, by using a detectably labeled antibody against the target. .

Activated antibodies can also be used for in situ imaging to confirm the activation of an activated antibody, for example, by protease digestion and binding to a specific target. In situ imaging is a technique that allows the localization of proteolytic activity and target in biological samples such as cell cultures or tissue sections. Using this method, both binding to a given target and proteolytic activity can be confirmed based on the presence of a detectable label (eg, a fluorescent label).

These methods can be used on any frozen cells or tissue obtained from a disease site (eg tumor tissue) or healthy tissue. These methods can also be used on fresh cell or tissue samples.

In these methods, the activated antibody is labeled with a detectable label. The detectable label can be a fluorescent dye (eg, fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (TRITC), near infrared (NIR) dye (eg, Qdot® nanocrystals), colloidal metal, hapten, radioactive marker, biotin and an enhancer such as streptavidin or an enzyme (eg horseradish peroxidase or alkaline phosphatase).

Detection of a label in a sample that has been incubated with a labeled, activated antibody indicates that the sample contains a target and contains a protease that is specific for the CM of the activated antibody. In some embodiments, the presence of a protease can be confirmed using a broad spectrum of protease inhibitors, such as those described herein, and/or using a protease-specific agent, e.g., an antibody such as A11, that is specific for the matriptase protease and inhibits proteolytic activity. matriptase; See, for example, International Publication No. WO 2010/129609, publ. Nov. 11, 2010 The same method using a broad spectrum of protease inhibitors such as those described herein and/or using a more selective inhibitory agent can be used to identify a protease or class of proteases specific for the CM of an activated antibody. In some embodiments, the presence of a target may be confirmed using a target-specific agent, such as another antibody, or a detectable label may compete with an unlabeled target. In some embodiments, an unlabeled, activated antibody can be used with detection by a labeled secondary antibody or a more sophisticated detection system.

Similar methods can also be used for in vivo imaging, where detection of a fluorescent signal in a subject, eg, a mammal, including a human, indicates that the disease site contains a target and contains a protease specific for the CM of the activated antibody.

These methods can also be used in kits and/or as reagents for detecting, identifying, or characterizing protease activity in a variety of cells, tissues, and organisms based on the protease-specific SM in an activated antibody.

In some embodiments, in situ imaging and/or in vivo imaging can be used in methods for identifying patients to be treated. For example, in in situ imaging, activated antibodies are used to screen samples from patients to identify patients having the appropriate protease(s) and target(s) at the appropriate location, such as at a tumor site.

In some embodiments, in situ imaging is used to identify or otherwise refine a patient population suitable for treatment with an activated antibody as described herein. For example, patients who test positive for both the target and

- 110 041958 for a protease that cleaves the substrate in the cleavable group (CM) of the test activated antibody (eg, with the accumulation of the activated antibody at the site of the disease) are identified as suitable candidates for treatment using such an activated antibody containing such CM. Similarly, patients who test negative for either or both of the target and the SM substrate-cleaving protease in the activated antibody tested using these methods are identified as eligible candidates for another form of therapy (i.e., not eligible for treatment using test activated antibody). In some embodiments, such patients who test negative for the first activated antibody may be tested with other activated antibodies containing other SMs until a suitable activated antibody for treatment is identified (e.g., an activated antibody containing a SM that is cleaved by the patient at the disease site). ).

In some embodiments, in vivo imaging is used to identify or otherwise refine a patient population suitable for treatment with an activated antibody as described herein. For example, patients who test positive for both the target and the substrate-cleaving protease in the cleavable group (CM) of the activated antibody being tested (e.g., with accumulation of the activated antibody at the disease site) are identified as suitable candidates for treatment with that activated antibody containing such a SM. Similarly, patients with a negative test are identified as suitable candidates for another form of therapy (ie, not suitable for treatment with the activated antibody being tested). In some embodiments, such patients who test negative for the first activated antibody can be tested with other activated antibodies containing other SMs until a suitable activated antibody for treatment is identified (e.g., an activated antibody containing a SM that is cleaved by the patient at the disease site) .

pharmaceutical compositions.

Antibodies, conjugated antibodies, activated antibodies, and/or conjugated activated antibodies as described herein (also referred to herein as active compounds), and derivatives, fragments, analogs, and homologues thereof, may be included in pharmaceutical compositions suitable for administration. Such compositions typically contain an antibody, a conjugated antibody, an activated antibody and/or a conjugated activated antibody, and a pharmaceutically acceptable carrier. As used herein, the term pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, the standard reference in the art, the contents of which are incorporated herein by reference.

Suitable examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers such as fatty oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional medium or agent is incompatible with the active compound, their use in the compositions is contemplated. Additional active compounds may also be included in the compositions.

The pharmaceutical composition of this disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous administration may include the following components:

a sterile diluent such as water for injection, saline, fatty oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents;

antibacterial agents such as benzyl alcohol or methyl parabens;

antioxidants such as ascorbic acid or sodium bisulfite;

chelating agents such as ethylenediaminetetraacetic acid (EDTA);

buffers such as acetates, citrates or phosphates; and tonicity agents such as sodium chloride or dextrose.

The pH can be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (when water-soluble) or dispersions and sterile powders for preparation shortly before administration of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline

- 111 041958 saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, through the use of a coating such as lecithin, through the maintenance of the required particle size in the case of a dispersion, and through the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, it may be desirable to include isotonic agents, eg sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in the composition. Prolonged absorption of injectable compositions can be accomplished by including in the composition an absorption delaying agent such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by administering the active compound in the required amount of a suitable solvent with one or a combination of the ingredients listed above, as appropriate, followed by sterile filtration. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and necessary ingredients other than those listed above. In the case of sterile powders to obtain sterile injectable solutions, the methods of preparation are vacuum drying and lyophilization to obtain a powder of the active ingredient plus any additional desired ingredient from its pre-filtered sterile solution.

Oral compositions typically include an inert diluent or edible carrier. They can also be enclosed in gelatin capsules or compressed into tablets. For purposes of oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using a liquid carrier for use in the form of a mouthrinse, wherein the compound in the liquid carrier is taken orally and rinsed and spit out or swallowed. Pharmaceutically compatible binders and/or adjuvants may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of a similar nature:

a binder such as microcrystalline cellulose, gum tragacanth or gelatin; a filler such as starch or lactose, a disintegrant such as alginic acid, Primogel or corn starch;

a lubricant such as magnesium stearate or sterotes;

a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or an orange flavor.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, for example a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be carried out by transmucosal or transdermal routes. For transmucosal or transdermal administration, penetration aids suitable for the barrier to be penetrated are used in the formulation. Such penetration aids are generally known in the art and include, for example, transmucosal agents, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be carried out using nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels or creams, as is well known in the art.

The compounds can also be prepared in the form of suppositories (eg, using conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are formulated with carriers that can protect the compound against rapid excretion from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such compositions are obvious to the person skilled in the art. These materials are also available commercially.

- 112 041958 based on Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells using monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. They can be obtained in accordance with methods known to those skilled in the art, for example, as described in US patent No. 4522811.

It is particularly advantageous to formulate oral or parenteral compositions in unit dosage form for ease of administration and uniformity of dosing. Unit dosage form, as used herein, refers to physically discrete units suitable as single doses for a subject to be treated, where each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The definition of unit dosage forms as described herein is dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved and the limitations inherent in the field of preparation of the compounds, such as the active compound for the treatment of individuals.

Pharmaceutical compositions may be enclosed in a container, pack or dispenser along with instructions for administration.

The invention is further described in the following examples, which do not limit the scope of the invention as described in the claims.

Examples

Example 1 Characterization of anti-CD 166 antibodies.

The assays presented herein are designed to evaluate the binding of anti-CD166 antibodies as described.

Binding of various anti-CD166 antibodies as described herein was confirmed by ELISA (FIG. 1). Tested antibodies against human CD166 containing the following sequence VH and VL.

Name of the antibody VH SEQ VL SEQ CD166-M9 _vKl/HcB SEQ ID NO: 121 SEQ ID NO: 123 CD166-M9_ _vK2/HcB SEQ ID NO: 121 SEQ ID NO: 124 CD166-M9 _vK3a/HcB SEQ ID NO: 121 SEQ ID NO: 125 CD166-M9_ _vK3b/HcB SEQ ID NO: 121 SEQ ID NO: 126 CD166-M9_ _vKl/HcC SEQ ID NO: 122 SEQ ID NO: 123 CD166-M9 _vK2/HcC SEQ ID NO: 122 SEQ ID NO: 124 CD166-M9 _vK3a/HcC SEQ ID NO: 122 SEQ ID NO: 125 CD166-M9_ _vK3b/HcC SEQ ID NO: 122 SEQ ID NO: 126

mAb M9 was obtained using mouse hybridoma technology and the remaining sequences were obtained by humanizing the sequence of mAb M9. One skilled in the art will recognize that the ability to induce anti-CD166 antibodies has been hampered for a long time because investigators have not been able to generate hybridomas and/or antibodies in mice injected with human CD166. In contrast, the anti-CD166 antibodies provided herein are from mice against human CD166.

Anti-CD166 antibody M9 containing VH of SEQ ID NO: 119 and VL of SEQ ID NO: 120 was used as a positive control, and an isotype control antibody was used as a negative control.

As shown in FIG. 1, all of the humanized anti-CD166 antibodies showed binding to CD166 comparable to M9. Using a standard ELISA protocol, human CD166 protein was absorbed onto ELISA plates and then incubated with the indicated concentration of antibody. Bound antibody was detected using an anti-human FAB secondary antibody with peroxidase.

Example 2. Study of disguise.

The studies presented herein are designed to identify and characterize masking groups for use in activated anti-CD166 antibodies of this disclosure.

Mab M9 mouse anti-CD166 antibody (VH of SEQ ID NO: 119 and VL of SEQ ID NO: 120) was used to screen a cysteine-restricted _X15 peptide library with a total diversity of 3x1011, where X is any amino acid using a method similar to with the method described in the international publication PCT No. WO 2010/081173, publ. July 15, 2010 Screening consisted of two rounds of MACS triage and four rounds of FACS triage. The sorting method is shown in Fig. 2.

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Individual clones from the M2F1.1, M2F2.1, M2F3.1 and M2F4.1 populations were sequenced and the results are shown in Table 1. 9.

Table 9

Masking peptide sequences

M2F1.1

JF16490 YLCQRHPLALKYCTN (SEQ ID NO: 135) JF16492 PLCVPTQLLRSCYNY (SEQ ID NO: 136) JF16493 AVCHPLANVETQCLD (SEQ ID NO: 137) JF16494 PHCHPLFNNTYCYRH (SEQ ID NO: 138) JF16495 PLCRPIELLASCPMK (SEQ ID NO: 139) JF16496 GAACVSAWGFFCECC (SEQ ID NO: 140) JF16498 DCAKDILHLMPHCSM (SEQ ID NO: 141) JF16501 NTCMHPLLLQGCKTY (SEQ ID NO: 142) JF16503 YLGCLLYAGPGCEGG (SEQ ID NO: 143) JF16506 ARCPHPLLLSICENN (SEQ ID NO: 144) JF16507 ELCPHPLPFGFCNNY (SEQ ID NO: 145) JF16508 ALYCHPPYIRCEEMT (SEQ ID NO: 146) M2F2.1 JF16534 TSLCHPVMIMYCKTG (SEQ ID NO: 147) JF16535 PLCHPLEQASWCNMD (SEQ ID NO: 148) JF16536 PHPCPRTGSRMCHFS (SEQ ID NO: 149) JF16537 SGCRHPLPLKACGTN (SEQ ID NO: 150) JF16538 GLCHPIRLHNTQCTI (SEQ ID NO: 151) JF16539 KCMHPLNLHNINCNH (SEQ ID NO: 152) JF16540 PICHPLREFMNTCFK (SEQ ID NO: 153) JF16541 NCHPLDWGWLGCMK (SEQ ID NO: 154) JF16542 YNNVCHPLFCSQHTY (SEQ ID NO: 155) JF16543 TFCHPLFSLNYCGHK (SEQ ID NO: 156) JF16544 FCHPLTLSNNKQCNR (SEQ ID NO: 157) JF16545 LSHCAVLLLRVCSGS (SEQ ID NO: 158) JF16546 KIHCHPLRLGTCLVG (SEQ ID NO: 159) JF16547 ETCAHPLDMRMCRHN (SEQ ID NO: 160) JF16548 PLCYPLILMSSCWLG (SEQ ID NO: 161) JF16549 YGICHPAPDLPCMQI (SEQ ID NO: 162) JF16550 TACHPLYNVEHLCEI (SEQ ID NO: 163) JF16551 TACNKSVCVAGCCLL (SEQ ID NO: 164) JF16552 LHPLCSYMKSCMKNN (SEQ ID NO: 165) JF16553 THCHCMVYFCPCRWS (SEQ ID NO: 166) M2F3.1 JF16554 PKCPHPLHLANCYAS (SEQ ID NO: 167) JF16555 KTCYHPTPVIAXNSY (SEQ ID NO: 168)

