Anti-CD71 antibody, activatable anti-CD71 antibody, and method of use thereof.

Abstract

To provide antibodies that bind CD71, activatable antibodies that specifically bind to CD71 and methods of making and using these anti-CD71 antibodies and anti-CD71 activatable antibodies in a variety of therapeutic, diagnostic and prophylactic indications.SOLUTION: The present invention discloses an isolated antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD71, where the AB specifically binds human CD71 and cynomolgus monkey CD71.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Related Applications This application claims the benefit under the provisions of U.S. Provisional Patent Application No. 62 / 156,838, filed on May 4, 2015; U.S. Provisional Patent Application No. 62 / 257,321, filed on November 19, 2015; U.S. Provisional Patent Application No. 62 / 257,484, filed on November 19, 2015; U.S. Provisional Patent Application No. 62 / 277,775, filed on January 12, 2016; U.S. Provisional Patent Application No. 62 / 310,553, filed on March 18, 2016; and U.S. Provisional Patent Application No. 62 / 315,276, filed on March 30, 2016. The entire content of each of these is hereby incorporated by reference into this specification.

[0002] Field of the Invention The present invention generally relates to antibodies that bind to CD71, activatable antibodies that specifically bind to CD71, and methods of making and using these anti-CD71 antibodies and anti-CD71 activatable antibodies in various therapeutic, diagnostic, and prophylactic applications.

Background Art

[0003] Background of the Invention Antibody-based therapies have been found to be effective treatments for some diseases, but in some cases, their therapeutic effectiveness is limited due to toxicity caused by widespread target expression. Additionally, antibody-based therapies have shown other limitations, such as being rapidly cleared from the circulation after administration.

[0004] In the field of small molecule therapies, strategies have been developed to provide prodrugs of active chemical entities. Such prodrugs are administered in a relatively inactive form (or a form with very low activity). Once administered, the prodrug is metabolized in vivo to the active compound. Such prodrug strategies can enhance the selectivity of the drug for the intended target and reduce side effects.

[0005] Therefore, in the field of antibody-based therapies, there is still a need for antibodies that mimic the desirable characteristics of small-molecule prodrugs. [Overview of the Initiative]

[0006] This disclosure provides an antibody or its antigen-binding fragment that specifically binds to CD71, also known as transferrin receptor protein 1 (TfR1).

[0007] In some embodiments, the antibody comprises an antibody or its antigen-binding fragment that specifically binds to CD71. In some embodiments, the antibody or its antigen-binding fragment that binds to CD71 is a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, an F(ab')2 fragment, scFv, scAb, dAb, a single-domain heavy chain antibody, or a single-domain light chain antibody. In some embodiments, such an antibody or its antigen-binding fragment that binds to CD71 is a mouse antibody, another rodent antibody, a chimeric antibody, a humanized antibody, or a fully human monoclonal antibody.

[0008] In some embodiments, the antibody or its antigen-binding fragment contains a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3-5.

[0009] In some embodiments, the antibody or its antigen-binding fragment contains a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 6-8.

[0010] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3-5, and a light chain variable region amino acid sequence or its antigen-binding fragment selected from the group consisting of SEQ ID NO: 2 and 6-8.

[0011] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5, and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8.

[0012] In some embodiments, the antibody or its antigen-binding fragment includes 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 SEQ ID NO: 1 and 3-5.

[0013] In some embodiments, the antibody or its antigen-binding fragment includes a light 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 SEQ ID NO: 2 and 6-8.

[0014] In some embodiments, the antibody or its antigen-binding fragment includes 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 SEQ ID NO: 1 and 3-5, and a light 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 SEQ ID NO: 2 and 6-8.

[0015] In some embodiments, the antibody or its antigen-binding fragment includes 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 SEQ ID NO: 3-5, and a light 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 SEQ ID NO: 6-8.

[0016] In some embodiments, the antibody or its antigen-binding fragment comprises a combination of a variable heavy chain complementarity-determining region 1 (VH CDR1, also known herein as CDRH1) sequence, a variable heavy chain complementarity-determining region 2 (VH CDR2, also known herein as CDRH2) sequence, a variable heavy chain complementarity-determining region 3 (VH CDR3, also known herein as CDRH3) sequence, a variable light chain complementarity-determining region 1 (VL CDR1, also known herein as CDRL1) sequence, a variable light chain complementarity-determining region 2 (VL CDR2, also known herein as CDRL2) sequence, and a variable light chain complementarity-determining region 3 (VL CDR3, also known herein as CDRL3) sequence, wherein at least one complementarity-determining region (CDR) sequence is an amino acid sequence VH CDR1 sequence containing TIFF0007887228000001.tif4128; amino acid sequence VH CDR2 sequence containing TIFF0007887228000002.tif4128; amino acid sequence ENWDPGFAF (SEQ ID NO: 11) VH CDR3 sequence containing; amino acid sequence VL CDR1 sequence containing TIFF0007887228000003.tif4128; amino acid sequence STSNLAS (SEQ ID NO: 14) VL CDR2 sequence containing; and amino acid sequence QQRRNYPYT (SEQ ID NO: 15) VL CDR3 sequence including Selected from the group consisting of

[0017] In some aspects, the antibody or its antigen-binding fragment comprises a combination of VH CDR1 sequence, VH CDR2 sequence, VH CDR3 sequence, VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence, wherein at least one CDR sequence is an amino acid sequence VH CDR1 sequences containing TIFF0007887228000004.tif4128 and sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical; amino acid sequences VH CDR2 sequences containing TIFF0007887228000005.tif4128 and sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical; amino acid sequences ENWDPGFAF (SEQ ID NO: 11) VH CDR3 sequences containing a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR3 sequence containing a sequence containing a VH CDR3 VL CDR1 sequences containing TIFF0007887228000006.tif4128 and sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical; amino acid sequences STSNLAS (SEQ ID NO: 14) VL CDR2 sequences containing a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR2 sequence containing a sequence containing a VL CDR2 QQRRNYPYT (SEQ ID NO: 15) VL CDR3 sequences containing a VL CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR3 sequence containing a Selected from the group consisting of

[0018] In some aspects, the antibody or its antigen-binding fragment comprises a combination of VH CDR1 sequence, VH CDR2 sequence, VH CDR3 sequence, VL CDR1 sequence, VL CDR2 sequence and VL CDR3 sequence, where the VH CDR1 sequence is an amino acid sequence Includes TIFF0007887228000007.tif4128; VH CDR2 sequence is amino acid sequence Includes TIFF0007887228000008.tif4128; VH CDR3 sequence is amino acid sequence ENWDPGFAF (SEQ ID NO: 11) Includes; VL CDR1 sequence is amino acid sequence Includes TIFF0007887228000009.tif4128; VL CDR2 sequence is amino acid sequence STSNLAS (SEQ ID NO: 14) Includes; and VL CDR3 sequence is amino acid sequence QQRRNYPYT (SEQ ID NO: 15) Includes.

[0019] In some aspects, the antibody or its antigen-binding fragment comprises a combination of VH CDR1 sequence, VH CDR2 sequence, VH CDR3 sequence, VL CDR1 sequence, VL CDR2 sequence and VL CDR3 sequence, where the VH CDR1 sequence is an amino acid sequence Contains a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007887228000010.tif4128; VH CDR2 sequence is an amino acid sequence Contains a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007887228000011.tif4128; VH CDR3 sequence is an amino acid sequence ENWDPGFAF (SEQ ID NO: 11) It includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR1 sequence of the amino acid sequence Contains a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007887228000012.tif4128; VL CDR2 sequence is an amino acid sequence STSNLAS (SEQ ID NO: 14) It includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR3 sequence of the amino acid sequence QQRRNYPYT (SEQ ID NO: 15) Includes sequences that are identical to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0020] In some embodiments, the antibody or its antigen-binding fragment is encoded by a nucleic acid sequence comprising 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: 1 and 3-5.

[0021] In some embodiments, the antibody or its antigen-binding fragment is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a light chain amino acid sequence containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 6-8.

[0022] In some embodiments, the antibody or its antigen-binding fragment is encoded by a nucleic acid sequence comprising 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: 1 and 3-5, and a nucleic acid sequence encoding a light chain amino acid sequence containing an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 6-8.

[0023] In some embodiments, the antibody or its antigen-binding fragment is encoded by a nucleic acid sequence comprising 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: 3 to 5, and a nucleic acid sequence encoding a light chain amino acid sequence containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8.

[0024] In some embodiments, the antibody or its antigen-binding fragment is encoded by a nucleic acid sequence that is 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 an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3-5.

[0025] In some embodiments, the antibody or its antigen-binding fragment is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence encoding a light chain amino acid sequence containing an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 6-8.

[0026] In some embodiments, the antibody or its antigen-binding fragment is encoded by a nucleic acid sequence that is 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 an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3-5, and a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence encoding a light chain amino acid sequence containing an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 6-8.

[0027] In some embodiments, the antibody or its antigen-binding fragment is encoded by a nucleic acid sequence that is 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 an amino acid sequence selected from the group consisting of SEQ ID NO: 3-5, and a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence encoding a light chain amino acid sequence containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6-8.

[0028] The Disclosure also provides a method for producing an antibody of the Disclosure by culturing cells containing the nucleic acid molecule or vector of the Disclosure under conditions that result in the expression of the antibody.

[0029] In some embodiments, an antibody or its antigen-binding fragment is incorporated into a multispecific antibody or its antigen-binding fragment, where at least one arm of the multispecific antibody or its antigen-binding fragment specifically binds to CD71. In some embodiments, an antibody or its antigen-binding fragment is incorporated into a bispecific antibody or its antigen-binding fragment, where at least one arm of the bispecific antibody or its antigen-binding fragment specifically binds to CD71.

[0030] In some embodiments, at least one arm of a multispecific antibody or its antigen-binding fragment, for example, a bispecific antibody or its antigen-binding fragment, includes a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3-5.

[0031] In some embodiments, at least one arm of a multispecific antibody or its antigen-binding fragment, for example, a bispecific antibody or its antigen-binding fragment, includes a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 6-8.

[0032] In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, includes a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3-5, and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 6-8.

[0033] In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, includes a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5 and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8.

[0034] In some embodiments, at least one arm of a multispecific antibody or its antigen-binding fragment, for example, a bispecific antibody or its antigen-binding fragment, includes 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 SEQ ID NO: 1 and 3-5.

[0035] In some embodiments, at least one arm of a multispecific antibody or its antigen-binding fragment, for example, a bispecific antibody or its antigen-binding fragment, includes a light 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 SEQ ID NO: 2 and 6-8.

[0036] In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, includes 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 SEQ ID NO: 1 and 3-5, and a light 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 SEQ ID NO: 2 and 6-8.

[0037] In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, includes 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 SEQ ID NO: 3-5, and a light 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 SEQ ID NO: 6-8.