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JF16556 AKCLPPLIQYCRCIK (SEQ ID NO: 169) JF16557 HACQHPLQLHTCKHN (SEQ ID NO: 170) JF16558 LCHPLVLSAWESCSSN (SEQ ID NO: 171) JF16559 WPLCSFGKSFCAQNA (SEQ ID NO: 172) JF16560 ECQSFEHFLTNNCHS (SEQ ID NO: 173) JF16561 SCKHPLVMPNLKCTR (SEQ ID NO: 174) JF16562 YPCHPLQLSIPHCTK (SEQ ID NO: 175) JF16563 ICHPLTHTMEYMCMN (SEQ ID NO: 176) JF16564 TLCHPLTFSVPTCTN (SEQ ID NO: 177) JF16565 PLCQPNRLLQACGNT (SEQ ID NO: 178) JF16566 TLCRHPLALDGCQNN (SEQ ID NO: 179) JF16567 QPMCYQPAHPLCNTI (SEQ ID NO: 180) JF16568 SNCHPLLFQHYHCML (SEQ ID NO: 181) JF16569 EKCYHPLTLAHCQNH (SEQ ID NO: 182) JF16571 NKCFVHPLAMPNCNS (SEQ ID NO: 183) JF16572 VNNCLLMTRAHCTSY (SEQ ID NO: 184) JF16573 LPCWAFAVNPLHCGD (SEQ ID NO: 185) M2F4.1 JS7503 VNNCLLMTRAHCTSY (SEQ ID 1 40: 186) JS7504 SSCPHPLGLTGCNDK (SEQ ID 1 40: 187) JS7505 NKCFVHPLAMPNCNS (SEQ ID 1 40: 188) JS7506 FVGCHSVYVSGCLRA (SEQ ID 1 40: 189) JS7507 NMCHPPHNIYSICNM (SEQ ID 1 40: 190) JS7509 LTCHLLPGLTLH-TK (SEQ ID 1 40: 191) JS7510 RTCHPLPGLTLHCTK (SEQ ID 1 40: 192) JS7511 HPLCFESMKNCFPNY (SEQ ID 1 40: 193) JS7513 TTCHPLSFTHNYCIT (SEQ ID 1 40: 194) JS7515 RDCGFDAVRADCLFG (SEQ ID 1 40: 195) JS7516 RTCSTHPLTMPQCNY (SEQ ID] 40: 196) JS7517 MKCHPLQLTGNTCSM (SEQ ID 1 40: 197) JS7518 SGCPHPLQLITCSTA (SEQ ID 1 40: 198) JS7519 KCFPAFHDGPLACAS (SEQ ID NO: 199) JS7520 LKCQHPLPMSHCQPQ (SEQ ID 1 40: 200) JS7521 AFCGFSVIHPLCSGA (SEQ ID 1 40: 201) JS7522 SVHCAVLKLDGCLGW (SEQ ID NO: 202) JS7523 TLPCHPIMVLGCTPM (SEQ ID 1 40: 203) JS7525 HYPCMKYNPLNCSMS (SEQ ID NO: 204) JS7526 LKCPHPLSLNGCTLK (SEQ ID NO: 205) JS7527 VYSCMANNPLDCFTQ (SEQ ID NO: 206) JS7528 PICHPLVTLMSYCNK (SEQ ID NO: 207) JS7529 DWCSFWAGQSVWCTS (SEQ ID NO: 208) JS7530 STCHPLTPFHDKCRY (SEQ ID NO: 209) JS7531 PVCPPLVTLMSYCNK (SEQ ID NO: 210) JS7532 STCHPLPTLMPYCNS (SEQ ID NO: 211) JS7533 FPLCGIGPAFCDTTV (SEQ ID NO: 212) JS7534 PTCHPLVLSVPCPKI (SEQ ID NO: 213) JS7537 GPLCDYFVFYSCRGS (SEQ ID NO: 214) JS7538 HTCYHPLKLGQCEMF (SEQ ID NO: 215) JS7539 RTCIHPLPLHQCHKP (SEQ ID NO: 216) JS7540 ACHPINFNSIVYCNN (SEQ ID NO: 217) JS7542 SHPCSWNLPGCEPD (SEQ ID NO: 218)

The masks were subjected to shortening and alanine scanning to obtain families of activated antibodies with varying masking efficiencies. The sequences are shown below in table.

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10 where a denotes the position of the alanine included as part of the scan. It is equivalent to A.

Table 10

Shortening and alanine scanning of masking peptides

7614 LCHPLVLSAWESCSS (SEQ ID NO: 219)

7614.4 LCaPLVLSAWESCSS (SEQ ID NO: 220) 7614.5 LCHaLVLSAWESCSS (SEQ ID NO: 221) 7614.6 LCHPaVLSAWESCSS (SEQ ID NO: 222) 7614.7 LCHPLaLSAWESCSS (SEQ ID NO: 223) 7614.8 LCHPLVaSAWESCSS (SEQ ID NO: 224) 7614.9 LCHPLVLSAaESCSS (SEQ ID NO: 225) 7614.10 ) LCHPLVLSAWaSCSS (SEQ i II 5NO: :226)

7614.11 CHPLVLSAWESC (SEQ ID NO: 227)

7614.12 HPLVL (SEQ ID NO: 228)

7614.13 HPL (SEQ ID NO: 229)

16522 LEGWCLHPLCLWGAG (SEP ID NO: 230)

16522.14 LEGaCLHPLCLWGAG (SEQ ID NO: 231) 16522.15 LEGWCaHPLCLWGAG (SEQ ID NO: 232) 16522.16 LEGWCLaPLCLWGAG (SEQ ID NO: 233) 16522.17 LEGWCLHaLCLWGAG (SEQ ID NO: 234) 16522.18 LEGWCLHPaCLWGAG (SEQ ID NO: 235) 16522.19 LEGWCLHPLCaWGAG (SEQ ID NO: 236) 16522.20 LEGWCLHPLCLaGAG (SEQ ID NO: 237)

16522.21 CLHPLC (SEQ ID NO: 238)

These masking peptides were used to generate activated anti-CD166 antibodies as described herein. The sequences for specific of these activated anti-CD166 antibodies are shown in Table 1 below. 11. In some embodiments, these CD166 activated antibodies comprise cleavage group 2001 (ISSGLLSGRSDNH; SEQ ID NO: 70), cleavage group 3001 (AVGLLAPPGGLSGRSDNH; SEQ ID NO: 76), cleavage group 2007 (ISSGLLSGRSDNH; SEQ ID NO: 342) , cleavage group 2008 (ISSGLLSGRSDQH; SEQ ID NO: 343), cleavage group 2011 (ISSGLLSGRSDNP; SEQ ID NO: 346), cleavage group 2012 (ISSGLLSGRSANP; SEQ ID NO: 347), cleavage group 2013 (ISSGLLSGRSANI; SEQ ID NO: 348); NO: 355) or cleavable group 3013 (AVGLLAPPGGLSGRSANI; SEQ ID NO: 356) as indicated.

While the specific sequences shown below contain the spacer sequence of SEQ ID NO: 305, one skilled in the art will appreciate that the CD166-activated antibodies of this disclosure may contain any suitable spacer sequence, such as, for example, a spacer sequence selected from a group consisting of QGQSGQG (SEQ ID NO: 305), QGQSGQ (SEQ ID NO: 88), QGQSG (SEQ ID NO: 306), QGQS (SEQ ID NO: 307), QGQ (SEQ ID NO: 308), QG (SEQ ID NO: 309), GQSGQG (SEQ ID NO: 359), QSGQG (SEQ ID NO: 360), SGQG (SEQ ID NO: 361), GQG (SEQ ID NO: 362), G or Q. In some embodiments, activated anti-CD166 antibodies as described herein may not have a spacer sequence attached to their N-terminus.

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Table 11 Anti-CD 166 Activated Antibody Sequences

Activated anti-CD166 antibody heavy chain (HuCD166_HcC): Amino acid sequence

QITLKESGPTLVKPTQTLTLTCTFSGFSLSTYGMGVGWIRQPPGKALEWLANIWWSEDK HYSPSLKSRLTITKDTSKNQVVLTITNVDPVDTATYYCVQIDYGNDYAFTYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 239)

Nucleotide sequence

CAGATCACCCTGAAAGAGTCCGGCCCCACCCTGGTGAAACCCACCCAGACCCTGACCCT GACATGCACCTTCTCCGGCTTCAGCCTGTCCACCTACGGCATGGGCGTGGGCTGGATCAGGCAG CCTCCTGGCAAGGCCCTGGAATGGCTGGCCAACATCTGGTGGTCCGAGGACAAGCACTACTCCC CCAGCCTGAAGTCCCGGCTGACCATCACCAAGGACACCTCCAAGAACCAGGTGGTGCTGACAAT CACAAACGTGGACCCCGTGGACACCGCCACCTACTACTGCGTGCAGATCGACTACGGCAACGAC TACGCCTTCACCTACTGGGGCCAGGGCACACTGGTGACAGTGTCCTCCGCCTCCACCAAGGGCC CCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGCACAGCTGCCCTGGGCTG CCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCAGC GGAGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGA CCGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAA CACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCCTGC CCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCTAAGGACACCC TGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGA AGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAG GAACAGTACAACTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGA ACGGCAAAGAGTACAAGTGCAAGGTGTCCA ACAAGGCCCTGCCTGCCCCCATCGAAAAGACCAT CTCCAAGGCCAAGGGCCAGCCCCGCGAGCCTCAGGTGTACACACTGCCCCCTAGCCGGGAAGAG ATGACCAAGAATCAGGTGTCCCTGACCTGTCTGGTGAAAGGCTTCTACCCCTCCGATATCGCCG TGGAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTC CGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAAC GTGTTCTCCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCC TGAGCCCCGGCAAG (SEQ ID NO: 241)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.6_2001 (SEQ ID NO: 310)] Amino acid sequence

[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSISSGLLSGRSDNHGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC] (SEQ ID NO: 242)

[spacer (SEQ ID NO: 480)] [HuCD166Lc1_7614.6_2001 (SEQ ID NO: 311)]

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Nucleotide sequence

[CAGGGCCAGTCTGGACAGGGC][CTGTGTCACCCTGCCGTGCTGTCTGCCTGGGAGTC CTGTTCCTCCGGCGGTGGCTCCTCTGGCGGCTCCATCTCCTCTGGCCTGCTGTCCGGCAGATCC GACAACCACGGCGGAGGCAGCGACATCGTGATGACCCAGTCCCCCCTGTCCCTGCCCGTGACAC CTGGCGAGCCTGCCTCCATCAGCTGCCGGTCCTCCAAGTCCCTGCTGCACTCCAACGGCATCAC CTACCTGTACTGGTATCTGCAGAAGCCCGGCCAGTCCCCTCAGCTGCTGATCTACCAGATGTCC AACCTGGCCTCCGGCGTGCCCGACAGATTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGA AGATCTCCCGGGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCGCCCAGAACCTGGAACTGCC CTACACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTC ATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTCGTGTGCCTGCTGAACA ACTTCTACCCTCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTC CCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACC CTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGA GCAGCCCCGTGACCAAGTCCTTCAACCGCGGCGAGTGC] (SEQ ID NO: 243)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.8_2001 (SEQ ID NO: 312)] Amino acid sequence

[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSISSGLLSGRSDNHGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC] (SEQ ID NO: 244)

[spacer (SEQ ID NO: 480)] [huCD166Lc1_7614,8_2001 (SEQ ID NO: 313)] Nucleotide sequence

[CAGGGCCAGTCTGGACAGGGC][CTGTGTCACCCTCTGGTGGCCTCTGCCTGGGAGTC CTGTTCCTCCGGCGGTGGCTCCTCTGGCGGCTCCATCTCCTCTGGCCTGCTGTCCGGCAGATCC GACAACCACGGCGGAGGCAGCGACATCGTGATGACCCAGTCCCCCCTGTCCCTGCCCGTGACAC CTGGCGAGCCTGCCTCCATCAGCTGCCGGTCCTCCAAGTCCCTGCTGCACTCCAACGGCATCAC CTACCTGTACTGGTATCTGCAGAAGCCCGGCCAGTCCCCTCAGCTGCTGATCTACCAGATGTCC AACCTGGCCTCCGGCGTGCCCGACAGATTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGA AGATCTCCCGCGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCGCCCAGAACCTGGAACTGCC CTACACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTC ATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTCGTGTGCCTGCTGAACA ACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTC CCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACC CTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGA GCAGCCCCGTGACCAAGTCCTTCAACCGCGGCGAGTGC] (SEQ ID NO: 245)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.6_3001 (SEQ ID NO: 314)] Amino acid sequence