[0038] In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, includes a combination of a variable heavy chain complementarity-determining region 1 (VH CDR1, also known herein as CDRH1) sequence, a variable heavy chain complementarity-determining region 2 (VH CDR2, also known herein as CDRH2) sequence, a variable heavy chain complementarity-determining region 3 (VH CDR3, also known herein as CDRH3) sequence, a variable light chain complementarity-determining region 1 (VL CDR1, also known herein as CDRL1) sequence, a variable light chain complementarity-determining region 2 (VL CDR2, also known herein as CDRL2) sequence, and a variable light chain complementarity-determining region 3 (VL CDR3, also known herein as CDRL3) sequence, wherein at least one complementarity-determining region (CDR) sequence is an amino acid sequence VH CDR1 sequence containing TIFF0007887228000013.tif4128; amino acid sequence VH CDR2 sequence containing TIFF0007887228000014.tif4128; amino acid sequence ENWDPGFAF (SEQ ID NO: 11) VH CDR3 sequence containing; amino acid sequence VL CDR1 sequence containing TIFF0007887228000015.tif4128; amino acid sequence STSNLAS (SEQ ID NO: 14) VL CDR2 sequence containing; and amino acid sequence QQRRNYPYT (SEQ ID NO: 15) VL CDR3 sequence including Selected from the group consisting of

[0039] In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, comprises a combination of VH CDR1 sequence, VH CDR2 sequence, VH CDR3 sequence, VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence, where at least one CDR sequence is an amino acid sequence VH CDR1 sequences containing TIFF0007887228000016.tif4128 and sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical; amino acid sequences VH CDR2 sequences containing TIFF0007887228000017.tif4128 and sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical; amino acid sequences ENWDPGFAF (SEQ ID NO: 11) VH CDR3 sequences containing a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR3 sequence containing a sequence containing a VH CDR3 VL CDR1 sequences containing TIFF0007887228000018.tif4128 and sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical; amino acid sequences STSNLAS (SEQ ID NO: 14) VL CDR2 sequences containing a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR2 sequence containing a sequence containing a VL CDR2 QQRRNYPYT (SEQ ID NO: 15) VL CDR3 sequences containing a VL CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR3 sequence containing a Selected from the group consisting of

[0040] In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, comprises a combination of VH CDR1 sequence, VH CDR2 sequence, VH CDR3 sequence, VL CDR1 sequence, VL CDR2 sequence and VL CDR3 sequence, where VH CDR1 sequence is an amino acid sequence Includes TIFF0007887228000019.tif4128; VH CDR2 sequence is amino acid sequence Includes TIFF0007887228000020.tif4128; VH CDR3 sequence is amino acid sequence ENWDPGFAF (SEQ ID NO: 11) Includes; VL CDR1 sequence is amino acid sequence Includes TIFF0007887228000021.tif4128; VL CDR2 sequence is amino acid sequence STSNLAS (SEQ ID NO: 14) Includes; and VL CDR3 sequence is amino acid sequence QQRRNYPYT (SEQ ID NO: 15) Includes.

[0041] In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, comprises a combination of VH CDR1 sequence, VH CDR2 sequence, VH CDR3 sequence, VL CDR1 sequence, VL CDR2 sequence and VL CDR3 sequence, where VH CDR1 sequence is an amino acid sequence Contains a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007887228000022.tif4128; VH CDR2 sequence is an amino acid sequence Contains a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007887228000023.tif4128; VH CDR3 sequence is an amino acid sequence ENWDPGFAF (SEQ ID NO: 11) It includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR1 sequence of the amino acid sequence Contains a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007887228000024.tif4128; VL CDR2 sequence is an amino acid sequence STSNLAS (SEQ ID NO: 14) It includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR3 sequence of the amino acid sequence QQRRNYPYT (SEQ ID NO: 15) Includes sequences that are identical to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0042] In some embodiments, at least one arm of a multispecific antibody or its antigen-binding fragment, for example, a bispecific antibody or its antigen-binding fragment, includes or contains a heavy chain or heavy chain variable region containing or derived from a heavy chain amino acid sequence or heavy chain variable region amino acid sequence shown in Table 12. In some embodiments, at least one arm of a multispecific antibody or its antigen-binding fragment, for example, a bispecific antibody or its antigen-binding fragment, includes or contains a light chain or light chain variable region containing or derived from a light chain amino acid sequence or light chain variable region amino acid sequence shown in Table 12. In some embodiments, at least one arm of a multispecific antibody or its antigen-binding fragment, for example, a bispecific antibody or its antigen-binding fragment, includes or contains a heavy chain or heavy chain variable region amino acid sequence containing or derived from a heavy chain amino acid sequence or heavy chain variable region amino acid sequence shown in Table 12, and a light chain or light chain variable region amino acid sequence containing or derived from a light chain amino acid sequence or light chain variable region amino acid sequence shown in Table 12.

[0043] In some embodiments, at least one arm of a multispecific antibody or its antigen-binding fragment, for example, a bispecific antibody or its antigen-binding fragment, includes 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 heavy chain variable region sequences shown in Table 12. In some embodiments, at least one arm of a multispecific antibody or its antigen-binding fragment, for example, a bispecific antibody or its antigen-binding fragment, includes an 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 light chain variable region sequences shown in Table 12. In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, includes an 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 heavy chain variable region sequences shown in Table 12, and a light 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 light chain variable region sequences shown in Table 12.

[0044] In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, includes a combination of a variable heavy chain complementarity determination region 1 (VH CDR1, also known herein as CDRH1) sequence, a variable heavy chain complementarity determination region 2 (VH CDR2, also known herein as CDRH2) sequence, a variable heavy chain complementarity determination region 3 (VH CDR3, also known herein as CDRH3) sequence, a variable light chain complementarity determination region 1 (VL CDR1, also known herein as CDRL1) sequence, a variable light chain complementarity determination region 2 (VL CDR2, also known herein as CDRL2) sequence, and a variable light chain complementarity determination region 3 (VL CDR3, also known herein as CDRL3) sequence, where at least one CDR sequence is the VH CDR1 sequence shown in Table 13; the VH CDR2 sequence shown in Table 13; the VH CDR3 sequence shown in Table 13; the VL The sequence is selected from the group consisting of CDR1 sequences; VL CDR2 sequences shown in Table 13; and VL CDR3 sequences shown in Table 13.

[0045] In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, comprises a combination of VH CDR1 sequence, VH CDR2 sequence, VH CDR3 sequence, VL CDR1 sequence, VL CDR2 sequence and VL CDR3 sequence, where at least one CDR sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR1 sequence shown in Table 13; a VH CD2 sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR2 sequence shown in Table 13; and the VH CDR1 sequence shown in Table 13. VH CDR3 sequences containing sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the CDR3 sequence; VL CDR1 sequences containing sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR1 sequences shown in Table 13; VL CDR2 sequences containing sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR2 sequences shown in Table 13; and VL CDR1 sequences shown in Table 13 The selection is made from a group of VL CDR3 sequences that include sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the CDR3 sequence.

[0046] In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, comprises a combination of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences, where the combination is a combination of the six CDR sequences (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) shown in a single column of Table 13.

[0047] In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, includes a light chain variable region comprising a combination of VL CDR1, VL CDR2, and VL CDR3 sequences, where the combination is a combination of the three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) shown in a single column of Table 13.

[0048] In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, includes a heavy chain variable region comprising a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, where the combination is a combination of the three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in a single column of Table 13.

[0049] In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, comprises a combination of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences, where each CDR sequence in the combination is 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 (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) shown in a single column of Table 13.

[0050] In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, includes a heavy chain variable region comprising a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, where each CDR sequence in the combination is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the corresponding CDR sequence in the combination of three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in a single column of Table 13.

[0051] In some embodiments, a multispecific antibody or its antigen-binding fragment, for example, at least one arm of a bispecific antibody or its antigen-binding fragment, includes a light chain variable region comprising a combination of VL CDR1, VL CDR2, and VL CDR3 sequences, where each CDR sequence in the combination is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the corresponding CDR sequence in the combination of three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) shown in a single column of Table 13.

[0052] Preferred anti-CD71 antibodies of this disclosure also include an antibody or an antigen-binding fragment that binds to the same epitope as an anti-CD71 antibody, which comprises a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3-5 and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 6-8 on human CD71 and / or cynomolgus monkey CD71.

[0053] The preferred anti-CD71 antibodies of this disclosure also have an amino acid sequence on human CD71 and / or cynomolgus monkey CD71. VH CDR1 sequence containing TIFF0007887228000025.tif4128; amino acid sequence VH CDR2 sequence containing TIFF0007887228000026.tif4128; amino acid sequence ENWDPGFAF (SEQ ID NO: 11) VH CDR3 sequence containing; amino acid sequence VL CDR1 sequence containing TIFF0007887228000027.tif4128; amino acid sequence STSNLAS (SEQ ID NO: 14) VL CDR2 sequence containing; and amino acid sequence QQRRNYPYT (SEQ ID NO: 15) VL CDR3 sequence including It contains an antibody or its antigen-binding fragment that binds to the same epitope as the anti-CD71 antibody.

[0054] Preferred anti-CD71 antibodies of this disclosure also include antibodies or antigen-binding fragments that cross-compete with anti-CD71 antibodies comprising a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3-5, and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 6-8, for binding to human CD71 and / or cynomolgus monkey CD71.

[0055] The preferred anti-CD71 antibodies of this disclosure also have an amino acid sequence for binding to human CD71 and / or cynomolgus monkey CD71. VH CDR1 sequence containing TIFF0007887228000028.tif4128; amino acid sequence VH CDR2 sequence containing TIFF0007887228000029.tif4128; amino acid sequence ENWDPGFAF (SEQ ID NO: 11) VH CDR3 sequence containing; amino acid sequence VL CDR1 sequence containing TIFF0007887228000030.tif4128; amino acid sequence STSNLAS (SEQ ID NO: 14) VL CDR2 sequence containing; and amino acid sequence QQRRNYPYT (SEQ ID NO: 15) VL CDR3 sequence including It contains an antibody or its antigen-binding fragment that cross-competes with an anti-CD71 antibody.

[0056] In some embodiments, the anti-CD71 antibody of the present disclosure comprises an isolated antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB specifically binds to human CD71 and cynomolgus monkey CD71. In some embodiments, the antibody or its antigen-binding fragment comprises a VH CDR1 sequence TIFF0007887228000031.tif4128; VH CDR2 sequence TIFF0007887228000032.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000033.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) This includes. In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8.

[0057] In some embodiments, the isolated antibody or its antigen-binding fragment is on human CD71 and / or cynomolgus monkey CD71, the VH CDR1 sequence. TIFF0007887228000034.tif4128; VH CDR2 sequence TIFF0007887228000035.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000036.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) The isolated antibody or its antigen-binding fragment binds to the same epitope as the isolated antibody, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 3-5 and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 6-8 on human CD71 and / or cynomolgus monkey CD71. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab fragments, F(ab')2 fragments, scFv, scAb, dAb, single-domain heavy chain antibodies, and single-domain light chain antibodies. In some embodiments, the antibody or its antigen-binding fragment binds specifically to human CD71.

[0058] In some embodiments, the isolated antibody or its antigen-binding fragment is VH CDR1 sequence TIFF0007887228000037.tif4128; VH CDR2 sequence TIFF0007887228000038.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000039.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) It cross-competes with isolated antibodies containing the following. In some embodiments, isolated antibodies or their antigen-binding fragments cross-compete with isolated antibodies containing a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3-5 and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6-8.

[0059] In some embodiments, the antibody or its antigen-binding fragment is conjugated to an active agent. In some embodiments, the isolated antibody or its antigen-binding fragment is conjugated to a VH CDR1 sequence. TIFF0007887228000040.tif4128; VH CDR2 sequence TIFF0007887228000041.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000042.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) It cross-competes with isolated antibodies containing the following. In some embodiments, isolated antibodies or their antigen-binding fragments cross-compete with isolated antibodies containing a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3-5 and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6-8. In some embodiments, the active substance is a toxin or a fragment thereof. In some embodiments, the active substance is a microtubule inhibitor. In some embodiments, the active substance is a nucleic acid damaging active substance. In some embodiments, the active substance is selected from the group consisting of drastatin or its derivatives, auristatin or its derivatives, meitansinoids or their derivatives, duocalmycin or its derivatives, calicheamycin or its derivatives, and pyrrolobenzodiazepines or their derivatives. In some embodiments, the active substance is auristatin E or its derivatives. In some embodiments, the active substance is monomethyl auristatin E (MMAE). In some embodiments, the active substance is monomethyl auristatin D (MMAD). In some embodiments, the active substance is a meitansinoid selected from the group consisting of DM1 and DM4. In some embodiments, the active substance is the meitansinoid DM4. In some embodiments, the active substance is duocalmycin. In some embodiments, the active substance is conjugated to AB via a linker. In some embodiments, the linker conjugating the active substance to AB comprises an SPDB portion, a vc portion, or a PEG2-vc portion. In some embodiments, the linker and the toxin conjugated to AB comprise an SPDB-DM4 portion, a vc-MMAD portion, a vc-MMAE portion, a vc-duocalmycin, or a PEG2-vc-MMAD portion. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the active substance is a detectable portion. In some embodiments, the detectable portion is a diagnostic active substance.