[[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDNHGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 246)

[spacer (SEQ ID NO: 481)] [huCD166Lc1_7614.6_3001 (SEQ ID NO: 315)]

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Nucleotide sequence

[CAGGGACAGTCTGGCCAGGGC][CTGTGTCACCCTGCTGTGCTGTCTGCCTGGGAGTC CTGTTCCAGCGGCGGAGGCTCCTCTGGCGGCTCTGCTGTGGGCCTGCTGGCTCCACCTGGCGGC CTGTCCGGCAGATCTGACAACCACGGCGGCTCCGACATCGTGATGACCCAGTCCCCCCTGTCCC TGCCCGTGACTCCTGGCGAGCCTGCCTCCATCTCCTGCCGGTCCTCCAAGTCCCTGCTGCACTC CAACGGCATCACCTACCTGTACTGGTATCTGCAGAAGCCCGGCCAGTCCCCTCAGCTGCTGATC TACCAGATGTCCAACCTGGCCTCCGGCGTGCCCGACAGATTCTCCGGCTCTGGCTCCGGCACCG ACTTCACCCTGAAGATCTCCCGGGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCGCCCAGAA CCTGGAACTGCCCTACACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGGACCGTGGCCGCT CCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTGGTCT GCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCA GTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCC TCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCC ACCAGGGACTGAGCAGCCCCGTGACCAAGTCCTTCAACCGGGGCGAGTGC] (SEQ ID NO: 247)

[spacer (SEQ ID NO: 309)] [huCD166Lc1_7614.8_3001 (SEQ ID NO: 316)] Amino acid sequence

[QG][LCHPLVASAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDNHGGSDIVMTQSPLSLPVTP GEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSGTDFTLK ISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC] (SEQ ID NO: 248)

[spacer (SEQ ID NO: 483)] [huCD166Lc1_7614.8_3001 (SEQ ID NO: 317)] Nucleotide sequence

[[CAGGGCC][TGTGTCACCCTCTGGTGGCCTCTGCCTGGGAGTCCTGTTCCTCCGGCGGAGGC TCCTCTGGCGGCTCTGCTGTGGGCCTGCTGGCTCCACCTGGCGGCCTGTCCGGCAGATCTGACA ACCACGGCGGCTCCGACATCGTGATGACCCAGTCCCCCCTGTCCCTGCCCGTGACTCCTGGCGA GCCTGCCTCCATCTCCTGCCGGTCCTCCAAGTCCCTGCTGCACTCCAACGGCATCACCTACCTG TACTGGTATCTGCAGAAGCCCGGCCAGTCCCCTCAGCTGCTGATCTACCAGATGTCCAACCTGG CCTCCGGCGTGCCCGACAGATTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGAAGATCTC CCGGGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCGCCCAGAACCTGGAACTGCCCTACACC TTCGGCCAGGGCACCAAGCTGGAAATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCC CACCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTCGTGTGCCTGCTGAACAACTTCTA CCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAA TCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCA AGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGAGCAGCCC CGTGACCAAGTCCTTCAACCGGGGCGAGTGC] (SEQ ID NO: 478)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.8_3001 (SEQ ID NO: 316)] Amino acid sequence

[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDNHGGSDIVMT QSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSG SGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 303)

[spacer (SEQ ID NO: 482)] [huCD166Lc1_7614.8_CM2 (SEQ ID NO: 479)]

- 119 041958

Nucleotide sequence

[CAGGGCCAGTCTGGCCAGGGC][CTGTGTCACCCTCTGGTGGCCTCTGCCTGGGAGTC CTGTTCCTCCGGCGGAGGCTCCTCTGGCGGCTCTGCTGTGGGCCTGCTGGCTCCACCTGGCGGC CTGTCCGGCAGATCTGACAACCACGGCGGCTCCGACATCGTGATGACCCAGTCCCCCCTGTCCC TGCCCGTGACTCCTGGCGAGCCTGCCTCCATCTCCTGCCGGTCCTCCAAGTCCCTGCTGCACTC CAACGGCATCACCTACCTGTACTGGTATCTGCAGAAGCCCGGCCAGTCCCCTCAGCTGCTGATC TACCAGATGTCCAACCTGGCCTCCGGCGTGCCCGACAGATTCTCCGGCTCTGGCTCCGGCACCG ACTTCACCCTGAAGATCTCCCGGGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCGCCCAGAA CCTGGAACTGCCCTACACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGGACCGTGGCCGCT CCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTCGTGT GCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCA GTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCC TCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCC ACCAGGGCCTGAGCAGCCCCGTGACCAAGTCCTTCAACCGGGGCGAGTGC (SEQ ID NO: 304)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.6_2001 VL domain (SEQ ID NO: 364)]

Amino acid sequence

[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSISSGLLSGRSDNHGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 363)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.8_2001 VL domain (SEQ ID NO: 366)]

Amino acid sequence

[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSISSGLLSGRSDNHGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 365)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.6_3001 VL domain (SEQ ID NO: 368)]

Amino acid sequence

[[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDNHGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 367)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.8_3001 VL domain (SEQ ID NO: 370)]

Amino acid sequence

[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDNHGGSDIVMT QSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSG SGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 369)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.6_2007 (SEQ ID NO: 372)] Amino acid sequence

[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSISSGLLSGRSDIHGGGSDIVMTQSPL

SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTKADYEKHKVECSSACEV NSFQ1)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.6_2007 VL domain (SEQ ID NO: 374)]

Amino acid sequence

- 120 041958

[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSISSGLLSGRSDIHGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 373)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.6_3007 (SEQ ID NO: 376)] Amino acid sequence

[[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDIHGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 375)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.6_3007 VL domain (SEQ ID NO: 378)]

Amino acid sequence

[[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDIHGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 377)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.6_2008 (SEQ ID NO: 380)] Amino acid sequence

[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSISSGLLSGRSDQHGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC] (SEQ ID NO: 379)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.6_2008 VL domain (SEQ ID NO: 382)]

Amino acid sequence

[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSISSGLLSGRSDQHGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 381)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.6_3008 (SEQ ID NO: 384)] Amino acid sequence

[[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDQHGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 383)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.6_3008 VL domain (SEQ ID NO: 386)]

Amino acid sequence

[[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDQHGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 385)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.6_2011 (SEQ ID NO: 388)] Amino acid sequence

[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSISSGLLSGRSDNPGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC] (SEQ ID NO: 387)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.6_2011 VL domain (SEQ ID NO: 390)]

Amino acid sequence

- 121 041958

[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSISSGLLSGRSDNPGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 389)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.6_3011 (SEQ ID NO: 392)] Amino acid sequence

[[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDNPGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 391)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.6_3011 VL domain (SEQ ID NO: 394)]

Amino acid sequence

[[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDNPGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 393)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.6_2012 (SEQ ID NO: 396)] Amino acid sequence

[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSISSGLLSGRSANPGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC] (SEQ ID NO: 395)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.6_2012 VL domain (SEQ ID NO: 398)]

Amino acid sequence

[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSISSGLLSGRSANPGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 397)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.6_3012 (SEQ ID NO: 400)] Amino acid sequence

[[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSANPGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 399)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.6_3012 VL domain (SEQ ID NO: 402)]

Amino acid sequence

[[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSANPGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 401)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.6_2013 (SEQ ID NO: 404)] Amino acid sequence

[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSISSGLLSGRSANIGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC] (SEQ ID NO: 403)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.6_2013 VL domain (SEQ ID NO: 406)]

Amino acid sequence

- 122 041958

[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSISSGLLSGRSANIGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 405)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.6_3013 (SEQ ID NO: 408)] Amino acid sequence

[[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSANIGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 407)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.6_3013 VL domain (SEQ ID NO: 410)]

Amino acid sequence

[[QGQSGQG][LCHPAVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSANIGGSDIVM

TQSPLSLPPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS

GSGSGTDFTLKISRVEEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 409) [spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.8_2007 (SEQ ID NO: 412)] Amino Acid Sequence [QGQSGQG][LCHPLVASAWESCSSGGDIGSGGISSGLLSGRS

SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTKADYEKHKVECSSACEV NSFQ1)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.8_2007 VL domain (SEQ ID NO: 414)]

Amino acid sequence

[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSISSGLLSGRSDIHGGGSDIVMTQSPL

SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 413)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.8_3007 (SEQ ID NO: 416)] Amino acid sequence

[[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDIHGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 415)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.8_3007 VL domain (SEQ ID NO: 418)]

Amino acid sequence

[[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDIHGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 417)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.8_2008 (SEQ ID NO: 420)] Amino acid sequence

[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSISSGLLSGRSDQHGGGSDIVMTQSPL

SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTKADYEKHKVECSSACEV NSFQ1)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.8_2008 VL domain (SEQ ID NO: 422)]

Amino acid sequence

- 123 041958

[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSISSGLLSGRSDQHGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 421)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.8_3008 (SEQ ID NO: 424)] Amino acid sequence

[[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDQHGGSDIVM

TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSS ID4]

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.8_3008 VL domain (SEQ ID NO: 426)]

Amino acid sequence

[[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDQHGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 425)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.8_2011 (SEQ ID NO: 428)] Amino acid sequence

[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSISSGLLSGRSDNPGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC] (SEQ ID NO: 427)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.8_2011 VL domain (SEQ ID NO: 430)]

Amino acid sequence

[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSISSGLLSGRSDNPGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 429)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.8_3011 (SEQ ID NO: 432)] Amino acid sequence

[[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDNPGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 431)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.8_3011 VL domain (SEQ ID NO: 434)]

Amino acid sequence

[[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDNPGGSDIVM

TQSPLSLPPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS

GSGSGTDFTLKISRVEEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 433)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.8_2012 (SEQ ID NO: 436)]

Amino acid sequence

[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSISSGLLSGRSANPGGGSDIVMTQSPL

SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG

TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV

VC L LNN FY PREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLS KADYE KHKVYAC EV

THQGLSSPVTKSFNRGEC] (SEQ ID NO: 435)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.8_2012 VL domain (SEQ ID NO: 438)]

Amino acid sequence

- 124 041958

[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSISSGLLSGRSANPGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 437)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.8_3012 (SEQ ID NO: 440)] Amino acid sequence

[[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSAVGLLAPPGGLSGRSANPGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 439)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.8_3012 VL domain (SEQ ID NO: 442)]

Amino acid sequence

[[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSAVGLLAPPGGLSGRSANPGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 441)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.8_2013 (SEQ ID NO: 444)] Amino acid sequence

[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSISSGLLSGRSANIGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC] (SEQ ID NO: 443)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614.8_2013 VL domain (SEQ ID NO: 446)]

Amino acid sequence

[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSISSGLLSGRSANIGGGSDIVMTQSPL

SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG

TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 445)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614,8_3013 (SEQ ID NO: 448)] Amino acid sequence

[[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSAVGLLAPPGGLSGRSANIGGSDIVM

TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV

YACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 447)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614.8_3013 VL domain (SEQ ID NO: 450)]

Amino acid sequence

[[QGQSGQG][LCHPLVASAWESCSSGGGSSGGSAVGLLAPPGGLSGRSANIGGSDIVM

TQSPLSLPPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS

GSGSGTDFTLKISRVEEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 449) [spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614_2001 (SEQ ID NO: 452)] Amino acid sequence [QGQSGQG][LCHPLVLSMTAWESCSSGDIGGSSGGSISSGLLSGRSDNHGSP

SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV

VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC] (SEQ ID NO: 451)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614_2001 VL domain (SEQ ID NO: 454)]

Amino acid sequence

- 125 041958

[QGQSGQG] [LCHPLVLSAWESCSSGGGSSGGSISSGLLSGRSDNHGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 453)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614_3001 (SEQ ID NO: 456)] Amino acid sequence

[[QGQSGQG][LCHPLVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDNHGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 455)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614_3001 VL domain (SEQ ID NO: 458)]

Amino acid sequence

[[QGQSGQG][LCHPLVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDNHGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 457)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614_2011 (SEQ ID NO: 460)] Amino acid sequence

[QGQSGQG][LCHPLVLSAWESCSSGGGSSGGSISSGLLSGRSDNPGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC] (SEQ ID NO: 459)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614_2011 VL domain (SEQ ID NO: 462)]

Amino acid sequence

[QGQSGQG][LCHPLVLSAWESCSSGGGSSGGSISSGLLSGRSDNPGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 461)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614_3011 (SEQ ID NO: 464)] Amino acid sequence

[[QGQSGQG][LCHPLVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDNPGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 463)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614_3011 VL domain (SEQ ID NO: 466)]

Amino acid sequence

[[QGQSGQG][LCHPLVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSDNPGGSDIVM TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 465)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614_2012 (SEQ ID NO: 468)] Amino acid sequence

[QGQSGQG][LCHPLVLSAWESCSSGGGSSGGSISSGLLSGRSANPGGGSDIVMTQSPL SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC] (SEQ ID NO: 467)

[spacer (SEQ ID NO: 305)] [HuCD166Lc1_7614_2012 VL domain (SEQ ID NO: 470)]

Amino acid sequence

- 126 041958

[QGQSGQG][LCHPLVLSAWESCSSGGGSSGGSISSGLLSGRSANPGGGSDIVMTQSPL

SLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSG

TDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 469)

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614_3012 (SEQ ID NO: 472)] Amino acid sequence

[[QGQSGQG][LCHPLVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSANPGGSDIVM

TQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVSFTHQSS ID4]

[spacer (SEQ ID NO: 305)] [huCD166Lc1_7614_3012 VL domain (SEQ ID NO: 474)]

Amino acid sequence

[[QGQSGQG][LCHPLVLSAWESCSSGGGSSGGSAVGLLAPPGGLSGRSANPGGSDIVM

TQSPLSLPPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS

GSGSGTDFTLKISRVEEAEDVGVYYCAQNLELPYTFGQGTKLEIK] (SEQ ID NO: 473)

Example 3. Obtaining activated antibodies against CD166.

The studies presented herein are designed to generate activated anti-CD166 antibodies as described herein.

Received activated antibodies against CD166 with different efficiency of masking (ie, an indicator of the ability of the MM of the activated antibody to block the binding of the AV activated antibody to its target). Peptides 16522 and 7614 were mutated by truncation and alanine scanning as described in Example 2 and these masking peptide variants were used to generate families of activated anti-CD166 antibodies with a masking fold range. The ability of the anti-CD166CD166 M9 antibody vK1/HcB (VH of SEQ ID NO: 121 and VL of SEQ ID NO: 123) and various activated anti-CD166 antibodies to bind human CD166 was assessed using a CD166 binding ELISA (Fig. 3A, 3B). The CD166 activated antibodies tested contained the heavy chain variable region sequence from SEQ ID NO: 121, the light chain variable region sequence from SEQ ID NO: 123, a cleavable group (CM) containing the amino acid sequence ISSGLLSGRSDNH (SEQ ID NO: 70) designated in herein as substrate 2001, and one of the masking groups shown in table. 10. Full-length sequences are shown above in table. eleven.

Using a standard ELISA protocol, human CD166 protein was absorbed onto ELISA plates and then incubated with the indicated concentration of antibody or activated antibody. Bound antibody or activated antibody was detected using an anti-human FAB secondary antibody with peroxidase. A summary of exemplary apparent dissociation constants (Kd) of in vitro activated antibodies of the present invention from a CD166 polypeptide, as determined by ELISA, is shown in Table 1 below. 17, as well as the corresponding increase in Kd relative to the original anti-CD166 M9 antibody (vk-1/HcB). With the exception of the original anti-CD166 M9 antibody, the mask for each activated antibody is as specified, as described herein, and the substrate sequence was 2001 (ISSGLLSGRSDNH (SEQ ID NO: 70)).

- 127 041958

Table 17

Apparent dissociation constants in an ELISA of activated antibodies against CD166

Antibody design Apparent Kd (nM) Magnification factor Kd CD166 М9 vkl/HcB 4.4 1 GDI 66-16522-2001 423.7 95 GDI 66-7614-2001 998.6 225 GDI 66-7614.5-2001 495.0 112 CD166-7614.6-2001 265.6 60 GDI 66-7614.7-2001 618.6 139 CD166-7614.8-2001 82.2 19 CD166-7614.9-2001 534.0 120 CD166-7614.10-2001 685, 2 154 CD166-7614.11-2001 544.0 123 CD166-7614.12-2001 331.9 75 CD166-7614.13-2001 77.7 18 CD166-16522.14-2001 422.1 95 CD166-16522.15-2001 447.7 101 CD166-16522.16-2001 47.4 eleven CD166-16522.17-2001 61.6 14 CD166-16522.18-2001 57.7 13 CD166-16522.19-2001 49, 2 eleven GDI 66-16522.20-2001 700.3 158 GDI 66-16522.21-2001 144.7 33

Example 4 Activation of Activated Anti-CD166 Antibodies.

The assays presented herein are designed to evaluate the activation of activated antibodies against CD166.

Fig. 4 is a graph showing the ability of various activated anti-CD166 antibodies of this description to bind human CD166 upon proteolytic activation. As shown in FIG. 4, activated anti-CD166 antibodies restore antibody binding upon proteolytic activation. The binding of an anti-CD166 antibody (huM9 HcC/vk-1; VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123), various CD166 activated antibodies as described herein, and uPA activated anti-CD166 antibodies were assessed using a binding ELISA. CD166. Activated anti-CD166 antibodies tested contain the heavy chain variable region sequence from SEQ ID NO: 122, the light chain variable region sequence from SEQ ID NO: 123, or a cleavable group (CM1, substrate 2001) containing the amino acid sequence ISSGLLSGRSDNH (SEQ ID NO: 70 ) or a cleavable group (CM2, substrate 3001) containing the amino acid sequence AVGLLAPPGGLSGRSDNH (SEQ ID NO: 76) and one of the masking groups shown in Table. 10. Full-length sequences are shown above in table. eleven.

Using a standard ELISA protocol, human CD166 protein was absorbed onto ELISA plates and then incubated with the indicated concentration of antibody or activated antibody. Bound antibody or activated antibody was detected using an anti-human FAB secondary antibody with peroxidase. Bound antibody or activated antibody was detected using an anti-human FAB secondary antibody with peroxidase.

In an exemplary study, the binding affinity of an anti-CD166 antibody (anti-CD166 antibody HcC/vk-1 as described herein and an activated anti-CD166 antibody (anti-CD166-7614.6-3001 antibody) as described herein) was determined with human cells (HCC1806 human breast cancer cells). ) and with human CD166. Under these conditions, the apparent illustrative binding affinities (Kd) by ELISA were 96.2 and 1.3 nM for the activated anti-CD166 antibody and the anti-CD166 antibody of the present disclosure, respectively. as measured by flow cytometry, the apparent binding affinities (Kd) were 372 and 3.2 nM for the activated anti-CD166 antibody and the anti-CD166 antibody of the present disclosure, respectively. 128 041958 binding affinity for the isolated CD166 and C polypeptide D166 on cells compared to an anti-CD166 antibody of the present disclosure.

Fig. 5 is a graph showing the ability of various anti-CD166 conjugated activated antibodies of this description to induce HCC1806 cell killing after proteolytic activation using uPA. These conjugated anti-CD166 antibodies are also referred to herein as CD166 AADC or Activatable Antibody Drug Conjugates. The CD166 conjugated activated antibodies tested contained the heavy chain variable region sequence from SEQ ID NO: 122, the light chain variable region sequence from SEQ ID NO: 123, a cleavable group (CM1, substrate 2001) containing the amino acid sequence ISSGLLSGRSDNH (SEQ ID NO: 70 ), one of the masking groups shown in Table. 10 and DM4 maytansinoid conjugated to an activated antibody via an SPDB linker. The full length sequences of the activated antibodies are shown in Table 1 above. 11. All conjugated activated antibodies described herein are from TCRS (The Chemistry Research Solution).

As shown in FIG. 5, various anti-CD166 conjugated activated antibodies of this description act similarly to isotype-DM4 in masking, but after proteolytic activation using uPA, these anti-CD166 conjugated activated antibodies show similar cell killing to huCD166 ADC. The ability of the drug conjugates to kill HCC1806 cells was assessed by adding the indicated concentration of ADC or AADC and incubating the cells for 3 days. Cell viability was measured using the CellTiter Glo assay. Similar results were observed when an activated antibody conjugated to a nucleic acid damaging agent was tested in such cell killing assays.

The following assays are designed to evaluate the activation and binding of activated anti-CD166 antibodies in tumor samples following exposure to one or more proteases.

Tumor xenograft samples and healthy tissue samples were analyzed using the immunohistochemistry (IHC) assay and in situ imaging assay described in PCT Publication No. WO 2014/107599, the entire contents of which are incorporated herein by reference. Briefly, this in situ assay uses an activated anti-CD166 antibody as described herein, the corresponding parent antibody, and a modified version of the activated anti-CD166 antibody in which CM is replaced by a non-cleavable linker, herein referred to generically as modified anti-CD166 antibody NSUB. The original antibody is used as a positive control, while the non-cleavable version of the activated anti-CD166 antibody is used as a negative control. Then, frozen xenograft tissue sections are placed on glass slides, washed twice with PBS-T, then with PBS, followed by tissue pretreatment for 30 min with a broad-spectrum protease inhibitor cocktail or buffer alone. A detectable label, such as Alexa Fluor-680®, is then conjugated to each of the activated anti-CD166 antibody and the modified anti-CD166 NSUB antibody. Then, the detectably labeled, activated anti-CD166 antibody and the modified anti-CD166 NSUB antibody are applied to the tissue and incubated for 1 hour in the dark (to prevent fluorescence fading). After incubation with 1 µg/ml, incubated tumor sections were washed with PBS-T, then with PBS, and counterstained with the nuclear marker DAPI for 1 min. Fluorescence microscopy analysis was then used to detect positive staining. Positive staining of activated anti-CD166 antibody terminated by pretreatment of tissue sections with protease inhibitors indicates that binding of activated anti-CD166 antibody to the tissue sample is the result of a proteolytic event. Positive staining of activated anti-CD166 antibody should also cease when the tissue is pretreated with an excess of unlabeled (cold) parent antibody. Moreover, incubation with tumor tissue should reveal positive staining for the parent antibody, which is not affected by pretreatment of the tissue with protease inhibitors, but which stops when the tissue is pretreated with unlabeled parent antibody. The signal of the modified anti-CD166 NSUB antibody should not be detected when detected in pre-treated tissue, in tissue not pre-treated with protease inhibitors, or in tissue pre-treated with unlabeled parent antibody.

Fig. 6A-6D are series of images showing that activated anti-CD166 antibody is activated (ie, cleaved) in colon cancer tissue samples and activated anti-CD166 antibody is not activated in healthy tissue samples. In FIG. 6A and 6C depict the results of IHC analysis of tumor and healthy tissue samples, and FIG. 6B and 6D depict the results of in situ imaging analysis of tumor and healthy tissue samples.

- 129 041958

Fig. 7A-7D are series of images showing that activated anti-CD166 antibody is activated (ie, cleaved) in lung cancer tissue samples and activated anti-CD166 antibody is not activated in healthy tissue samples. In FIG. 7A and 7C depict the results of IHC analysis of tumor and healthy tissue samples, and FIG. 7B and 7D depict the results of in situ imaging analysis of tumor and healthy tissue samples.

Fig. 8A-8D are a series of images showing that the activated anti-CD166 antibody is not activated in healthy tissue samples. In FIG. 8A and 8C depict the results of IHC analysis of healthy tissue samples, and FIG. 8B and 8D depict the results of an in situ imaging analysis of healthy tissue samples.

Example 5 Activity of Conjugated Antibodies Against CD166.

This example shows that an anti-CD166 conjugate antibody of this specification (CD166 ADC) exhibits in vitro killing activity compared to a control antibody conjugate (control ADC).

Fig. 9 is a series of graphs showing the activity of an anti-CD166 conjugated antibody of this description against a breast cancer cell line, a prostate cancer cell line, a pancreatic cancer cell line, a head and neck squamous cell line, and a CD166 cell line as a negative control.

Example 6 Efficacy of Conjugated Anti-CD166 Activated Antibodies in Tumor Models.

This example demonstrates the efficacy of a CD166 conjugated, activable antibody of this specification (CD166 AADC or Activatable Antibody Drug Conjugates) in various tumor models.

Fig. 10 is a graph showing the efficacy of an AADC containing an activated anti-CD166 antibody of this specification conjugated to DM4 maytansinoid compared to a DM4-conjugated isotype control and an ADC containing a DM4-conjugated variant of the parent antibody for an activated anti-CD166 antibody. Efficacy is measured as mean tumor volume measured at various time points after administration (5 mg/kg IV on days 1 and 8) in a breast cancer model.

As shown in FIG. 10, the performance of the AADC is equivalent to that observed for the ADC.

Fig. 11 is a graph showing the potency of activated anti-CD166 antibodies of this specification compared to a DM4-conjugated isotype control and a DM4-conjugated CD166 ADC parental anti-CD166 antibody. Efficacy is measured as mean tumor volume measured at various time points after administration (5 mg/kg IV on days 1 and 8) in the H292 non-small cell lung cancer (NSCLC) model.