[0060] In some embodiments, the conjugated antibody is (a) an antibody or antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB is (i) a VH CDR1 sequence TIFF0007887228000043.tif4128; VH CDR2 sequence TIFF0007887228000044.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000045.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) (ii) an antibody or its antigen-binding fragment (AB) comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8; (b) a conjugated antibody comprising an active substance conjugated to AB, selected from the group consisting of auristatin E, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), meitansinoid DM4, meitansinoid DM1, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, and duocalmycin.

[0061] This disclosure also provides an activatable antibody comprising an antibody or antigen-binding fragment that specifically binds to CD71, coupled to a masking moiety (MM), such that the coupling of the MM reduces the ability of the antibody or antigen-binding fragment to bind to CD71. In some embodiments, the MM is coupled via a sequence comprising a substrate of a protease, e.g., a protease that is active in the affected tissue and / or a protease that co-localizes with CD71 at the treatment site of the subject. The activatable anti-CD71 antibody provided herein, also interchangeably referred to herein as anti-CD71 activatable antibody or CD71 activatable antibody, is stable in circulating blood, is activated at the treatment and / or diagnostic site of the subject but not in normal, e.g., healthy tissue or other tissue not targeted for treatment and / or diagnosis, and, once activated, exhibits binding to CD71 at least comparable to that of the corresponding unmodified antibody, also referred herein as the parent antibody.

[0062] The present invention also provides a method for treating, preventing, and / or delaying the onset or progression of, or alleviating, symptoms associated with abnormal expression and / or activity of CD71 in a subject, using an activatable antibody that binds to CD71, in particular an activatable antibody that binds to and neutralizes CD71, or otherwise inhibits at least one of the biological activity and / or CD71-mediated signaling pathways of CD71.

[0063] The present invention also provides a method for treating, preventing, and / or delaying the onset or progression of, or alleviating, symptoms in a subject that are associated with the presence, growth, proliferation, metastasis and / or activity of cells expressing or abnormally expressing CD71, using an activatable antibody that binds to CD71, in particular an activatable antibody that binds to, targets, neutralizes, kills, or otherwise inhibits at least one biological activity of cells expressing or abnormally expressing CD71.

[0064] The present invention also provides a method for treating, preventing, and / or delaying the onset or progression of, or alleviating, symptoms in a subject that are associated with the presence, growth, proliferation, metastasis, and / or activity of CD71-expressing cells, using activatable antibodies that bind to CD71, in particular activatable antibodies that bind to, target, neutralize, kill, or otherwise inhibit at least one biological activity of CD71-expressing cells.

[0065] The present invention also provides a method for treating, preventing, and / or delaying the onset or progression of, or alleviating, symptoms in a subject that are associated with the presence, growth, proliferation, metastasis, and / or activity of cells that abnormally express CD71, using an activatable antibody that binds to CD71, in particular an activatable antibody that binds to, targets, neutralizes, kills, or otherwise inhibits at least one biological activity of cells that abnormally express CD71.

[0066] In its activated state, the activatable antibody binds to CD71 and comprises (i) an antibody or antigen-binding fragment (AB) that specifically binds to CD71, (ii) a masking moiety (MM) that inhibits the binding of AB to CD71 when the activatable antibody is in an uncleaved state, and (c) a cleavable moiety (CM) coupled to AB, wherein the CM is a polypeptide that functions as a substrate for a protease.

[0067] In some embodiments, the activatable antibody, in its uncleaved state, has a structural configuration such as MM-CM-AB or AB-CM-MM from the N-terminus to the C-terminus.

[0068] In some embodiments, the activatable antibody includes a linked peptide between MM and CM.

[0069] In some embodiments, the activatable antibody includes a linked peptide between CM and AB.

[0070] In some embodiments, the activatable antibody comprises a first linked peptide (LP1) and a second linked peptide (LP2), which, in the uncleaved state, have a structural arrangement such as MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM from the N-terminus to the C-terminus. In some embodiments, the two linked peptides do not need to be identical to each other.

[0071] In some embodiments, at least one of LP1 or LP2 is The amino acid sequence is selected from the group consisting of TIFF0007887228000046.tif4159, and n is at least an integer of 1.

[0072] In some embodiments, at least one of LP1 or LP2 is It contains an amino acid sequence selected from the group consisting of TIFF0007887228000047.tif18145.

[0073] In some aspects, LP1 is an amino acid sequence Includes TIFF0007887228000048.tif18139.

[0074] In some embodiments, LP2 is an amino acid sequence Includes TIFF0007887228000049.tif10141.

[0075] In some embodiments, AB has a dissociation constant of about 100 nM or less for binding to mammalian CD71. In some embodiments, AB has a dissociation constant of about 10 nM or less for binding to mammalian CD71. In some embodiments, AB has a dissociation constant of about 5 nM or less for binding to CD71. In some embodiments, AB has a dissociation constant of about 1 nM or less for binding to CD71. In some embodiments, AB has a dissociation constant of about 0.5 nM or less for binding to CD71. In some embodiments, AB has a dissociation constant of about 0.1 nM or less for binding to CD71. In some aspects, AB has shown binding to mammalian CD71 in the following ranges: 0.01 nM~100 nM, 0.01 nM~10 nM, 0.01 nM~5 nM, 0.01 nM~1 nM, 0.01~0.5 nM, 0.01 nM~0.1 nM, 0.01 nM~0.05 nM, 0.05 nM~100 nM, 0.05 nM~10 nM, 0.05 nM~5 nM, 0.05 nM~1 nM, 0.05~0.5 nM, 0.05 nM~0.1 nM, 0.1 nM~100 nM, 0.1 nM~10 nM, 0.1 nM~5 nM, 0.1 nM~1 nM, 0.1~0.5 nM, 0.5 nM~100 nM, 0.5 nM~10 It has a dissociation constant of nM, 0.5 nM to 5 nM, 0.5 nM to 1 nM, 1 nM to 100 nM, 1 nM to 10 nM, 1 nM to 5 nM, 5 nM to 100 nM, 5 nM to 10 nM, or 10 nM to 100 nM.

[0076] In some embodiments, the activatable antibody comprises an antibody or its antigen-binding fragment (AB) that specifically binds to CD71. In some embodiments, the antibody or its antigen-binding fragment that binds to CD71 is a monoclonal antibody, a domain antibody, a single-chain antibody, a Fab fragment, an F(ab')2 fragment, scFv, scAb, dAb, a single-domain heavy-chain antibody, or a single-domain light-chain antibody. In some embodiments, such an antibody or its antigen-binding fragment that binds to CD71 is a mouse antibody, another rodent antibody, a chimeric antibody, a humanized antibody, or a fully human monoclonal antibody.

[0077] In some embodiments, the uncleaved, activatable antibody specifically binds to mammalian CD71 with dissociation constants of ≤1 nM, ≤5 nM, ≤10 nM, ≤15 nM, ≤20 nM, ≤25 nM, ≤50 nM, ≤100 nM, ≤150 nM, ≤250 nM, ≤500 nM, ≤750 nM, ≤1000 nM, and ≤122 / or ≤2000 nM.

[0078] In some embodiments, the activatable antibody in the non-cleaved state is at a concentration of 1 nM to 2000 nM, 1 nM to 1000 nM, 1 nM to 750 nM, 1 nM to 500 nM, 1 nM to 250 nM, 1 nM to 150 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 25 nM, 1 nM to 15 nM, 1 nM to 10 nM, 1 nM to 5 nM, 5 nM to 2000 nM, 5 nM to 1000 nM, 5 nM to 750 nM, 5 nM to 500 nM, 5 nM to 250 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 25 nM, 5 nM to 15 nM, 5 nM to 10 nM, 10 nM to 2000 nM, 10 nM to 1000 nM, 10 nM to 750 nM, 10 nM to 500 nM, 10 nM to 250 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 25 nM, 10 nM to 15 nM, 15 nM to 2000 nM, 15 nM to 1000 nM, 15 nM to 750 nM, 15 nM to 500 nM, 15 nM to 250 nM, 15 nM to 150 nM, 15 nM to 100 nM, 15 nM to 50 nM, 15 nM to 25 nM, 25 nM to 2000 nM, 25 nM to 1000 nM, 25 nM to 750 nM, 25 nM to 500 nM, 25 nM to 250 nM, 25 nM to 150 nM, 25 nM to 100 nM, 25 nM to 50 nM, 50 nM to 2000 nM, 50 nM to 1000 nM, 50 nM to 750 nM, 50 nM to 500 nM, 50 nM to 250 nM, 50 nM to 150 nM, 50 nM to 100 nM, 100 nM to 2000 nM, 100 nM to 1000 nM, 100 nM to 750 nM, 100 nM to 500 nM, 100 nM to 250 nM, 100 nM to 150 nM, 150 nM to 2000 nM, 150 nM to 1000 nM, 150 nM to 750 nM, 150 nM to 500 nM, 150 nM to 250 nM, 250 nM to 2000 nM, 250 nM to 1000 nM, 250 nM to 750 nM, 250 nM to 500 nM, 500 nM to 2000 nM, 500 nM to 1000 nM, 500 nM to 750It specifically binds to mammalian CD71 with dissociation constants in the ranges of nM, 500 nM~500 nM, 500 nM~250 nM, 500 nM~150 nM, 500 nM~100 nM, 500 nM~50 nM, 750 nM~2000 nM, 750 nM~1000 nM, or 1000 nM~2000 nM.

[0079] In some embodiments, the activated, activatable antibody specifically binds to mammalian CD71 with dissociation constants of 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, or 10 nM or less.

[0080] In some embodiments, the activatable antibody in the activated state is 0.01 nM to 100 nM, 0.01 nM to 10 nM, 0.01 nM to 5 nM, 0.01 nM to 1 nM, 0.01 to 0.5 nM, 0.01 nm to 0.1 nM, 0.01 nm to 0.05 nM, 0.05 nM to 100 nM, 0.05 nM~10 nM, 0.05 nM~5 nM, 0.05 nM~1 nM, 0.05~0.5 nM, 0.05 nM~0.1 nM, 0.1 nM~100 nM, 0.1 nM~10 nM, 0.1 nM~5 nM, 0.1 nM~1 nM, 0.1~0.5 nM, 0.5 nM~100 nM, 0.5 nM~10 nM, 0.5 It specifically binds to mammalian CD71 with dissociation constants in the ranges of nM~5 nM, 0.5 nM~1 nM, 1 nM~100 nM, 1 nM~10 nM, 1 nM~5 nM, 5 nM~100 nM, 5 nM~10 nM, or 10 nM~100 nM.

[0081] In some embodiments, mammalian CD71 is selected from the group consisting of human CD71, mouse CD71, rat CD71, and cynomolgus monkey CD71. In some embodiments, AB specifically binds to human CD71, mouse CD71, or cynomolgus monkey CD71 with a dissociation constant of less than 1 nM. In some embodiments, mammalian CD71 is human CD71.

[0082] In some embodiments, AB has one or more of the following features: (a) AB specifically binds to human CD71; and (b) AB specifically binds to both human CD71 and cynomolgus monkey CD71.

[0083] In some embodiments, AB has one or more of the following characteristics: (a) AB specifically binds to human CD71 and cynomolgus monkey CD71; (b) AB inhibits the binding of transferrin to mammalian CD71; (c) AB inhibits the binding of human transferrin to human CD71; and (d) AB inhibits the binding of cynomolgus monkey transferrin to cynomolgus monkey CD71.

[0084] In some embodiments, AB blocks the ability of innate ligands to bind to mammalian CD71 at EC50 values ​​of 5 nM or less, 10 nM or less, 50 nM or less, 100 nM or less, 500 nM or less, and / or 1000 nM or less. In some embodiments, AB blocks the ability of transferrin to bind to mammalian CD71 at EC50 values ​​of 5 nM or less, 10 nM or less, 50 nM or less, 100 nM or less, 500 nM or less, and / or 1000 nM or less. In some embodiments, the innate ligand for CD71 is transferrin.

[0085] In some aspects, AB blocks the ability of natural ligands to bind to mammalian CD71 at EC50 in the following ranges: 5 nM~1000 nM, 5 nM~500 nM, 5 nM~100 nM, 5 nM~50 nM, 5 nM~10 nM, 10 nM~1000 nM, 10 nM~500 nM, 10 nM~100 nM, 10 nM~500 nM, 50 nM~500 nM, 50 nM~100 nM, 100 nM~1000 nM, 100 nM~500 nM, and 500 nM~1000 nM. In some embodiments, AB blocks the ability of transferrin to bind to mammalian CD71 at EC50 in the following ranges: 5 nM–1000 nM, 5 nM–500 nM, 5 nM–100 nM, 5 nM–50 nM, 5 nM–10 nM, 10 nM–1000 nM, 10 nM–500 nM, 10 nM–100 nM, 10 nM–50 nM, 50 nM–1000 nM, 50 nM–500 nM, 50 nM–1000 nM, 100 nM–1000 nM, 100 nM–500 nM, and 500 nM–1000 nM. In some embodiments, the natural ligand for CD71 is transferrin.

[0086] In some embodiments, ABs of the present disclosure inhibit or reduce the growth, proliferation, and / or translocation of cells expressing mammalian CD71. While not intended to be bound by any theory, ABs of the present disclosure may inhibit or reduce the growth, proliferation, and / or translocation of cells expressing mammalian CD71 by specifically binding to CD71 and inhibiting, blocking, and / or preventing the binding of the native ligand to mammalian CD71. In some embodiments, the native ligand for mammalian CD71 is transferrin.

[0087] In some embodiments, the activatable antibody contains a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3-5.

[0088] In some embodiments, the activatable antibody contains a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 6-8.

[0089] In some embodiments, the activatable antibody comprises a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3-5, and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 6-8.

[0090] In some embodiments, the activatable antibody comprises a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5, and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8.

[0091] In some embodiments, the activatable antibody includes 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 SEQ ID NO: 1 and 3-5.

[0092] In some embodiments, the activatable antibody includes a light 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 SEQ ID NO: 2 and 6-8.

[0093] In some embodiments, the activatable 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 an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3-5, and a light 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 SEQ ID NO: 2 and 6-8.

[0094] In some embodiments, the activatable antibody includes 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 SEQ ID NO: 3-5, and a light 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 SEQ ID NO: 6-8.

[0095] In some embodiments, the activatable antibody comprises a combination of a variable heavy chain complementarity determination region 1 (VH CDR1, also known herein as CDRH1) sequence, a variable heavy chain complementarity determination region 2 (VH CDR2, also known herein as CDRH2) sequence, a variable heavy chain complementarity determination region 3 (VH CDR3, also known herein as CDRH3) sequence, a variable light chain complementarity determination region 1 (VL CDR1, also known herein as CDRL1) sequence, a variable light chain complementarity determination region 2 (VL CDR2, also known herein as CDRL2) sequence, and a variable light chain complementarity determination region 3 (VL CDR3, also known herein as CDRL3) sequence, wherein at least one CDR sequence is an amino acid sequence VH CDR1 sequence containing TIFF0007887228000050.tif4128; amino acid sequence VH CDR2 sequence containing TIFF0007887228000051.tif4128; amino acid sequence ENWDPGFAF (SEQ ID NO: 11) VH CDR3 sequence containing; amino acid sequence VL CDR1 sequence containing TIFF0007887228000052.tif4128; amino acid sequence STSNLAS (SEQ ID NO: 14) VL CDR2 sequence containing; and amino acid sequence QQRRNYPYT (SEQ ID NO: 15) VL CDR3 sequence including Selected from the group consisting of

[0096] In some aspects, the activatable antibody comprises a combination of VH CDR1 sequence, VH CDR2 sequence, VH CDR3 sequence, VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence, where at least one CDR sequence is an amino acid sequence VH CDR1 sequences containing TIFF0007887228000053.tif4128 and sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical; amino acid sequences VH CDR2 sequences containing TIFF0007887228000054.tif4128 and sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical; amino acid sequences ENWDPGFAF (SEQ ID NO: 11) VH CDR3 sequences containing a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR3 sequence containing a sequence containing a VH CDR3 VL CDR1 sequences containing TIFF0007887228000055.tif4128 and sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical; amino acid sequences STSNLAS (SEQ ID NO: 14) VL CDR2 sequences containing a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR2 sequence containing a sequence containing a VL CDR2 QQRRNYPYT (SEQ ID NO: 15) VL CDR3 sequences containing a VL CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR3 sequence containing a Selected from the group consisting of

[0097] In some aspects, the activatable antibody comprises a combination of VH CDR1 sequence, VH CDR2 sequence, VH CDR3 sequence, VL CDR1 sequence, VL CDR2 sequence and VL CDR3 sequence, where VH CDR1 sequence is an amino acid sequence Includes TIFF0007887228000056.tif4128; VH CDR2 sequence is amino acid sequence Includes TIFF0007887228000057.tif4128; VH CDR3 sequence is amino acid sequence ENWDPGFAF (SEQ ID NO: 11) Includes; VL CDR1 sequence is amino acid sequence Includes TIFF0007887228000058.tif4128; VL CDR2 sequence is amino acid sequence STSNLAS (SEQ ID NO: 14) Includes; and VL CDR3 sequence is amino acid sequence QQRRNYPYT (SEQ ID NO: 15) Includes.

[0098] In some aspects, the activatable antibody comprises a combination of VH CDR1 sequence, VH CDR2 sequence, VH CDR3 sequence, VL CDR1 sequence, VL CDR2 sequence and VL CDR3 sequence, where VH CDR1 sequence is an amino acid sequence Contains a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007887228000059.tif4128; VH CDR2 sequence is an amino acid sequence Contains a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007887228000060.tif4128; VH CDR3 sequence is an amino acid sequence ENWDPGFAF (SEQ ID NO: 11) It includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR1 sequence of the amino acid sequence Contains a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TIFF0007887228000061.tif4128; VL CDR2 sequence is an amino acid sequence STSNLAS (SEQ ID NO: 14) It includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR3 sequence of the amino acid sequence QQRRNYPYT (SEQ ID NO: 15) Includes sequences that are identical to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0099] In some embodiments, the AB of the activatable anti-CD71 antibody includes a heavy chain variable region amino acid sequence selected from the group consisting of heavy chain variable region sequences shown in Table 12. In some embodiments, the AB of the activatable anti-CD71 antibody includes a light chain variable region amino acid sequence selected from the group consisting of light chain variable region sequences shown in Table 12. In some embodiments, the AB of the activatable anti-CD71 antibody includes a heavy chain variable region amino acid sequence selected from the group consisting of heavy chain variable region sequences shown in Table 12, and a light chain variable region amino acid sequence selected from the group consisting of light chain variable region sequences shown in Table 12.

[0100] In some embodiments, the AB of the activatable anti-CD71 antibody includes 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 heavy chain variable region sequences shown in Table 12. In some embodiments, the AB of the activatable anti-CD71 antibody includes a light 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 light chain variable region sequences shown in Table 12. In some embodiments, the AB of the activatable anti-CD71 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 an amino acid sequence selected from the group consisting of heavy chain variable region sequences shown in Table 12, and a light 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 light chain variable region sequences shown in Table 12.

[0101] In some embodiments, the activatable antibody comprises a combination of a variable heavy chain complementarity determination region 1 (VH CDR1, also known herein as CDRH1) sequence, a variable heavy chain complementarity determination region 2 (VH CDR2, also known herein as CDRH2) sequence, a variable heavy chain complementarity determination region 3 (VH CDR3, also known herein as CDRH3) sequence, a variable light chain complementarity determination region 1 (VL CDR1, also known herein as CDRL1) sequence, a variable light chain complementarity determination region 2 (VL CDR2, also known herein as CDRL2) sequence, and a variable light chain complementarity determination region 3 (VL CDR3, also known herein as CDRL3) sequence, wherein at least one CDR sequence is one of the VH CDR1 sequences shown in Table 13; VH CDR2 sequences shown in Table 13; VH CDR3 sequences shown in Table 13; VL CDR1 sequences shown in Table 13; VL CDR2 sequences shown in Table 13; The group consisting of VL CDR3 sequences shown in Table 13 is selected.

[0102] In some embodiments, the activatable antibody comprises a combination of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences, where at least one CDR sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR1 sequence shown in Table 13; a VH CDR2 sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR2 sequence shown in Table 13; and the VH CDR1 sequence shown in Table 13. VH CDR3 sequences containing sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the CDR3 sequence; VL CDR1 sequences containing sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR1 sequences shown in Table 13; VL CDR2 sequences containing sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR2 sequences shown in Table 13; and VL CDR1 sequences shown in Table 13 The selection is made from a group of VL CDR3 sequences that include sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the CDR3 sequence.

[0103] In some aspects, the activatable antibody comprises a combination of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences, where the combination is a combination of the six CDR sequences (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) shown in a single column in Table 13.

[0104] In some embodiments, the activatable antibody comprises a heavy chain containing a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, where the combination is a combination of the three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in a single column in Table 13.

[0105] In some embodiments, the activatable antibody comprises a light chain containing a combination of VL CDR1, VL CDR2, and VL CDR3 sequences, where the combination is a combination of the three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) shown in a single column in Table 13.

[0106] In some embodiments, the activatable antibody comprises a combination of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences, where each CDR sequence in the combination is 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 (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) shown in a single column of Table 13.

[0107] In some embodiments, the activatable antibody comprises a heavy chain variable region including a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, where each CDR sequence in the combination is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the corresponding CDR sequence in the combination of three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in a single column of Table 13.

[0108] In some embodiments, the activatable antibody comprises a light chain variable region including a combination of VL CDR1, VL CDR2, and VL CDR3 sequences, where each CDR sequence in the combination is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the corresponding CDR sequence in the combination of three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) shown in a single column of Table 13.

[0109] In some embodiments, MM has a dissociation constant for binding to AB that is greater than the dissociation constant of AB for CD71.

[0110] In some embodiments, MM has a dissociation constant for binding to AB that is less than or equal to the dissociation constant of AB for CD71.

[0111] In some embodiments, MM has a dissociation constant for binding to AB that is smaller than the dissociation constant of AB for CD71.

[0112] In some embodiments, the dissociation constant of MM with respect to AB (K d) is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or less than or greater than the dissociation constant of AB with respect to the target, or 1-5, 5-10, 10-100, 10-10,000, 10-10,000, 10-10,000,000, 10-10,000,000 00, 100~1,000, 100~10,000, 100~100,000, 100~1,000,000, 100~10,000,000, 1,000~10,000, 1,000~100,000, 1,000~1,000,000, 10,000~100,000, 10,000~100,000, 10,000~10,000,000, 100,000~1,000,000, or 100,000~10,000,000 times or more.

[0113] In some embodiments, when the activatable antibody is in a cleaved state, MM does not interfere with or compete with AB for binding to CD71.

[0114] In some embodiments, MM is a polypeptide with a length of approximately 2 to 40 amino acids. In some embodiments, MM is a polypeptide with a length of up to approximately 40 amino acids.

[0115] In some embodiments, the polypeptide sequence of MM differs from the polypeptide sequence of CD71. In some embodiments, the identity of the polypeptide sequence of MM to any natural binding partner of AB is 50% or less. In some embodiments, the polypeptide sequence of MM differs from the polypeptide sequence of CD71, and the identity of the polypeptide sequence to any natural binding partner of AB is 40% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less.

[0116] In some embodiments, the dissociation constant (K) of AB when coupled to MM is given to CD71.d ) is at least twice the K of AB when not coupled to MM with respect to CD71. d By coupling MM to AB such that the dissociation constant (K) of AB with respect to CD71 when coupled to MM is at least twice the K of AB when not coupled to MM with respect to CD71, the ability of AB to bind to CD71 is reduced.