H292 tumor xenografts were treated with the isotype control-DM4, huCD166-DM4 ADC, huCD166_7614.6_CM2-DM4 AADC, or huCD166_7614.6_CM1-DM4 ADDC, where CM1 and CM2 are the 2001 and 3001 substrates described herein, respectively. Tumors were grown to an average volume of 150 mm ³ , mice were then randomly assigned to groups of eight and dosed with the indicated test substances on days 1 and 8. Mean tumor volume±SEM was plotted.

As shown in FIG. 11, the performance of both AADCs tested is equivalent to that observed for the ADCs.

Fig. 12 is a graph showing the potency of CD166 AADC, a DM4-conjugated activated anti-CD166 antibody, also referred to herein as huCD166_7614.6_CM2-DM4 AADC (where CM2 is substrate 3001), compared to a DM4-conjugated isotype control, and CD166 ADC conjugated to DM4 parent anti-CD166 antibody. Efficacy is measured as mean tumor volume measured at various time points after administration (5 mg/kg IV on days 1 and 8) in the H1975 model of non-small cell lung cancer (NSCLC).

As shown in FIG. 12, the performance of the AADC is equivalent to that observed for the ADC.

Example 7 Tolerability Assay of Activated CD166 Antibodies.

Anti-CD166 antibodies of this specification have been characterized by their species specificity for binding to human CD166 and other closely related proteins.

As shown in FIG. 13, anti-CD166 antibodies of this description bind human and cynomolgus monkey CD166 with equal affinity. None of the anti-CD166 antibodies tested as described herein bound to rat or mouse CD166. The Kd for binding of anti-CD166 antibody to human CD166 and cynomolgus CD166 in these exemplary binding assays is 1.3 nM for both.

CD166 expression levels were then analyzed in different types of normal human and cynomolgus monkey tissues. As shown in Table. 3 below, CD166 expression levels were almost identical in

- 130 041958 human and cynomolgus macaque tissue samples analyzed:

Table 3

CD166 Expression Levels in Human and Cynomolgus Macaque Normal Tissue Samples

Fabric type Javanese macaque Human Breast ++ ++ Brain - - Colon ++ ++ Esophagus - - Heart - - Bud + + Liver ++ ++ Lung ++ + Nerve - - Ovary ++ + Pancreas ++ ++ Prostate + + + + + + Leather N/A -/+ Small intestine ++ ++ Salivary gland ++ ++ Spleen - - Stomach + + + + + + Striated/skeletal muscle - - Testicle - - Uterus ++ ++

Significant CD 166 expression was detected in both human and cynomolgus monkey liver tissue samples.

In initial pharmacokinetic studies, conjugated activated anti-CD166 antibodies of this description were found to avoid antigen-dependent clearance (ie rapid clearance of antibodies due to the prevalence of naturally expressed CD166 antigen in the body).

In FIG. 14 shows the results of a tolerability study in cynomolgus monkeys using a 5 mg/kg administration of a CD166 conjugated activated antibody as described. These studies were performed using huCD166 7614.6 CM2-DM4 AADC (where CM2 is substrate 3001). 5 mg/kg was chosen because this is the therapeutic dose used for other DM4 antibody conjugates containing an SPDB bond.

The pharmacokinetics of huCD166-7614.6-CM1 DM4 drug conjugate (where CM1 is the 2001 substrate) and unconjugated anti-huCD166 antibody were evaluated in cynomolgus monkeys after a single dose of 5 or 3 mg/kg, respectively. Total serum levels of human IgG were measured using sandwich ELISA against human IgG. Consistent with elimination by targeting, anti-CD166 AADC showed a significantly stronger effect than antibody. No toxicity was observed in monkeys treated with the huCD166-7614.6-CM1 DM4 drug conjugate. After 21 days, there were no clinical observations, signs of toxicity due to the target and signs of liver toxicity. As shown in FIG. 14, the recovery of anti-CD166 DM4 conjugate was compared with the parent antibody. Antigen binding was below the level of quantitation.

Fig. 15 is a graph showing that the CD166 conjugated activated antibody is well tolerated at the intended therapeutic dose. These studies were performed using huCD166 7614.6 CM2-DM4 AADC (where CM2 is substrate 3001).

Thus, in contrast to conventional ADC therapy, there was no evidence of liver injury in cynomolgus monkeys following administration of the anti-CD166 conjugated, activated antibody of this description.

Example 8 Protease-Dependent Activation of Activated Anti-CD166 Antibodies.

The exemplary assays presented herein are designed to evaluate protease-dependent activation of activated antibodies and conjugated activated antibodies.

- 131 041958 against CD166 according to this description.

Fig. 16A, 16B and 16C are graphs showing the ability of various conjugated anti-CD166 activated antibodies as described herein to bind human CD166 on HCC1806 cells in the presence or absence of protease-dependent activation. These conjugated activated anti-CD166 antibodies are also referred to herein as CD166 AADC or Activatable Antibody Drug Conjugates. As shown in FIG. 16A, anti-CD166 conjugated activated antibodies to CD166 are blocked on HCC1806 cells. In contrast, as shown in FIG. 16B and 16C, conjugated activated anti-CD166 antibodies restore antibody binding activity similar to that of unmasked anti-CD166 conjugated antibody and unmasked anti-CD166 antibody when AADCs are proteolytically activated using matriptase (MT-SP1) or matrix metalloproteinase 14 (MMP-14). .

The binding of an anti-CD166 antibody (VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123), various conjugated and unconjugated anti-CD166 activated antibodies of this description, and protease-activated conjugated activated anti-CD166 antibodies of this description were assessed using a binding assay for based on flow cytometry. The conjugated and non-conjugated CD166 activated antibodies tested contained the heavy chain variable region sequence from SEQ ID NO: 122, the light chain variable region sequence from SEQ ID NO: 123, a cleavable group (CM1, substrate 2001) containing the amino acid sequence ISSGLLSGRSDNH (SEQ ID NO : 70) or a cleavable group (CM2, substrate 3001) containing the amino acid sequence AVGLLAPPGGLSGRSDNH (SEQ ID NO: 76) and one of the masking groups shown in Table. 10. Full-length sequences are shown above in table. 11. Conjugated anti-CD166 antibodies and conjugated activated anti-CD166 antibodies contained DM4 maytansinoid conjugated to the activated antibody via an SPDB linker. In a typical assay, HCC1806 cells were incubated with the indicated concentrations of antibody, activated antibody, conjugated antibody, or activated anti-CD166 antibody in PBS+2% FBS for 1 hour on ice. After washing 2X with PBS + 2% FBS, cells were incubated with secondary goat anti-human IgG conjugated to AlexaFluor 647 (Jackson ImmunoResearch) for 30-45 min on ice. Cells were then washed 2X with PBS+2% FBS and fixed with 1% formaldehyde. Bound antibody was detected using a Guava EasyCyte cytometer and the median fluorescence intensity (MFI) of the cell population was measured.

As shown in FIG. 16A, anti-CD166 conjugated activated antibodies to CD166 are blocked on HCC1806 cells. As shown in FIG. 16B and 16C, various conjugated and unconjugated anti-CD166 antibodies of this description act similarly to each other when masked, but when conjugated activated anti-CD166 antibodies are proteolytically activated using a protease, they show binding similar to that of the unmasked parental huCD166 antibody and the unmasked huCD166ADC.

Example 9 Binding of Activated CD166 Antibodies and Antibody Drug Conjugates to Human Tissues.

The illustrative studies in this example demonstrate the binding properties of anti-CD166 antibody drug conjugates (ADC) and activated anti-CD166 antibody drug conjugates (AADC) of the present disclosure to human tissues.

In this study, frozen tissue sections derived from normal human prostate, ovary, breast, pancreas, and right atrium (cat. nos. T1234201, T1234086, T1234183, T1234188, and T1234127, respectively; BioChain, Newark, CA) were prepared and blocked with using standard protocols, and then incubated with 0.4 μg/ml anti-CD166 ADC of the present description (anti-CD166-spdb-DM4), anti-CD166 AADC of the present description (anti-CD166-7614.6-3001-spdb-DM4), activated anti-CD166 AADCs of the present specification (anti-CD166-7614.6-3001-spdb-DM4, incubated with purified uPA for 16 h at 37°C) or ADC isotype control (chKTI-spdb-DM4, a chimeric human IgG1 antibody against a soybean trypsin inhibitor conjugated to spdb-DM4). After incubation with the test substances, the sections were treated with 2.5 μg/ml mouse anti-DM4 monoclonal antibody (Immunogen). DM4 loading was detected by incubation with an anti-mouse antibody conjugated to an HRP polymer (EnvisionTM+ System-HRP Labeled polymer anti-mouse, Dako, K4006) followed by the addition of the substrate 3,3'-diaminobenzidine (DAB Plus, Dako, K3467) . The tissues were counterstained with hematoxylin and images were obtained on an Olympus VS120 virtual slice scanner.

Illustrative results of this study show that the anti-CD166 AADC of the present specification (anti-CD166-7614.6-3001-spdb-DM4) showed no immunostaining on each of the five tissue sections consistent with their masked state and similar to those observed for the ADC isotype control. . Activation of anti-CD166 AADC as described herein with uPA restored binding of activated anti-CD166 AADC in the two highest expressed tissues, prostate and breast. The intensity and distribution of staining of the uPA-activated anti-CD166 AADC of the present disclosure was similar to the original anti-CD166 antibody drug conjugate (anti-CD166spdb-DM4).

Example 10 Binding of Anti-CD166 Antibodies to Human Cancers.

This example shows that CD166 is expressed in a variety of patient-derived tumors by immunohistochemical (IHC) staining using an anti-CD166 antibody.

In this study, formalin-fixed, paraffin-embedded (FFPE) tumor samples in tissue microarrays (US Biomax) were prepared and blocked using standard protocols, and then incubated with 5 μg/ml of anti-CD166 rabbit monoclonal antibody EPR2759[2] (Abcam, ab109215 ). Detection of anti-CD166 antibody was performed by incubation with 5 μg/ml biotin-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch, 711-065-152) followed by the addition of ABC-HRP Elite Standard (Vector Laboratories, PK-6100) to form an avidin complex. -biotin-HRP followed by the addition of 3,3'-diaminobenzidine substrate (DAB Plus, Dako, K3467). The tissues were counterstained with hematoxylin and images were obtained on an Olympus VS120 virtual slice scanner.

Each nuclear tissue was assessed by IHC score as negative (no staining), mild (intensity 1+ in <70% of tumor cells or 2+ in <30% of tumor cells), moderate (intensity 1+ in >70% of tumor cells or 2+ in >30% -<70% of tumor cells or 3+ in <30% of tumor cells) or strong (intensity of 2+ in >70% of tumor cells or 3+ in >30% of tumor cells), and the percentage of samples tested for which shows moderate or strong IHC staining for antibodies against CD166, shown in table. 4.1 below.

Table 4.1

IHC Analysis of CD166 Expression in Patient-Derived Cancers

Fabric (total number of samples) Type of malignant tumor % of Samples with Moderate or Strong IHC Prostate (119) Adenocarcinoma 98.3 Breast (392) duct carcinoma 87.5 Easy ( 213) Non-small cell cancer lung 83.1 head and (122) neck Squamous carcinoma 81.1 endometrium (147) Adenocarcinoma 75.5 ovaries 129) Adenocarcinoma 70.5 Biliary (177) Cholangiocarcinoma 56.5

Example 11 Binding of anti-CD166 antibodies to human and cynomolgus monkey cells.

The exemplary assays presented herein are designed to evaluate activated anti-CD166 antibodies of this description for binding to human and cynomolgus monkey cells in a flow cytometry assay.

Fig. 17 is a graph showing the ability of anti-CD166 antibodies (antihuCD166) of this description to bind human H292 cells or cynomolgus monkey primary kidney epithelial cells as measured by flow cytometry. As shown in FIG. 17, the anti-CD166 antibodies of the present disclosure show comparable affinity for CD166 on the cell surface of both human and cynomolgus monkey cells.

The binding of an anti-CD166 antibody (huM9 vk-1/HcC; VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123) as described herein to human H292 cells or cynomolgus monkey primary kidney epithelial cells was assessed using a flow-through binding assay. cytometry. In a typical assay, H292 cells or cynomolgus monkey primary kidney epithelial cells were incubated with the indicated concentrations of anti-CD166 antibody in PBS+2%FBS for 1 hour on ice. After washing 2X with PBS + 2% FBS, cells were incubated with secondary goat anti-human IgG conjugated to AlexaFluor 647 (Jackson ImmunoResearch) for 30-45 min on ice. Cells were then washed 2X with PBS + 2% FBS and fixed with 1%

- 133 041958 formaldehyde. Bound antibody was detected using a Guava EasyCyte cytometer and the median fluorescence intensity (MFI) of the cell population was measured.