[0117] In some embodiments, the dissociation constant (K) of AB when coupled to MM with respect to CD71 d ) is at least five times the K of AB when not coupled to MM with respect to CD71. d By coupling MM to AB such that the dissociation constant (K) of AB with respect to CD71 when coupled to MM is at least five times the K of AB when not coupled to MM with respect to CD71, the ability of AB to bind to CD71 is reduced.

[0118] In some embodiments, the dissociation constant (K) of AB when coupled to MM with respect to CD71 d ) is at least ten times the K of AB when not coupled to MM with respect to CD71. d By coupling MM to AB such that the dissociation constant (K) of AB with respect to CD71 when coupled to MM is at least ten times the K of AB when not coupled to MM with respect to CD71, the ability of AB to bind to CD71 is reduced.

[0119] In some embodiments, the dissociation constant (K) of AB when coupled to MM with respect to CD71 d ) is at least twenty times the K of AB when not coupled to MM with respect to CD71. d By coupling MM to AB such that the dissociation constant (K) of AB with respect to CD71 when coupled to MM is at least twenty times the K of AB when not coupled to MM with respect to CD71, the ability of AB to bind to CD71 is reduced.

[0120] In some embodiments, the dissociation constant (K) of AB when coupled to MM with respect to CD71 d ) is at least forty times the K of AB when not coupled to MM with respect to CD71. d By coupling MM to AB such that the dissociation constant (K) of AB with respect to CD71 when coupled to MM is at least forty times the K of AB when not coupled to MM with respect to CD71, the ability of AB to bind to CD71 is reduced.

[0121] In some embodiments, the dissociation constant (K) of AB when coupled to MM with respect to CD71 d) is the K of AB when not coupled to MM for CD71. d The coupling of MM to AB reduces AB's ability to bind to CD71 by at least 100 times.

[0122] In some embodiments, the dissociation constant (K) of AB when coupled to MM is given to CD71. d ) is the K of AB when not coupled to MM for CD71. d The coupling of MM to AB reduces AB's ability to bind to CD71 by at least 1000 times.

[0123] In some embodiments, the dissociation constant (K) of AB when coupled to MM is given to CD71. d ) is the K of AB when not coupled to MM for CD71. d The coupling of MM to AB reduces AB's ability to bind to CD71 by at least 10,000 times.

[0124] In some embodiments, when assayed in vitro using a target displacement assay, for example, the assay described in PCT publication number WO2010 / 081173, the ability of AB to bind to CD71 by MM in the presence of CD71 is reduced by at least 90% when CM is not cleaved compared to when CM is cleaved. The contents of WO2010 / 081173 are incorporated herein by reference in their entirety.

[0125] In some embodiments, MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16-295 and 297-314.

[0126] In some embodiments, the CM-cleaving protease is active in the affected tissue, for example, upregulated or otherwise unregulated, and cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease.

[0127] In some embodiments, the protease coexists with CD71 in the tissue and, when the activatable antibody is exposed to the protease, cleaves the CM in the activatable antibody.

[0128] In some embodiments, when the activatable antibody is in an uncleaved state, the binding of the activatable antibody to CD71 is reduced to occur with a dissociation constant at least twice that of the binding of unmodified AB to CD71; however, when it is in a cleaved state (i.e., when the activatable antibody is in a cleaved state), the CM is positioned on the activatable antibody so that AB binds to CD71.

[0129] In some embodiments, when the activatable antibody is in an uncleaved state, the binding of the activatable antibody to CD71 is reduced to a dissociation constant at least five times that of the binding of unmodified AB to CD71; however, when it is in a cleaved state (i.e., when the activatable antibody is in a cleaved state), the CM is positioned on the activatable antibody so that AB binds to CD71.

[0130] In some embodiments, when the activatable antibody is in an uncleaved state, the binding of the activatable antibody to CD71 is reduced to a dissociation constant at least 10 times that of the binding of unmodified AB to CD71; however, when it is in a cleaved state (i.e., when the activatable antibody is in a cleaved state), the CM is positioned on the activatable antibody so that AB binds to CD71.

[0131] In some embodiments, when the activatable antibody is in an uncleaved state, the binding of the activatable antibody to CD71 is reduced to a dissociation constant at least 20 times that of the binding of unmodified AB to CD71; however, when it is in a cleaved state (i.e., when the activatable antibody is in a cleaved state), the CM is positioned on the activatable antibody so that AB binds to CD71.

[0132] In some embodiments, when the activatable antibody is in an uncleaved state, the binding of the activatable antibody to CD71 is reduced to occur with a dissociation constant at least 40 times that of the binding of unmodified AB to CD71; however, when it is in a cleaved state, CM is positioned on the activatable antibody so that AB binds to CD71.

[0133] In some embodiments, when the activatable antibody is in an uncleaved state, the binding of the activatable antibody to CD71 is reduced to occur with a dissociation constant at least 50 times that of the binding of unmodified AB to CD71; however, when it is in a cleaved state, CM is positioned on the activatable antibody so that AB binds to CD71.

[0134] In some embodiments, when the activatable antibody is in an uncleaved state, the binding of the activatable antibody to CD71 is reduced to a dissociation constant at least 100 times that of the binding of unmodified AB to CD71; however, when it is in a cleaved state, CM is positioned on the activatable antibody so that AB binds to CD71.

[0135] In some embodiments, when the activatable antibody is in an uncleaved state, the binding of the activatable antibody to CD71 is reduced to occur with a dissociation constant at least 200 times greater than that of the binding of unmodified AB to CD71; however, when it is in a cleaved state, CM is positioned on the activatable antibody so that AB binds to CD71.

[0136] In some embodiments, CM is a polypeptide with a length of up to 15 amino acids.

[0137] In some embodiments, CM is a polypeptide comprising a first cleavable portion (CM1) which is a substrate for at least one matrix metalloproteinase (MMP) and a second cleavable portion (CM2) which is a substrate for at least one serine protease (SP). In some embodiments, the CM1 and CM2 substrate sequences of the CM1-CM2 substrate are each independently polypeptides up to 15 amino acids in length.

[0138] In some aspects, CM is a substrate of at least one protease that is upregulated or unregulated in cancer, or is thought to be upregulated or unregulated in cancer.

[0139] In some embodiments, CM is a substrate of at least one protease selected from the group consisting of matrix metalloproteinases (MMPs), thrombin, neutrophil elastase, cysteine ​​proteases, legumain, and serine proteases, such as matryptase (MT-SP1), and urokinase (uPA). While not bound by theory, these proteases are thought to be upregulated or unregulated in at least one of the cancers.

[0140] Exemplary substrates include, but are not limited to, substrates that can be cleaved by one or more of the enzymes or proteases listed in Table 4.

[0141] In some embodiments, the CM is selected for use with a specific protease, such as a protease known to coexist with the target of an activatable antibody.

[0142] In some embodiments, CM is a substrate of at least one MMP. Examples of MMPs include those listed in Table 4. In some embodiments, CM is a substrate of a protease selected from the group consisting of MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. In some embodiments, CM is a substrate of MMP9. In some embodiments, CM is a substrate of MMP14.

[0143] In some embodiments, the CM is an array This substrate contains TIFF0007887228000062.tif101147.

[0144] In some embodiments, CM is an amino acid sequence LSGRSDNH (SEQ ID NO: 359) It includes. In some embodiments, CM is an amino acid sequence TGRGPSWV (SEQ ID NO: 356) It includes. In some embodiments, CM is an amino acid sequence PLTGRSGG (SEQ ID NO: 362) It includes. In some embodiments, CM is an amino acid sequence Includes TIFF0007887228000063.tif4128. In some embodiments, CM is an amino acid sequence Includes TIFF0007887228000064.tif4128. In some embodiments, CM is an amino acid sequence Includes TIFF0007887228000065.tif4128. In some embodiments, CM is an amino acid sequence PLGL (SEQ ID NO: 375) It includes. In some embodiments, CM is an amino acid sequence SARGPSRW (SEQ ID NO: 357) It includes. In some embodiments, CM is an amino acid sequence TARGPSFK (SEQ ID NO: 358) It includes. In some embodiments, CM is an amino acid sequence GGWHTGRN (SEQ ID NO: 360) It includes. In some embodiments, CM is an amino acid sequence HTGRSGAL (SEQ ID NO: 361) It includes. In some embodiments, CM is an amino acid sequence AARGPAIH (SEQ ID NO: 363) It includes. In some embodiments, CM is an amino acid sequence RGPAFNPM (SEQ ID NO: 364) It includes. In some embodiments, CM is an amino acid sequence SSRGPAYL (SEQ ID NO: 365) It includes. In some embodiments, CM is an amino acid sequence RGPATPIM (SEQ ID NO: 366) It includes. In some embodiments, CM is an amino acid sequence RGPA (SEQ ID NO: 367) It includes. In some embodiments, CM is an amino acid sequence LSGRSGNH (SEQ ID NO: 789) It includes. In some embodiments, CM is an amino acid sequence SGRSANPRG (SEQ ID NO: 790) It includes. In some embodiments, CM is an amino acid sequence LSGRSDDH (SEQ ID NO: 791) It includes. In some embodiments, CM is an amino acid sequence LSGRSDIH (SEQ ID NO: 792) It includes. In some embodiments, CM is an amino acid sequence LSGRSDQH (SEQ ID NO: 793) It includes. In some embodiments, CM is an amino acid sequence LSGRSDTH (SEQ ID NO: 794) It includes. In some embodiments, CM is an amino acid sequence LSGRSDYH (SEQ ID NO: 795) It includes. In some embodiments, CM is an amino acid sequence LSGRSDNP (SEQ ID NO: 796) It includes. In some embodiments, CM is an amino acid sequence LSGRSANP (SEQ ID NO: 797) It includes. In some embodiments, CM is an amino acid sequence LSGRSANI (SEQ ID NO: 798) It includes. In some embodiments, CM is an amino acid sequence LSGRSDNI (SEQ ID NO: 799) It includes. In some embodiments, CM is an amino acid sequence MIAPVAYR (SEQ ID NO: 800) It includes. In some embodiments, CM is an amino acid sequence RPSPMWAY (SEQ ID NO: 801) It includes. In some embodiments, CM is an amino acid sequence WATPRPMR (SEQ ID NO: 802) It includes. In some embodiments, CM is an amino acid sequence FRLLDWQW (SEQ ID NO: 803) It includes. In some embodiments, CM is an amino acid sequence ISSGL (SEQ ID NO: 804) It includes. In some embodiments, CM is an amino acid sequence ISSGLLS (SEQ ID NO: 805) Includes. In some embodiments, CM is an amino acid sequence and / or ISSGLL (SEQ ID NO: 806) Includes.

[0145] In some embodiments, CM is a substrate for MMP, and sequence Includes TIFF0007887228000066.tif45147.

[0146] In some embodiments, CM is an amino acid sequence ISSGLSS (SEQ ID NO: 376) It includes. In some embodiments, CM is an amino acid sequence QNQALRMA (SEQ ID NO: 377) It includes. In some embodiments, CM is an amino acid sequence AQNLLGMV (SEQ ID NO: 378) It includes. In some embodiments, CM is an amino acid sequence STFPFGMF (SEQ ID NO: 379) It includes. In some embodiments, CM is an amino acid sequence PVGYTSSL (SEQ ID NO: 380) It includes. In some embodiments, CM is an amino acid sequence DWLYWPGI (SEQ ID NO: 381) It includes. In some embodiments, CM is an amino acid sequence ISSGLLSS (SEQ ID NO: 382) It includes. In some embodiments, CM is an amino acid sequence LKAAPRWA (SEQ ID NO: 383) It includes. In some embodiments, CM is an amino acid sequence GPSHLVLT (SEQ ID NO: 384) It includes. In some embodiments, CM is an amino acid sequence LPGGLSPW (SEQ ID NO: 385) It includes. In some embodiments, CM is an amino acid sequence MGLFSEAG (SEQ ID NO: 386) It includes. In some embodiments, CM is an amino acid sequence SPLPLRVP (SEQ ID NO: 387) It includes. In some embodiments, CM is an amino acid sequence RMHLRSLG (SEQ ID NO: 388) It includes. In some embodiments, CM is an amino acid sequence LAAPLGLL (SEQ ID NO: 389) It includes. In some embodiments, CM is an amino acid sequence AVGLLAPP (SEQ ID NO: 390) It includes. In some embodiments, CM is an amino acid sequence LLAPSHRA (SEQ ID NO: 391) It includes. In some embodiments, CM is an amino acid sequence PAGLWLDP (SEQ ID NO: 392) Includes.

[0147] In some embodiments, CM is a substrate of thrombin. In some embodiments, CM is a substrate of thrombin, and the sequence GPRSFGL (SEQ ID NO: 393) or GPRSFG (SEQ ID NO: 394) It includes. In some embodiments, CM is an amino acid sequence GPRSFGL (SEQ ID NO: 393) It includes. In some embodiments, CM is an amino acid sequence GPRSFG (SEQ ID NO: 394) Includes.

[0148] In some forms, CM is It contains an amino acid sequence selected from the group consisting of TIFF0007887228000067.tif31145.

[0149] In some embodiments, CM is an amino acid sequence Includes TIFF0007887228000068.tif4128. In some embodiments, CM is an amino acid sequence Includes TIFF0007887228000069.tif4128. In some embodiments, CM is an amino acid sequence Includes TIFF0007887228000070.tif4128. In some aspects, CM is an amino acid sequence TSGRSANP (SEQ ID NO: 398) It includes. In some embodiments, CM is an amino acid sequence VAGRSMRP (SEQ ID NO: 399) It includes. In some embodiments, CM is an amino acid sequence VVPEGRRS (SEQ ID NO: 400) It includes. In some embodiments, CM is an amino acid sequence ILPRSPAF (SEQ ID NO: 401) It includes. In some embodiments, CM is an amino acid sequence MVLGRSLL (SEQ ID NO: 402) It includes. In some embodiments, CM is an amino acid sequence QGRAITFI (SEQ ID NO: 403) It includes. In some embodiments, CM is an amino acid sequence SPRSIMLA (SEQ ID NO: 404) It includes. In some embodiments, CM is an amino acid sequence SMLRSMPL (SEQ ID NO: 405) Includes.

[0150] In some embodiments, CM is a substrate of neutrophil elastase. In some embodiments, CM is a substrate of serine proteases. In some embodiments, CM is a substrate of uPA. In some embodiments, CM is a substrate of legmaine. In some embodiments, CM is a substrate of matryptase. In some embodiments, CM is a substrate of cysteine ​​proteases. In some embodiments, CM is a substrate of cysteine ​​proteases such as cathepsin.

[0151] In some embodiments, CM is a CM1-CM2 substrate, and sequence Includes TIFF0007887228000071.tif176148.

[0152] In some embodiments, the CM1-CM2 substrate is sequenced It includes TIFF0007887228000072.tif4128, which is also referred to as substrate 2001 in this specification. In some embodiments, the CM1-CM2 substrate is sequence It contains TIFF0007887228000073.tif4128, which is also referred to herein as substrate 1001 / LP' / 0001, where LP' used in this CM1-CM2 substrate is an amino acid sequence GGSGGS (SEQ ID NO: 1037) In some embodiments, the CM1-CM2 substrate is sequenced This includes TIFF0007887228000074.tif4128, which is also referred to herein as substrate 2015 and / or substrate 1004 / LP' / 0003, where LP' used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate is sequence It contains TIFF0007887228000075.tif4128, which is also referred to herein as substrate 0003 / LP' / 1004, where LP' used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate is sequence It contains TIFF0007887228000076.tif4128, which is also referred to herein as substrate 1003 / LP' / 0003, where LP' used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate is sequence It contains TIFF0007887228000077.tif4128, which is also referred to herein as substrate 0003 / LP' / 1003, where LP' used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate is sequence This includes TIFF0007887228000078.tif4128, which is also referred to herein as substrate 3001 and / or substrate 1004 / LP' / 0001, where LP' used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate is sequence It contains TIFF0007887228000079.tif4128, which is also referred to herein as substrate 0001 / LP' / 1004, where LP' used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate is sequence It contains TIFF0007887228000080.tif4128, which is also referred to herein as substrate 1003 / LP' / 0001, where LP' used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate is sequence It contains TIFF0007887228000081.tif4128, which is also referred to herein as substrate 0001 / LP' / 1003, where LP' used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate is sequence It contains TIFF0007887228000082.tif4128, which is also referred to herein as substrate 0001 / LP' / 1001, where LP' used in this CM1-CM2 substrate is an amino acid sequence GGSGGS (SEQ ID NO: 1037) In some embodiments, the CM1-CM2 substrate is sequenced It includes TIFF0007887228000083.tif4128, which is also referred to herein as substrate 0002 / LP' / 1001, where LP' used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 1037) and is. In some embodiments, the CM1-CM2 substrate has the sequence It includes TIFF0007887228000084.tif4128, which is also referred to herein as substrate 1001 / LP' / 0002, where LP' used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 1037) and is. In some embodiments, the CM1-CM2 substrate has the sequence It includes TIFF / 0007887228000085.tif4128, which is also referred to herein as substrate 0001 / LP' / 1002, where LP' used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 1037) and is. In some embodiments, the CM1-CM2 substrate has the sequence It includes TIFF00D7887228000086.tif4128, which is also referred to herein as substrate 1002 / LP' / 0001, where LP' used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 1037) and is. In some embodiments, the CM1-CM2 substrate has the sequence It includes TIFF0007887228000087.tif4128, which is also referred to herein as substrate 0002 / LP' / 1002, where LP' used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 1037)[[ID=2二十七]] and is. In some embodiments, the CM1-CM2 substrate has the sequence Contains TIFF0007887228000088.tif4128, which is also referred to herein as substrate 1002 / LP' / 0002, where LP' used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 1037) and is. In some embodiments, the CM1-CM2 substrate is the sequence Contains TIFF0007887228000089.tif4128, which is also referred to herein as substrate 2002. In some embodiments, the CM1-CM2 substrate is the sequence Contains TIFF0007887228000090.tif4128, which is also referred to herein as substrate 2003. In some embodiments, the CM1-CM2 substrate is the sequence Contains TIFF0007887228000091.tif4128, which is also referred to herein as substrate 2004. In some embodiments, the CM1-CM2 substrate is the sequence <0​​​​​​​​​​It includes TIFF0007887228000097.tif4128, which is also referred to as substrate 2010 in this specification. In some embodiments, the CM1-CM2 substrate is sequence It includes TIFF0007887228000098.tif4128, which is also referred to as substrate 2011 in this specification. In some embodiments, the CM1-CM2 substrate is sequence It includes TIFF0007887228000099.tif4128, which is also referred to as substrate 2012 in this specification. In some embodiments, the CM1-CM2 substrate is sequence It includes TIFF0007887228000100.tif4128, which is also referred to as substrate 2013 in this specification. In some embodiments, the CM1-CM2 substrate is sequence It includes TIFF0007887228000101.tif4128, which is also referred to as substrate 3006 in this specification. In some embodiments, the CM1-CM2 substrate is sequence It includes TIFF0007887228000102.tif4128, which is also referred to as substrate 3007 in this specification. In some embodiments, the CM1-CM2 substrate is sequence It includes TIFF0007887228000103.tif4128, which is also referred to herein as substrate 3008. In some embodiments, the CM1-CM2 substrate is sequence It includes TIFF0007887228000104.tif4128, which is also referred to as substrate 3009 in this specification. In some embodiments, the CM1-CM2 substrate is sequence It includes TIFF0007887228000105.tif4128, which is also referred to as substrate 3010 in this specification. In some embodiments, the CM1-CM2 substrate is sequence It includes TIFF0007887228000106.tif4128, which is also referred to as substrate 3011 in this specification. In some embodiments, the CM1-CM2 substrate is sequence It includes TIFF0007887228000107.tif4128, which is also referred to as substrate 3012 in this specification. In some embodiments, the CM1-CM2 substrate is sequence It includes TIFF0007887228000108.tif4128, which is also referred to as substrate 3013 in this specification. In some embodiments, the CM1-CM2 substrate is sequence It includes TIFF0007887228000109.tif4128, which is also referred to as substrate 2014 in this specification. In some embodiments, the CM1-CM2 substrate is sequence It includes TIFF0007887228000110.tif4128, which is also referred to as substrate 3014 in this specification. In some embodiments, the CM1-CM2 substrate is sequence It contains TIFF0007887228000111.tif4128, which is also referred to herein as substrate 0001 / LP' / 1004, where LP' used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate is sequence This includes TIFF0007887228000112.tif4128, which is also referred to herein as substrate 0001 / LP' / 1003, where LP' used in this CM1-CM2 substrate is the amino acid sequence GG.

[0153] In some embodiments, CM is a substrate of at least two proteases. In some embodiments, each protease is selected from the group of proteases shown in Table 4. In some embodiments, CM is a substrate of at least two proteases, one of which is selected from the group of MMP, thrombin, neutrophil elastase, cysteine ​​protease, uPA, legmine, and matryptase, and the other protease is selected from the group of proteases shown in Table 4. In some embodiments, CM is a substrate of at least two proteases selected from the group of MMP, thrombin, neutrophil elastase, cysteine ​​protease, uPA, legmine, and matryptase.

[0154] In some embodiments, the activatable antibody comprises at least a first CM and a second CM. In some embodiments, the first CM and the second CM are polypeptides with a length of 15 amino acids or less. In some embodiments, the first CM and the second CM in the uncleaved activatable antibody have a structural arrangement such as MM-CM1-CM2-AB or AB-CM2-CM1-MM from the N-terminus to the C-terminus. 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, legmine, and matryptase. In some embodiments, the first CM is cleaved by a first cleavage agent selected from the group consisting of MMP, thrombin, neutrophil elastase, cysteine ​​protease, uPA, legmine, and matryptase, and the second CM is cleaved by a second cleavage agent located in the target tissue. In some embodiments, the other protease is selected from the group of proteases shown in Table 4. In some embodiments, the first and second cleaving agents are the same protease selected from the group of MMP, thrombin, neutrophil elastase, cysteine ​​protease, uPA, regmine, and matryptase, and the first CM and the second CM are different substrates of this enzyme. In some embodiments, the first and second cleaving agents are the same protease selected from the group of proteases shown in Table 4. In some embodiments, the first and second cleaving agents are different proteases. In some embodiments, the first and second cleaving agents coexist in the target tissue. In some embodiments, the first and second CM are cleaved by at least one cleaving agent present in the target tissue.

[0155] In some embodiments, the activatable antibody is exposed to and cleaved by the protease such that, after the protease cleaves the CM, the activated antibody contains a light chain amino acid sequence that includes at least a portion of the LP2 and / or CM sequence in the activated or cleaved state.

[0156] The preferred activatable anti-CD71 antibodies of this disclosure also include an antibody or an antigen-binding fragment thereof that binds to the same epitope as an anti-CD71 antibody comprising a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3-5 and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 6-8 on human CD71 and / or cynomolgus monkey CD71.

[0157] The preferred activatable anti-CD71 antibodies of this disclosure also have an amino acid sequence on human CD71 and / or cynomolgus monkey CD71. VH CDR1 sequence containing TIFF0007887228000113.tif4128; amino acid sequence VH CDR2 sequence containing TIFF0007887228000114.tif4128; amino acid sequence ENWDPGFAF (SEQ ID NO: 11) VH CDR3 sequence containing; amino acid sequence VL CDR1 sequence containing TIFF0007887228000115.tif4128; amino acid sequence STSNLAS (SEQ ID NO: 14) VL CDR2 sequence containing; and amino acid sequence QQRRNYPYT (SEQ ID NO: 15) The antibody or its antigen-binding fragment contains an anti-CD71 antibody that binds to the same epitope as the VL CDR3 sequence containing the VL CDR3 sequence.