As shown in FIG. 17, the anti-CD166 antibody of the present disclosure bound to human and cynomolgus monkey cells with comparable affinity (EC ₅₀ 3.1 nM for human cells and EC ₅₀ 1/7 nM for cynomolgus monkey cells).

Example 12 Inhibition of Cell Binding to CD6 by Anti-CD166 Antibodies.

In this exemplary assay, anti-CD166 antibodies of the present disclosure are shown to block human lymphoma cell adhesion to immobilized CD6, the receptor for which CD166 is a ligand.

Fig. 18 is a graph showing the ability of anti-CD166 antibodies (anti-huCD166; vk-1/HcC) of the present disclosure to block the adhesion of HuT-78 human lymphoma cells to immobilized human CD166. In this assay, CD166-expressing HuT-78 cells were fluorescently labeled and incubated with recombinant CD6 protein immobilized on plastic plates. After several washes, bound HuT-78 cells were detected by fluorescence measurement and recorded as a percentage of total fluorescence (before washing). MAB656 is a murine anti-CD166 monoclonal antibody reported to inhibit cell adhesion in this assay (R&D Systems) presumably by interfering with the interaction of CD166 with its receptor, CD6. In this exemplary assay, anti-CD166 antibody of the present disclosure and MAB656 show similar levels of inhibition of cell adhesion to CD6. Both antibodies showed a close EC ₅₀ of approximately 3.7 nM. The antibody isotype control showed no inhibition of cell adhesion.

Example 13 Protease-Dependent Activation of Activated Anti-CD166 Antibodies.

The exemplary assays provided herein are designed to evaluate protease-dependent activation of activated antibodies and anti-CD166 conjugated activated antibodies as described herein.

Fig. 19A is a graph showing in vitro binding to CD166 in an ELISA assay of anti-CD166 antibody (anti-huCD166 HcC/vk-1, CD166 Ab), anti-CD166 antibody drug conjugate (anti-CD166-spdb-DM4, CD166-ADC ), an activated anti-CD166 antibody (anti-CD166-7614.6-3001, CD166 Pb), an activated antibody-drug conjugate (anti-CD166-7614,6-3001-spdb-DM4, CD166-AADC). As indicated, assays were also performed with a protease-activated anti-CD166-activated antibody drug conjugate using urokinase (uPA), matriptase (MT-SP1), or matrix metalloproteinase 14 (MMP14) as indicated. Using a standard ELISA protocol, human CD166 protein was absorbed onto ELISA plates and then incubated with the indicated concentration of antibody. Bound antibody was detected using a horseradish peroxidase-conjugated anti-human IgG secondary antibody.

Fig. 19B and 19C are graphs showing binding to human HCC1806 or H292 cells in flow cytometry analysis of an anti-CD166 antibody (anti-huCD166 HcC/vk-1, CD166 Ab), an anti-CD166 antibody drug conjugate (anti-CD166-spdb- DM4, CD166-ADC), activated anti-CD166 antibody (anti-CD166-7614.6-3001, CD166 Pb), activated antibody-drug conjugate (anti-CD166-7614.6-3001-spdb-DM4, CD166AADC). As indicated, assays were also performed with a protease-activated anti-CD166-activated antibody drug conjugate using urokinase (uPA), matriptase (MT-SP1) or matrix metalloproteinase 14 (MMP14) as indicated. An IgG1 isotype control antibody conjugated to spdb-DM4 (IgG1 isotype-ADC) was also tested as a control. In a typical assay, H292 cells or HCC1806 cells were incubated with the indicated concentrations of anti-CD166 antibody in PBS+2% FBS for 1 hour on ice. After washing 2X with PBS + 2% FBS, cells were incubated with secondary goat anti-human IgG conjugated to AlexaFluor 647 (Jackson ImmunoResearch) for 30-45 min on ice. Cells were then washed 2X with PBS+2% FBS and fixed using 1% formaldehyde. Bound antibody was detected using a Guava EasyCyte cytometer and the median fluorescence intensity (MFI) of the cell population was measured.

- 134 041958

Table 14

Binding of Anti-CD166 Antibody Constructs to CD166 and Human Cells

Antibody design ELISA CD166, Kd (nM) Flow cytometry HCC1806, Kd (nM) Flow cytometry H2 92, Kd (nM)

CD166-AADC 2.9 >300 >200 CD166-AADC, cleaved and RA 0.18 9, 9 5, 5 CD166-AADC cleaved with matriptase 0.17 9, 9 5, 3 CD166-AADC cleaved by MMP-14 0.16 12 6, 9 CD166-ADC 0.15 6, 1 3.3 CD166-Pb 3, 05 >300 >200

As shown by the illustrative results in table. 14, uncleaved CD166-AADC shows an increased apparent Kd compared to unmasked CD166-ADC in ELISA and flow cytometry assays. Activation of CD166-AADC by all three proteases appeared to restore the binding affinity of the activated anti-CD166 antibody drug conjugate to a level comparable to that of unmasked CD166-ADC.

Fig. 20A and 20B are graphs showing in vitro cytotoxicity for human HCC1806 or H292 cells of anti-CD166 antibody (anti-huCD166 HcC/vk-1, CD166 Ab), anti-CD166 antibody drug conjugate (anti-CD166-spdb-DM4, CD166-ADC), activated anti-CD166 antibody (anti-CD166-7614.6-3001, CD166 Pb), activated antibody-drug conjugate (anti-CD166-7614.6-3001-spdb-DM4, CD166-AADC). As indicated, assays were also performed with a protease-activated anti-CD166-activated antibody drug conjugate using urokinase (uPA), matriptase (MT-SP1), or matrix metalloproteinase 14 (MMP14) as indicated. An IgG1 isotype control antibody conjugated to spdb-DM4 (IgG1 isotype-ADC) was also tested as a control. In a typical assay, H292 cells or HCC1806 cells were incubated with the indicated concentrations of antibody, activated antibody, activated antibody drug conjugate, or activated antibody drug conjugate treated with protease, and the cells were incubated for 3-5 days. Cell viability was measured using the CellTiter Glo assay. The results of these cytotoxicity assays are summarized in Table 1 below. 15.

Table 15

In Vitro Cytotoxicity of Anti-CD 166 Antibody Constructs to Human Cells

Antibody design H292 EC50 (nM) HC1806 EC50 (nM) CD166-AADC 2.2 0.9 CD166-AADC cleaved by uPA 0.5 0.3 CD166-AADC cleaved with matriptase 0.5 0.2 CD166-AADC cleaved by MMP-14 0.5 0.2 CD166-ADC 0.3 0, 1

These illustrative results from Table. 15 and FIG. 20A and 20B show that activated anti-CD166 antibody drug conjugates (CD166-AADC) of the present disclosure, proteolytically activated using a substrate sequence (SM)-recognizing protease, show a several-fold increase in cytotoxicity to human cells compared to with corresponding uncleaved anti-CD166 AADCs. In addition, activated anti-CD166 AADCs have shown cytotoxicity comparable to unmasked anti-CD166 ADCs.

- 135 041958

CD166.

Example 14 Immunological risk analysis of activated anti-CD166 antibodies.

The exemplary assays provided herein are designed to evaluate the ability of the activated anti-CD166 antibody drug conjugates (CD166AADC) of the present disclosure to trigger an immunological response in an in vitro assay.

In these illustrative studies, human peripheral blood mononuclear cells (PBMCs) were tested in in vitro assays for cytokine release and proliferation in response to treatment with an activated anti-CD166 antibody drug conjugate (anti-CD1667614.6-3001-spdb-DM4, CD166-AADC ), an anti-CD166 antibody drug conjugate (anti-CD166 (vk-1/HcC)-spdb-DM4, CD166-ADC), a positive control anti-CD3 antibody (OKT3 antibody), and an isotype control antibody drug conjugate (isotype ) in the following concentrations.

no. Antibody (nM) no. Antibody (nM) 1 No processing 10 Isotype (0.67 nM) 2 Anti-SBZ (0.67 nM) eleven Isotype (6.7 nM) 3 Anti-SBZ (6.7 nM) 12 Isotype (67 nM) 4 Anti-SBZ (67 nM) 13 Isotype (670 nM) 5 Anti-SBZ (670 nM) 14 CD166-ADC (0.67 nM) 6 CD166-AADC (0.67 nM) 15 CD166-ADC (6.7 nM) 7 CD166-AADC (6.7 nM) 16 CD166-ADC (67 nM) 8 CD166-AADC (67 nM) 17 CD166-ADC (670 nM) 9 CD166-AADC (670 nM)

In this illustrative study, samples from five normal, healthy donors were evaluated in two separate formats, wet coat and soluble test substance. The level of a standard panel of Th1 and Th2 cytokines, including IL-2, IL-4, IL-6, IL-10, TNFa, and IFNy, was measured 24 hours after treatment. As shown in FIG. 21A-21D, for dose titration of either CD166-AADC or the corresponding CD166-ADC, no significant release of cytokines from human PBMC is shown compared to positive control (anti-CD3, OKT3) and negative control (isotype-ADC) antibodies. In addition, in contrast to the OKT3 positive control antibody, neither CD166-AADC nor CD166-ADC induced PBMC proliferation as assessed on day 8 post-treatment.

Example 16 Antibody-Dependent Cellular Cytotoxicity of Anti-CD166 Conjugated Activated Antibodies.

The exemplary assays provided herein are designed to evaluate in vitro antibody-dependent cellular cytotoxicity (ADCC) for human cells of activated anti-CD166 antibody drug conjugates of the present disclosure.

Fig. 22 is a graph showing an in vitro ADCC assay on human ovarian adenocarcinoma (SKOV3) cells using an activated anti-CD166 antibody drug conjugate of the present disclosure (anti-huCD166 (HcC/vk-1)-7614.6-3001-spdb-DM4 , CD166-AADC), anti-CD166 antibody drug conjugate (anti-CD166-spdbDM4, CD166-ADC), anti-EGFR positive control and IgG1-spdb-DM4 isotype control (IgG1 isotype-ADC)

ADCC activity in this exemplary assay was assessed using the biological ADCC reporter assay (Promega). In this assay, Jurkat T cells stably transfected with FcRyIIIa and NF-AT inducible luciferase reporter were incubated with CD166-expressing SKOV3 cells in the presence of CD166-AADC or control antibodies. Luciferase activity is stimulated as a function of antibody binding to target cells, recruitment of the FcRYIIIa receptor, and downstream signaling in effector cells. SKOV3 cells expressing high levels of CD166 and EGFR when incubated with CD166-AADC or CD166-ADC showed activity similar to the IgG1 ADC isotype control and lower than that seen with a positive anti-EGFR control. The dose of CD166-AADC or CD166-ADC was titrated in this assay. These data suggest that CD166-AADC, like CD166-ADC, have limited potential for ADCC.

Example 17 Efficacy of Conjugated Activated Anti-CD166 Antibodies Against Cell- and Patient-Derived Tumor Xenograft Models.

The exemplary studies provided herein are designed to evaluate the in vivo efficacy of activated anti-CD166 antibody drug conjugates of the present disclosure against cell-derived and patient-derived xenografts in mouse tumor models.

In these illustrative studies, a variety of cell line-derived and patient-derived xenograft (PDX) models representing different types of cancer were tested.

- 136 041958 internal tumors. Two models are derived from xenograft cell lines (H292 cells of non-small cell lung carcinoma (NSCLC) and HCC1806 cells of triple negative breast carcinoma), and two xenograft models are derived from patients (PDX) for ovarian carcinoma (CTG-0791) and cholangiocarcinoma (CTG-0941) ). For each model, tumor-bearing mice were randomized into groups and treated with an activated anti-CD166 antibody drug conjugate of the present disclosure (anti-huCD166 (HcC/vk-1)-7614.6-3001spdb-DM4, CD166-AADC), isotype control SPDB-DM4 conjugate or vehicle control as indicated. Test substances were administered intravenously on study days 0 and 7 at 3 or 5 mg/kg as indicated.

As shown in FIG. 23, treatment with CD166-AADC, at or below the expected human therapeutic dose, resulted in tumor regression and sustained responses in most mice. Other similar illustrative studies are summarized in Table 1. 16 below.

Table 16

Efficacy of Activated Anti-CD166 Antibody Drug Conjugates

Model tumors Type of malignant tumor Antitumor response to CD166-AADC H292 NSCLC Yes H1975 NSCLC Yes CTG-0166 NSCLC (squamous) Yes NSS1806 Triple negative breast cancer Yes CTG-0791 ovaries Yes CTG-0941 Cholangiocarcinoma Yes

Example 18 In Vitro Cytotoxicity of Conjugated Anti-CD166 Activated Antibodies Against Cell Lines Derived from Endometrial Cancers.