[0158] Preferred activatable anti-CD71 antibodies of this disclosure also include an antibody or an antigen-binding fragment thereof that binds to the same epitope as an anti-CD71 antibody comprising a heavy chain variable region amino acid sequence selected from the group consisting of heavy chain variable region sequences shown in Table 12 and a light chain variable region amino acid sequence selected from the group consisting of light chain variable region sequences shown in Table 12 on human CD71 and / or cynomolgus monkey CD71.

[0159] Preferred activatable anti-CD71 antibodies of this disclosure also include an antibody or its antigen-binding fragment that binds to the same epitope as an anti-CD71 antibody comprising a combination of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences on human CD71 and / or cynomolgus monkey CD71, wherein the combination is a combination of the six CDR sequences (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) shown in a single column of Table 13.

[0160] The preferred activatable anti-CD71 antibodies of this disclosure also include an antibody or its antigen-binding fragment that cross-compete with an anti-CD71 antibody comprising a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3-5 and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 6-8 for binding to human CD71 and / or cynomolgus monkey CD71.

[0161] The preferred activatable anti-CD71 antibodies of this disclosure also have an amino acid sequence for binding to human CD71 and / or cynomolgus monkey CD71. VH CDR1 sequence containing TIFF0007887228000116.tif4128; amino acid sequence VH CDR2 sequence containing TIFF0007887228000117.tif4128; amino acid sequence ENWDPGFAF (SEQ ID NO: 11) VH CDR3 sequence containing; amino acid sequence The VL CDR1 sequence containing TIFF0007887228000118.tif4128; amino acid sequence STSNLAS (SEQ ID NO: 14) The VL CDR2 sequence containing; and amino acid sequence QQRRNYPYT (SEQ ID NO: 15) It includes an antibody or an antigen-binding fragment thereof that cross-competes with an anti-CD71 antibody containing a VL CDR3 sequence.

[0162] Suitable activatable anti-CD71 antibodies of the present disclosure also cross-compete for binding to human CD71 and / or cynomolgus CD71, like an anti-CD71 antibody comprising a heavy-chain variable region amino acid sequence selected from the group consisting of the heavy-chain variable region sequences 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. It includes an antibody or an antigen-binding fragment thereof.

[0163] Suitable activatable anti-CD71 antibodies of the present disclosure also cross-compete for binding to human CD71 and / or cynomolgus CD71, like an anti-CD71 antibody comprising a combination of VH CDR1 sequence, VH CDR2 sequence, VH CDR3 sequence, VL CDR1 sequence, VL CDR2 sequence and VL CDR3 sequence. It includes an antibody or an antigen-binding fragment thereof, where the combination is a combination of the six CDR sequences (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3) shown in a single row of Table 13.

[0164] In some embodiments, an activatable anti-CD71 antibody is an antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB specifically binds to human CD71 and cynomolgus monkey CD71; a masking moiety (MM) that inhibits the binding of AB to CD71 when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) coupled to AB, the CM being a polypeptide that functions as a substrate for a protease, wherein the activatable antibody binds to CD71 in an activated state.

[0165] In some embodiments, MM has a dissociation constant greater than that of AB for CD71 for binding to AB. In some embodiments, when the activatable antibody is in a cleaved state, MM does not interfere with or compete with AB for binding to CD71. In some embodiments, MM is a polypeptide with a length of 40 amino acids or less. In some embodiments, the polypeptide sequence of MM is different from the polypeptide sequence of human CD71. In some embodiments, the identity of the MM polypeptide sequence to any natural binding partner of AB is 50% or less. In some embodiments, MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-295 and 297-314. In some embodiments, MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17 and 297-314.

[0166] In some embodiments, CM is a substrate of an active protease in the affected tissue. In some embodiments, CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-423, 680-698, 713, 714, and 789-808. In some embodiments, CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 406-423, 680-698, 713, 714, and 807-808.

[0167] In some embodiments, the activatable antibody comprises its antigen-binding fragment selected from the group consisting of Fab fragments, F(ab')2 fragments, scFv, scAb, dAb, single-domain heavy chain antibodies, and single-domain light chain antibodies. In some embodiments, AB of the activatable antibody specifically binds to human CD71. In some embodiments, AB binds to the VH CDR1 sequence TIFF0007887228000119.tif4128; VH CDR2 sequence TIFF0007887228000120.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000121.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) This includes. In some embodiments, AB includes a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8 and 809 to 908.

[0168] In some embodiments, AB is linked to CM. In some embodiments, AB is directly linked to CM. In some embodiments, AB is linked to CM via a linking peptide. In some embodiments, MM is linked to CM such that the uncleaved activatable antibody has a structural configuration such as MM-CM-AB or AB-CM-MM from the N-terminus to the C-terminus. In some embodiments, the activatable antibody contains a linking peptide between MM and CM. In some embodiments, the activatable antibody contains a linking peptide between CM and AB. In some embodiments, the activatable antibody contains a first linking peptide (LP1) and a second linking peptide (LP2), and the uncleaved activatable antibody has a structural configuration such as MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM from the N-terminus to the C-terminus. In some embodiments, the two linking peptides do not need to be identical to each other. In some embodiments, each of LP1 and LP2 is a peptide about 1 to 20 amino acids long.

[0169] In some embodiments, the activatable antibody includes a heavy chain sequence with SEQ ID NO: 325 or 699 and a light chain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 327, 329, 331, 333, 335, 337, 650, 652, 654, 656, 658, 660, 670-673, 701-712, 721-788, 809-836, and 841-908.

[0170] In some embodiments, the activatable antibody comprises a combination of amino acid sequences, the combination of amino acid sequences selected from a single column in Table D, where for a given combination, (a) the heavy chain of AB comprises the amino acid sequence of a VH CDR sequence corresponding to the given combination in a single column listed in Table D, (b) the light chain of AB comprises the amino acid sequence of a VL CDR sequence corresponding to the given combination in a single column listed in Table D, (c) MM comprises the amino acid sequence of a mask sequence (MM) corresponding to the given combination in a single column listed in Table D, and (d) CM comprises the amino acid sequence of a substrate sequence (CM) corresponding to the given combination in a single column listed in Table D.

[0171] In some embodiments, the activatable antibody comprises a combination of amino acid sequences, where for a given combination of amino acid sequences: (a) the heavy chain of AB comprises an amino acid sequence of a VH sequence or VH CDR sequence selected from the group consisting of VH sequences or VH CDR sequences listed in the corresponding column of Table E; (b) the light chain of AB comprises an amino acid sequence of a VL sequence or VL CDR sequence selected from the group consisting of VL sequences or VL CDR sequences listed in the corresponding column of Table E; (c) MM comprises an amino acid sequence of a mask sequence (MM) selected from the group consisting of MM sequences listed in the corresponding column of Table E; and (d) CM comprises an amino acid sequence of a substrate sequence (CM) selected from the group consisting of CM sequences listed in the corresponding column of Table E.

[0172] In some embodiments, the activatable anti-CD71 antibody comprises an antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, MM, and CM, wherein the activatable antibody comprises a heavy chain sequence with SEQ ID NO: 325 or 699; and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 327, 329, 331, 333, 335, 337, 650, 652, 654, 656, 658, 660, 670-673, 701-712, 721-788, 809-836, and 841-908. In some embodiments, MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 17, and 297-314, and CM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 406-423, 680-698, 713, 714, and 789-808. In some aspects, AB is a VH CDR1 sequence TIFF0007887228000122.tif4128; VH CDR2 sequence TIFF0007887228000123.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000124.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) This includes. In some embodiments, AB includes a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8 and 809 to 908.

[0173] In some embodiments, the activatable anti-CD71 antibody comprises an antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, an MM containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-295 and 297-314, and a CM containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-423, 680-698, 713, 714, and 789-808. In some embodiments, the MM contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, and 297-314, and the CM contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 406-423, 680-698, 713, 714, and 807-808. In some embodiments, the AB is a VH CDR1 sequence TIFF0007887228000125.tif4128; VH CDR2 sequence TIFF0007887228000126.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000127.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) This includes. In some embodiments, AB includes a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8 and 809 to 908.

[0174] In some embodiments, the activatable anti-CD71 antibody comprises an antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB specifically binds to the same human CD71 and / or cynomolgus monkey CD71 epitope as the isolated antibody of the Disclosure; a masking moiety (MM) that inhibits the binding of the AB to CD71 when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) coupled to the AB, which is a polypeptide that functions as a substrate for a protease.

[0175] In some embodiments, the anti-CD71 activatable antibody of the present disclosure comprises an isolated antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB specifically binds to human CD71 and cynomolgus monkey CD71. In some embodiments, the antibody or its antigen-binding fragment comprises a VH CDR1 sequence TIFF0007887228000128.tif4128; VH CDR2 sequence TIFF0007887228000129.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000130.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) This includes. In some embodiments, the activatable antibody or its antigen-binding fragment includes a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8.

[0176] In some embodiments, the activatable anti-CD71 antibody comprises an antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB specifically cross-competes with the isolated antibody of this disclosure for binding to human CD71 and / or cynomolgus monkey CD71; a masking moiety (MM) that inhibits the binding of the AB to CD71 when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.

[0177] In some embodiments, the anti-CD71 activatable antibody of the present disclosure comprises an isolated antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB specifically binds to human CD71 and cynomolgus monkey CD71. In some embodiments, the antibody or its antigen-binding fragment comprises a VH CDR1 sequence TIFF0007887228000131.tif4128; VH CDR2 sequence TIFF0007887228000132.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000133.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) This includes. In some embodiments, the activatable antibody or its antigen-binding fragment includes a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8.

[0178] In some embodiments, the activatable antibody also includes an active agent conjugated to AB. In some embodiments, AB of the active agent conjugated to AB or the activatable antibody is a therapeutic active agent. In some embodiments, the active agent is an antineoplastic active agent. In some embodiments, the active agent is a toxin or a fragment thereof. As used herein, a fragment of a toxin is a fragment that retains toxic activity. In some embodiments, the active agent is conjugated to AB via a cleavable linker. In some embodiments, the active agent is conjugated to AB via a linker comprising at least one CM1-CM2 substrate sequence. In some embodiments, the active agent is conjugated to AB via a non-cleavable linker. In some embodiments, the active agent is conjugated to AB via a linker that is cleavable in an intracellular or lysosomal environment. In some embodiments, the active agent is a microtubule inhibitor. In some embodiments, the active agent is a nucleic acid damaging active agent, such as a DNA alkylating agent, a DNA cleaving agent, a DNA crosslinking agent, a DNA intercalator, or other DNA damaging active agent. In some embodiments, the active substance is an active substance selected from the group listed in Table 5. In some embodiments, the active substance is drastatin. In some embodiments, the active substance is auristatin or a derivative thereof. In some embodiments, the active substance is auristatin E or a derivative thereof. In some embodiments, the active substance is monomethyl auristatin E (MMAE). In some embodiments, the active substance is monomethyl auristatin D (MMAD). In some embodiments, the active substance is a meitansinoid or a meitansinoid derivative. In some embodiments, the active substance is DM1 or DM4. In some embodiments, the active substance is duocalmycin or a derivative thereof. In some embodiments, the active substance is calitiamycin or a derivative thereof. In some embodiments, the active substance is pyrrolobenzodiazepine. In some embodiments, the active substance is a pyrrolobenzodiazepine dimer.

[0179] In some embodiments, an activatable antibody is conjugated to one or more equivalents of a certain active substance. In some embodiments, an activatable antibody is conjugated to one equivalent of a certain active substance. In some embodiments, an activatable antibody is conjugated to two, three, four, five, six, seven, eight, nine, ten, or more than ten equivalents of a certain active substance. In some embodiments, an activatable antibody is part of a mixture of activatable antibodies having a uniform number of equivalents of the multiple conjugated active substances. In some embodiments, an activatable antibody is part of a mixture of activatable antibodies having a non-uniform number of equivalents of the multiple conjugated active substances. In some embodiments, a mixture of activatable antibodies is such that the average number of active substances conjugated to each activatable antibody is 0-1, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, and 10 or more. In some embodiments, the mixture of activatable antibodies is such that the average number of active substances conjugated to each activatable antibody is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0180] In some embodiments, the activatable antibody includes one or more site-specific amino acid sequence modifications such that the number of lysine residues and / or cysteine ​​residues is increased or decreased compared to the original amino acid sequence of the activatable antibody, and therefore, in some embodiments, the number of active substances that can be conjugated to the activatable antibody is increased or decreased accordingly, or in some embodiments, site-specific conjugation of active substances to the activatable antibody is restricted. In some embodiments, the activatable antibody to be modified is site-specifically modified using one or more non-natural amino acids, and therefore, in some embodiments, conjugation of active substances is limited to the sites of non-natural amino acids only.