The exemplary studies provided herein are designed to evaluate the in vitro efficacy of the activated anti-CD166 antibody drug conjugates of the present disclosure against cell lines derived from endometrial cancers.

As shown in FIG. 24A-24C, these illustrative studies showed in vitro cytotoxicity for a cell line derived from human endometrial cancers (HEC-1-A, AN3-CA, and KLE cell lines), an anti-CD166 antibody drug conjugate (anti-CD166- spdb-DM4, CD166-ADC) and activated antibody drug conjugates (anti-CD166-7614.6-3001-spdb-DM4, CD166-7614.6-AADC; and anti-CD166-7614.8-3001-spdb-DM4, CD166-7614.8 -AADC). An ADC isotype control (chKTI-spdb-DM4) was used as a control. In a typical assay, cells are incubated with the indicated concentrations of antibody drug conjugate or activated antibody drug conjugate and the cells are incubated for 3-5 days. Cell viability was measured using the CellTiter Glo assay. These exemplary results indicated that anti-CD166-AADC and anti-CD166-ADC of the present disclosure showed cytotoxicity against all cell lines compared to the negative control. These illustrative results also showed that uncleaved anti-CD166-AADCs showed lower cytotoxicity than anti-CD166-ADCs as described herein due to their lower target affinity due to the masking substrate. The relative sensitivity of the cell lines to the anti-CD166 agents of the present disclosure seemed to correlate with the level of CD166 expression in each cell. As shown in FIG. 24D, to determine the relative amount of CD166 expressed on the surface of each endometrial cell line, flow cytometry analysis was performed using anti-CD166 antibodies of the present disclosure.

Example 18 In Vitro Cytotoxicity of Activated CD166 Antibody Drug Conjugates Against Cell Lines Derived from Various Cancers.

The exemplary studies provided herein are designed to evaluate the in vitro efficacy of the activated anti-CD166 antibody drug conjugates of the present disclosure against cell lines derived from endometrial cancers.

In these exemplary studies, the in vitro cytotoxicity of anti-CD166 antibody drug conjugates (anti-CD166 (vk-1/HcC)-spdb-DM4, CD166-ADC) was tested against cell lines derived from a variety of cancers. In a typical assay, cells were incubated for 3-5 days with CD166-ADC at various concentrations (0.1 to 50 nM). life

- 137 041958 cell capacity was measured using CellTiter Glo assay. The cytotoxicity of CD166-ADC was compared with a negative isotype control (chKTI-spdb-DM4). For the PC3 prostate cancer cell line and the head and neck squamous cell carcinoma SAS cell line, the test substance was anti-CD166-vc-MMAD and the negative control was the palivizumab-vc-MMAD isotype control. The results of these cytotoxicity assays are summarized in Table 1 below. 8.

Table 8

In Vitro Cytotoxicity of Anti-CD166 Activated Antibody-Drug Conjugates Against Human Cancer Cells

cell type Type of malignant tumor Cytotoxicity CD166-ADC? ZR75-1 Human breast ductal carcinoma (estrogen receptor positive) Yes ZR75-30 Human breast ductal carcinoma (estrogen receptor positive) Yes MDA-MB-361 Human breast ductal carcinoma (estrogen receptor positive) Yes NSS1954 Human breast ductal carcinoma (triple negative) Yes NSS1143 Human breast cancer (triple negative) Yes RSH adenocarcinoma of the prostate human Yes SAS Squamous cell carcinoma of the head and neck human Yes

Example 19 In Vivo Imaging of Activated Anti-CD166 Antibodies in a Mouse Lung Cancer Xenograft Model.

This example shows that the activated anti-CD166 antibodies of the present disclosure show tumor-associated protease dependent in vivo activation and binding to CD166 expressed in a lung cancer xenograft (NSCLC) mouse model by in vivo fluorescence imaging.

In FIG. 25A shows in vivo imaging of live mice with lung tumor xenografts (H292 cells) using fluorescently labeled anti-CD166 antibodies and activated anti-CD166 antibodies of the present disclosure. In this study, 7-8 week old female nu/nu mice (n=3) were implanted subcutaneously in the right hind groin with 5 x 10 ⁶ H292 cells, a cell line derived from human non-small cell lung cancer. After tumors have grown to 300-380 mm ³ , anti-CD166 antibody (vk-1/HcC) of the present description (CD166-Ab), activated anti-CD166 antibodies with different masking sequences and substrate sequences of the present description (anti-CD166 -7614.6-3001, anti-CD166-7614.8-2001), or a masked anti-CD166 antibody of the present specification lacking a CM domain (CD166 7614.6-NSUB) was administered as a pre-blocked reagent to each of the mice at a dose of 5 mg/kg. As a control, an isotype control antibody (palivizumab) was administered in a similar manner. 48 hours later, mice were injected with an anti-CD166 antibody labeled with AlexaFluor 750 (anti-CD166-AF750). Mice were subjected to in vivo fluorescence imaging at 24, 72 and 96 hours after anti-CD166-AF750 administration using an excitation signal at 745 nm and detection of an emission signal at 800 nm. Depicted are representative mice visualized 96 hours later. The scale shows the relative amplitude of the detected fluorescent signal. The mean tumor to background ratio (TBR) for each test substance, as measured in mice, is shown in FIG. 25V.

The results of this exemplary study show that, based on the fluorescent signals from the unmasked anti-CD166 antibody of the present disclosure and the activated anti-CD166 antibodies of the present disclosure, they were able to bind CD166 in the xenograft, thus blocking subsequent binding of the fluorescently labeled anti-CD166 antibody. In contrast, the corresponding masked anti-CD166 antibody, but lacking the

- 138 -

Claims (42)

protease (CM) cleavage did not block the subsequent binding of CD166-AF750 to an extent comparable to the isotype control. Without wishing to be bound by any particular theory, this exemplary study has shown that the CD166-activated antibodies of the present disclosure can be activated in vivo by cleavage with a tumor-associated protease, thereby allowing the activated-activated CD166 antibody to bind CD166 in a tumor xenograft to a degree comparable to unmasked anti-CD166 antibody of the present disclosure. A masked anti-CD166 antibody lacking the protease cleavage (CM) domain of the present disclosure was not amenable to activation in the same manner, and thus did not bind significantly to the tumor xenograft. Example 20 Efficacy of conjugated anti-CD166 activated antibodies against cell-derived tumor xenograft models. The exemplary studies presented herein are designed to evaluate the in vivo efficacy of the presently described activated anti-CD166 antibody drug conjugates (AADC) against cell-derived xenografts in mouse tumor models. In these exemplary studies, activated anti-CD166 antibody drug conjugates of the present disclosure were tested for efficacy against mouse tumor xenograft models for lung cancer (H292 non-small cell lung carcinoma (NSCLC) cells). In this study, tumor-bearing mice were treated with anti-CD166 antibody drug conjugates and an activated anti-CD166 antibody drug conjugate (AADC) of the present disclosure, including anti-CD166-7614-2001-spdb-DM4 (CD166-7614-2001-DM4 ), anti-CD166-7614.6-2001-spdb-DM4 (CD166-7614.6-2001-DM4), anti-CD166-7614.8-3001-spdb-DM4 (CD166-7614.8-3001-DM4), anti-huCD166-spdb-DM4 ( CD166-DM4) and an isotype control (palivizumab-spdb-DM4; Isotype-DM4). 5 mg/kg of the test substances were intravenously administered on study days 1 and 8 to each mouse, and the mean tumor volume (MTV) of the H292 xenograft was measured on the indicated days. As shown in FIG. 26, treatment with activated anti-CD166 antibody drug conjugates of the present disclosure resulted in a decrease in MTV over time to a degree comparable to that observed with an unmasked anti-CD166-DM4 drug conjugate. Ahtu-CD166-DM4 AADC, which has a significantly higher masking effect on reducing the binding affinity of the parent antibody to CD166 (see, for example, Table 17), appears to have significantly lower efficacy than anti-CD166 AADCs, which have lower masking. . Other embodiments. While the invention has been described with respect to its detailed description, the foregoing description is intended to be illustrative and not to limit the scope of the invention as defined by the scope of the appended claims. Other aspects, advantages, and modifications are included within the scope of the following. CLAIM 1. An isolated antibody or antigen-binding fragment (AB) thereof that specifically binds to mammalian CD166, where AB specifically binds to human CD166 and cynomolgus monkey CD166, where AB contains the amino acid sequence of complementarity determining region 1 (CDR1) of the heavy chain variable region (VH) GFSLSTYGMGVG (SEQ ID NO: 127); the amino acid sequence of complementarity determining region 2 (CDR2) VH NIWWWSEDKH (SEQ ID NO: 128); the amino acid sequence of complementarity determining region 3 (CDR3) VH IDYGNDYAFTY (SEQ ID NO: 129); the light chain variable region (VL) CDR1 amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131); the amino acid sequence of CDR2 VL QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133); and the amino acid sequence of CDR3 VL AQNLELPYT (SEQ ID NO: 134). 2. An activated antibody that, when activated, binds to mammalian CD166, comprising an antibody or antigen-binding fragment (AB) thereof, that specifically binds to mammalian CD166, where AB specifically binds to human CD166 and cynomolgus CD166, which contains the amino acid sequence CDR1 VH GFSLSTYGMGVG (SEQ ID NO: 127), CDR2 amino acid sequence VH NIWWSEDKH (SEQ ID NO: 128), amino acid sequence CDR3 VH IDYGNDYAFTY (SEQ ID NO: 129), amino acid sequence - 139 041958 CDR1 VL RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131), CDR2 VL amino acid sequence QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133), and CDR3 VL amino acid sequence AQNLELPYT (SEQ ID NO: 134); a masking group (MM) that inhibits AB binding to mammalian CD166 if the antibody to be activated is in an uncleaved state; and a cleavable group (CM) attached to AB, where CM is a polypeptide that acts as a substrate for the protease. 3. The activated antibody of claim 2, wherein the MM has a dissociation constant for binding to AB that is higher than the dissociation constant of AB from mammalian CD166. 4. An activated antibody according to claim 2 or 3, wherein the MM does not interfere with or compete with AB for binding to mammalian CD166 if the activated antibody is in a cleaved state. 5. An activated antibody according to any one of claims 2 to 4, wherein MM is a polypeptide no more than 40 amino acids in length. 6. An activated antibody according to any one of claims 2 to 5, wherein the MM polypeptide sequence differs from the human CD166 sequence. 7. An activated antibody according to any one of claims 2-6, wherein the MM polypeptide sequence is not more than 50% identical to any natural AB binding partner. 8. An activated antibody according to any one of claims 2-7, wherein SM is a substrate for a protease that is active in diseased tissue. 9. An activated antibody according to any one of claims 2-8, wherein the AB is attached to the CM. 10. An activated antibody according to any one of claims 2-9, wherein the AB is directly attached to the CM. 11. An activated antibody according to any one of claims 2-10, wherein the AB is attached to the CM via a binding peptide. 12. An activated antibody according to any one of claims 2-11, wherein the MM is attached to the CM such that the activated antibody in the uncleaved state contains the following structural arrangement from the N-terminus to the C-terminus: MM-CM-AB or AB-CM- MM. 13. An activated antibody according to any one of claims 2-12, wherein the activated antibody comprises a binding peptide between MM and CM. 14. An activated antibody according to any one of claims 2-13, wherein the activated antibody comprises a binding peptide between CM and AB. 15. An activated antibody according to any one of paragraphs.2-14, where the activated antibody contains the first binding peptide (LP1) and the second binding peptide (LP2); and where the activated antibody in the uncleaved state has the following structural arrangement from the N-terminus to the C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM; where the two binding peptides do not have to be identical to each other; and/or where each of LP1 and LP2 is a peptide of approximately 1-20 amino acids in length. 16. Activated antibody according to any one of paragraphs.2-15, containing a heavy chain containing the amino acid sequence of SEQ ID NO: 121, 122 or 239; and a light chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 123-126, 242, 244, 246, 248, 303, 310, 312, 314, 316 and 363-474. 17. An activated antibody according to any one of claims 2-16, where MM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 135-238; And The CM contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-87 and 318-358. 18. An activated antibody according to claim 17, where MM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 219-238; And The SM contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-87 and 336-358. 19. Conjugated activated antibody, which in the activated state binds to CD166 of a mammal, containing an activated antibody according to any one of paragraphs.2-18; and an AB-conjugated agent, wherein the agent is a toxin, or a fragment thereof, or a detectable group. 20. A conjugated antibody, or a conjugated activated antibody, comprising an antibody according to claim 1 conjugated to an agent, or an activated antibody according to claim 19, where (i) when the agent is a toxin or a fragment thereof, the agent is selected from the group consisting of an inhibitor microtubules, nucleic acid damaging agent, dolastatin or a derivative thereof, auristatin or a derivative thereof, maytansinoid or a derivative thereof, duocarmycin or a derivative thereof, calicheamicin or a derivative thereof, pyrrolobenzodiazepine or a derivative thereof - 140 041958 derivative, auristatin E or its derivative, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), monomethylauristatin D (MMAD), maytansinoid selected from the group consisting of DM1 and DM4, maytansinoid DM4, maytansinoid DM1, duocarmycin, pyrrolebenzodiazepine and a dimer of pyrrolobenzodiazepine; or (ii) when the agent is a detectable group, the detectable group is a diagnostic agent. 21. An isolated antibody or an antigen-binding fragment thereof according to claim 1, or an activated antibody according to any one of claims 2-18, or a conjugated antibody or a conjugated activated antibody according to any one of claims 19 or 20, where its antigen-binding fragment is selected from the group consisting of from Fab fragment, F(ab')2 fragment, scFv and scAb. 22. An isolated antibody or an antigen-binding fragment thereof according to claim 1, or an activated antibody according to any one of claims 2-18, or a conjugated antibody or a conjugated activated antibody according to any one of claims 19 or 20, where AB contains a heavy chain variable region containing an amino acid sequence of SEQ ID NO: 121 or 122, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 123-126. 23. An isolated antibody or antigen-binding fragment thereof according to claim 1, or an activated antibody according to any one of claims 2-18, or a conjugated antibody or a conjugated activated antibody according to any one of claims 19 or 20, wherein the AB specifically binds to human CD166. 24. An activated antibody according to any one of claims 2-18 or 21-23, or a conjugated antibody or a conjugated activated antibody according to any one of claims 19-23, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing an amino acid sequence selected from a group consisting of SEQ ID NOs: 121 and 122, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 123-126, 363-370, 373, 374, 377, 378, 381, 382 385 386 389 390 393 394 397 398 401 402 405 406 409 410 413 414 417 418 421 422 425 426 429 430 433, 434, 437, 438, 441, 442, 445, 446, 449, 450, 453, 454, 457, 458, 461, 462, 465, 466, 469, 470, 473 and 474. 25. An activated antibody according to any one of claims 2-18, 23 or 24 or a conjugated antibody or a conjugated activated antibody according to any one of claims 19-23, where (i) the activated antibody contains a combination of amino acid sequences, where the combination of amino acid sequences is selected from ( a) an AB heavy chain that contains amino acid sequences from VH CdR sequences containing SEQ ID NOs: 127, 128 and 129, (b) an AB light chain that contains amino acid sequences from VL CdR sequences containing SEQ ID NOs: 130, 132 and 134, (c) MM which contains an amino acid sequence from masking sequences (MM) selected from the group consisting of SEQ ID NOS: 219, 222, 224 and 230, and (d) CM which contains an amino acid sequence from substrate sequences (CM) selected from the group consisting of SEQ ID NOs: 21, 32, 39, 44, 52, 61, 70, 71, 76, 87, 318 and 338-358; and/or (ii) the activated antibody comprises a combination of amino acid sequences, where for a given combination of amino acid sequences (a), the AB heavy chain comprises amino acid sequences from the VH sequence of SEQ ID NO: 121 or 122 or the VH CDR sequences containing SEQ ID NO: 127 , 128 and 129, (b) the AB light chain contains amino acid sequences from the VL sequence of SEQ ID NOs: 123, 124, 125, or 126, or VL CDR sequences containing either SEQ ID NOs: 130, 132, and 134, or SEQ ID NO: 131, 133, or 134, (c) MM contains an amino acid sequence from masking sequences (MM) selected from the group consisting of SEQ ID NOs: 219-225 and 227-238, and (d) CM contains an amino acid sequence from substrate sequences (SM) selected from the group consisting of SEQ ID NOs: 18, 20, 21, 24, 32, 39-41, 44, 51, 52, 54, 59-71, 76 and 318-358; and/or (iii) the activated antibody comprises a heavy chain containing an amino acid sequence from SEQ ID NOs: 121, 122, or 239 and a light chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 123-126, 242, 244, 246, 248, 303, 310, 312, 314, 316 and 363-474. 26. A conjugated antibody comprising (a) an antibody or antigen-binding fragment (AB) thereof that specifically binds to mammalian CD166, wherein the AB contains (i) the amino acid sequence of CDR1 VH GFSLSTYGMGVG (SEQ ID NO: 127), the amino acid sequence of CDR2 VH NIWWSEDKH ( SEQ ID NO: 128), CDR3 amino acid sequence VH IDYGNDYAFTY (SEQ ID NO: 129), CDR1 amino acid sequence VL RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131), amino acids- - 141 041958 a CDR2 VL sequence of QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133) and a CDR3 VL amino acid sequence of AQNLELPYT (SEQ ID NO: 134), or (ii) a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 121 and 122, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 123-126, 363-370, 373, 374, 377, 378 , 381, 382, 385, 386, 389, 390, 393, 394, 397, 398, 401, 402, 405, 406, 409, 410, 413, 414, 417, 418, 421, 422, 425, 426, 429 or (iii ) a heavy chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 121, 122 and 239, and a light chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 123-126, 242, 244, 246, 248, 303, 310, 31 2, 314, 316 and 363-474; and (b) an AB conjugated agent, wherein the agent is selected from the group consisting of auristatin E, monomethylauristatin F (MMAF), monomethylauristatin E (MMAE), monomethylauristatin D (MMAD), maytansinoid DM4, maytansinoid DM1, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, and duocarmycin. 27. A conjugated, activated antibody that, when activated, binds to mammalian CD166, comprising an antibody or antigen-binding fragment (AB) thereof that specifically binds to mammalian CD166, wherein AB specifically binds to human CD166 and cynomolgus CD166; a masking group (MM) that inhibits the binding of AB to mammalian CD166 if the antibody to be activated is in an uncleaved state; a cleavable group (CM) attached to AB, where CM is a polypeptide that acts as a substrate for the protease; and an AB conjugated agent, wherein the AB contains (i) CDR1 VH amino acid sequence GFSLSTYGMGVG (SEQ ID NO: 127), CDR2 VH amino acid sequence NIWWSEDKH (SEQ ID NO: 128), CDR3 VH amino acid sequence IDYGNDYAFTY (SEQ ID NO: 129 ), CDR1 VL amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 130) or RSSQSLLHSNGITYLY (SEQ ID NO: 131), CDR2 VL amino acid sequence QMSNLAS (SEQ ID NO: 132) or QMSNRAS (SEQ ID NO: 133), and CDR3 VL amino acid sequence AQNLELPYT (SEQ ID NO: 134); or (ii) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 121 and 122 and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 123-126 , 363-370,373,374,377,378,381,382,385,386,389,390,393,394,397,398,401,402,405,406,409,410,413,414,417 , 418, 421, 422, 425, 426, 429, 430, 433, 434, 437, 438, 441, 442, 445, 446, 449, 450, 453, 454, 457, 458, 461, 462, 465, 466 , 469, 470, 473 and 474; or (iii) a heavy chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 121, 122 and 239 and a light chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 123-126, 242 , 244, 246, 248, 303, 310, 312, 314, 316 and 363-474; and wherein the agent is selected from the group consisting of auristatin E, monomethylauristatin F (MMAF), monomethylauristatin E (MMAE), monomethylauristatin D (MMAD), maytansinoid DM4, maytansinoid DM1, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer and duocarmycin. 28. An activated antibody according to any one of paragraphs 2-18 or 21-23 or an activated antibody according to any one of paragraphs 19-25, where MM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 135-238, or selected from the group consisting of SEQ ID NOs: 219-238, or selected from the group consisting of SEQ ID NOs: 18-87 and 318-358, or selected from the group consisting of SEQ ID NOs: 70-87 and 336-358 . 29. The conjugated antibody or conjugated activated antibody of any one of claims 19-28, wherein the agent is conjugated to AB via a linker, wherein the linker by which the agent is conjugated to AB contains an SPDB group, a vc group, or a PEG2-VC group; and/or wherein the linker and the AB-conjugated toxin comprises an SPDB-DM4 group, a vc-MMAD group, a vc-MMAE group, a vc-duocarmycin group, or a PEG2-VC-MMAD group; or where the linker is a cleavable linker or a non-cleavable linker. 30. A conjugated activated antibody or a conjugated antibody comprising an antibody or antigen-binding fragment (AB) thereof that, when activated, binds to mammalian CD166; and a toxin conjugated to AB via a linker, wherein the conjugated activated antibody or conjugated antibody contains amino acid sequences, a linker, and a toxin selected from - 142 041958 (a) AB, which contains a heavy chain containing the amino acid sequence of the heavy chain or the sequence of the variable domain of the heavy chain, containing SEQ ID NO: 122 or 239; (b) an AB that contains a light chain comprising a light chain amino acid sequence or a light chain variable domain sequence selected from the group consisting of SEQ ID NOs: 123, 240, 242, 244, 246, 252, 303, 310, 312, 314, 316, 363-370, 387-402, 427-442 and 451-474; and (c) a linker and a toxin which comprise a linker and a toxin selected from the group consisting of vcMMAD, PEG2-vc-MMAD, vc-MMAE, vc-duocarmycin and spdb-DM4. 31. A pharmaceutical composition comprising an antibody according to any one of claims 1 or 21, an activated antibody according to any one of claims 2-18, 21-25, 27 or 28 or a conjugated antibody or a conjugated activated antibody according to any one of claims 19-30 and carrier. 32. A pharmaceutical composition according to claim 31, containing an additional agent, where the additional agent is a drug. 33. An isolated nucleic acid molecule encoding an isolated antibody or antigen-binding fragment thereof according to any one of claims 1 and 23-25. 34. An isolated nucleic acid molecule encoding an activated antibody according to any one of claims 2-18, 21-25, 27 and 28. 35. A vector containing an isolated nucleic acid molecule according to claim 33 or 34. 36. A method for producing an antibody or an activated antibody by culturing a cell under conditions for expression of the antibody or an activated antibody, wherein the cell contains the nucleic acid molecule according to claim 33 or 34 or the vector according to claim 35. 37. A method of making an activated antibody that binds to mammalian CD166 in an activated state, wherein the method comprises (a) culturing a cell containing a nucleic acid construct that encodes an activated antibody under conditions that allow expression of the activated antibody, wherein the activated antibody comprises an activated antibody according to any of claims 2-18, 21-25 or 28; and (b) isolating the activated antibody. 38. The method of claim 37, which further comprises conjugating the agent with an isolated, activated antibody, wherein the agent is a toxin, or a fragment thereof, or a detectable group; and/or where (i) the agent is a microtubule inhibitor; or (ii) the agent is a nucleic acid damaging agent; or (iii) the agent is selected from the group consisting of dolastatin or a derivative thereof, auristatin or a derivative thereof, maytansinoid or a derivative thereof, duocarmycin or a derivative thereof, calicheamicin or a derivative thereof, and pyrrolobenzodiazepine or a derivative thereof; or (iv) the agent is auristatin E or a derivative thereof, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), monomethylauristatin D (MMAD), maytansinoid selected from the group consisting of DM1 and DM4, maytansinoid DM4, maytansinoid DM1, duocarmycin , pyrrolebenzodiazepine or pyrrolobenzodiazepine dimer; and/or (v) the agent is a detectable group; and/or (vi) the detectable group is a diagnostic agent. 39. A method of treating, alleviating a symptom, or delaying the progression of a disorder or disease associated with cells expressing mammalian CD166, comprising administering to a patient a therapeutically effective amount of an antibody according to any one of claims 1 or 21, an activated antibody according to any one of claims 2-18, 21 -25 and 28, a conjugated antibody or a conjugated activated antibody according to any one of claims 19-30 or a pharmaceutical composition according to claim 31 or 32 to the patient. 40. The method of claim 39, wherein the disorder or disease is a disorder or disease in which the affected cells express mammalian CD166. 41. The method of claim 39 or 40, wherein (i) the disorder or disease is a cancer, wherein the cancer is adenocarcinoma, bile duct cancer, bladder cancer, bone cancer, breast cancer, endometrial cancer, carcinoid cancer. tumor, cervical cancer, cholangiocarcinoma, colorectal cancer, colon cancer, glioma, head and neck cancer, leukemia, liver cancer, lung cancer, non-small cell lung cancer, lymphoma, melanoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer metastatic castration-resistant prostate carcinoma, kidney cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, genitourinary cancer, or urothelial cancer. 42. The method of claim 41 wherein the breast cancer is Her2 negative breast cancer, triple negative breast cancer, or estrogen receptor positive breast cancer. - 143 -