[0181] In some embodiments, the active substance is an anti-inflammatory substance.

[0182] In some embodiments, the activatable antibody also includes a detectable portion. In some embodiments, the detectable portion is a diagnostic agent.

[0183] In some embodiments, the activatable antibody also includes a signal peptide. In some embodiments, the signal peptide is conjugated to the activatable antibody via a spacer. In some embodiments, the spacer is conjugated to the activatable antibody in the absence of the signal peptide. In some embodiments, the spacer is directly ligated to the MM of the activatable antibody. In some embodiments, the spacer is directly ligated to the MM of the activatable antibody in a structural configuration of spacer-MM-CM-AB from the N-terminus to the C-terminus. An example of a spacer directly ligated to the N-terminus of the MM of the activatable antibody is QGQSGQ (SEQ ID NO: 424). Other examples of spacers directly ligated to the N-terminus of the MM of the activatable antibody include: Examples include TIFF0007887228000134.tif11143 and Q. Other examples of spacers directly ligated to the N-terminus of the MM of an activatable antibody include: Examples include TIFF0007887228000135.tif18140 and G. In some embodiments, the spacer is not ligated to the N-terminus of MM. In some embodiments, the spacer comprises at least the amino acid sequence QGQSGQ (SEQ ID NO: 424). In some embodiments, the spacer comprises at least the amino acid sequence QGQSGQG (SEQ ID NO: 645). In some embodiments, the spacer comprises at least the amino acid sequence QGQSG (SEQ ID NO: 646). In some embodiments, the spacer comprises at least the amino acid sequence QGQS (SEQ ID NO: 647). In some embodiments, the spacer comprises at least the amino acid sequence QGQ (SEQ ID NO: 648). In some embodiments, the spacer comprises at least the amino acid sequence QG (SEQ ID NO: 649). In some embodiments, the spacer comprises at least the amino acid residue Q. In some embodiments, the spacer comprises at least the amino acid sequence GQSGQG (SEQ ID NO: 666). In some embodiments, the spacer includes at least the amino acid sequence QSGQG (SEQ ID NO: 667). In some embodiments, the spacer includes at least the amino acid sequence SGQG (SEQ ID NO: 668). In some embodiments, the spacer includes at least the amino acid sequence GQG (SEQ ID NO: 669). In some embodiments, the spacer includes at least the amino acid sequence G. In some embodiments, the spacer is absent.

[0184] In some embodiments, the activatable antibody AB naturally contains one or more disulfide bonds. In some embodiments, AB can be manipulated to contain one or more disulfide bonds.

[0185] In some embodiments, an activatable antibody or its antigen-binding fragment is conjugated to an active substance. In some embodiments, the activatable antibody is a VH CDR1 sequence TIFF0007887228000136.tif4128; VH CDR2 sequence TIFF0007887228000137.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000138.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) The active agent comprises an antibody or its antigen-binding fragment that cross-competes with an isolated antibody containing the active agent. In some embodiments, the activatable antibody comprises an antibody or its antigen-binding fragment that cross-competes with an isolated antibody comprising a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3-5 and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6-8. In some embodiments, the active agent is a toxin or a fragment thereof. In some embodiments, the active agent is a microtubule inhibitor. In some embodiments, the active agent is a nucleic acid damaging active agent. In some embodiments, the active agent is selected from the group consisting of drastatin or its derivatives, auristatin or its derivatives, meitansinoid or its derivatives, duocalmycin or its derivatives, calicheamycin or its derivatives, and pyrrolobenzodiazepine or its derivatives. In some embodiments, the active agent is auristatin E or its derivatives. In some embodiments, the active agent is monomethyl auristatin E (MMAE). In some embodiments, the active agent is monomethyl auristatin D (MMAD). In some embodiments, the active substance is a maytansinoid selected from the group consisting of DM1 and DM4. In some embodiments, the active substance is the maytansinoid DM4. In some embodiments, the active substance is duocalmycin. In some embodiments, the active substance is conjugated to AB via a linker. In some embodiments, the linker conjugating the active substance to AB includes an SPDB portion, a vc portion, or a PEG2-vc portion. In some embodiments, the linker and the toxin conjugated to AB include an SPDB-DM4 portion, a vc-MMAD portion, a vc-MMAE portion, a vc-duocalmycin, or a PEG2-vc-MMAD portion. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the active substance is a detectable portion. In some embodiments, the detectable portion is a diagnostic active substance.

[0186] In some embodiments, the conjugated activatable antibody comprises an antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB specifically binds to human CD71 and cynomolgus monkey CD71; a masking moiety (MM) that inhibits the binding of AB to CD71 when the activatable antibody is in an uncleaved state; a cleavable moiety (CM) coupled to AB, which is a polypeptide that functions as a substrate for a protease; and an active substance conjugated to AB, the conjugated activatable antibody that binds to CD71 in an activated state. In some embodiments, the active substance is selected from the group consisting of drastatin or its derivatives, auristatin or its derivatives, meitansinoids or their derivatives, duocalmycin or its derivatives, calicheamicin or its derivatives, and pyrrolobenzodiazepines or its derivatives. In some embodiments, the active substance is selected from the group consisting of auristatin E, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), meitansinoid DM4, meitansinoid DM1, duocalmycin, pyrrolobenzodiazepine, and pyrrolobenzodiazepine dimers. In some embodiments, the active substance is conjugated to AB via a linker. In some embodiments, the linker conjugating the active substance to AB comprises an SPDB portion, a vc portion, or a PEG2-vc portion. In some embodiments, the linker and the toxin conjugated to AB comprise an SPDB-DM4 portion, a vc-MMAD portion, a vc-MMAE portion, a vc-duocalmycin, or a PEG2-vc-MMAD portion. In some embodiments, AB of the conjugated activatable antibody or its antigen-binding fragment comprises a VH CDR1 sequence TIFF0007887228000139.tif4128; VH CDR2 sequence TIFF0007887228000140.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000141.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) In some embodiments, the AB of the conjugated activatable antibody or its antigen-binding fragment includes a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3-5, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6-8 and 809-908. In some embodiments, MM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 16-295 and 297-314. In some embodiments, MM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 17, and 297-314. In some embodiments, CM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 356-423, 680-698, 713, 714, and 789-808. In some embodiments, CM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 406-423, 680-698, 713, 714, and 807-808. In some embodiments, the activatable antibody comprises a combination of amino acid sequences, the combination of amino acid sequences selected from a single column in Table D, where for a given combination, (a) the heavy chain of AB comprises the amino acid sequence of a VH CDR sequence corresponding to the given combination in a single column listed in Table D, (b) the light chain of AB comprises the amino acid sequence of a VL CDR sequence corresponding to the given combination in a single column listed in Table D, (c) MM comprises the amino acid sequence of a mask sequence (MM) corresponding to the given combination in a single column listed in Table D, and (d) CM comprises the amino acid sequence of a substrate sequence (CM) corresponding to the given combination in a single column listed in Table D.In some embodiments, the activatable antibody comprises a combination of amino acid sequences, where for a given combination of amino acid sequences: (a) the heavy chain of AB comprises an amino acid sequence of a VH sequence or VH CDR sequence selected from the group consisting of VH sequences or VH CDR sequences listed in the corresponding column of Table E; (b) the light chain of AB comprises an amino acid sequence of a VL sequence or VL CDR sequence selected from the group consisting of VL sequences or VL CDR sequences listed in the corresponding column of Table E; (c) MM comprises an amino acid sequence of a mask sequence (MM) selected from the group consisting of MM sequences listed in the corresponding column of Table E; and (d) CM comprises an amino acid sequence of a substrate sequence (CM) selected from the group consisting of CM sequences listed in the corresponding column of Table E. In some embodiments, the activatable antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 325 or 699; and a light chain containing the amino acid sequence selected from the group consisting of SEQ ID NO: 327, 329, 331, 333, 335, 337, 650, 652, 654, 656, 658, 660, 670-673, 701-712, 721-788, 809-836, and 841-908.

[0187] In some aspects, the conjugated activatable antibody includes the following conjugated activatable antibodies: An antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB specifically binds to human CD71 and cynomolgus monkey CD71; a masking moiety (MM) that inhibits the binding of AB to CD71 when the activatable antibody is in an uncleaved state; a cleavable moiety (CM) coupled to AB, which is a polypeptide that functions as a substrate for a protease; and a conjugated active substance conjugated to AB, wherein the AB binds to CD71 in an activated state, and the AB is (i) a VH CDR1 sequence TIFF0007887228000142.tif4128; VH CDR2 sequence TIFF0007887228000143.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000144.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) (ii) a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8, or (iii) a heavy chain containing an amino acid sequence of SEQ ID NO: 325 or 699, and a light chain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 327, 329, 331, 333, 335, 337, 650, 652, 654, 656, 658, 660, 670 to 673, 701 to 712, 721 to 788, 809 to 836 and 841 to 908, wherein the active substance is auristatin E, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), monomethyl auristatin D The activatable antibody is selected from the group consisting of (MMAD), meitansinoid DM4, meitansinoid DM1, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, and duocalmycin. In some embodiments, MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16-295 and 297-314. In some embodiments, MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 17, and 297-314. In some embodiments, CM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 356-423, 680-698, 713, 714, and 789-808. In some embodiments, CM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 406-423, 680-698, 713, 714, and 807-808. In some embodiments, the active substance is conjugated to AB via a linker, and the linker conjugating the active substance to AB comprises an SPDB portion, a vc portion, or a PEG2-vc portion. In some embodiments, the toxin conjugated to the linker and AB comprises an SPDB-DM4 portion, a vc-MMAD portion, a vc-MMAE portion, a vc-duocalmycin, or a PEG2-vc-MMAD portion.

[0188] In some embodiments, the conjugated activatable antibody comprises a conjugated activatable antibody or conjugated antibody comprising an antibody or its antigen-binding fragment (AB) that binds to CD71 in the activated state; and a toxin conjugated to AB via a linker, wherein the conjugated activatable antibody or conjugated antibody comprises an amino acid sequence, a linker, and a toxin selected from a single column of Table F, wherein for a given combination: (a) AB comprises a heavy chain comprising an amino acid sequence of a heavy chain sequence or heavy chain variable domain sequence corresponding to a given combination in a single column listed in Table F; (b) AB comprises a light chain comprising an amino acid sequence of a light chain sequence or light chain variable domain sequence corresponding to a given combination in a single column listed in Table F; and (c) the linker and toxin comprises a linker and a toxin corresponding to a given combination in a single column listed in Table F.

[0189] In some embodiments, the activatable 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 NO: 1 and 3-5.

[0190] In some embodiments, the activatable 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 NO: 2 and 6-8.

[0191] In some embodiments, the activatable 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 NO: 1 and 3-5, and a nucleic acid sequence encoding a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 6-8.

[0192] In some embodiments, the activatable 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 NO: 3 to 5, and a nucleic acid sequence encoding a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8.

[0193] In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence encoding a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3-5.

[0194] In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence encoding a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 6-8.

[0195] In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence encoding a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3-5, and a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence encoding a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 6-8.

[0196] In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence encoding a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 3-5, and a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence encoding a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 6-8.

[0197] In some embodiments, the activatable 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 heavy chain variable region sequences shown in Table 12. In some embodiments, the activatable 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 light chain variable region sequences shown in Table 12. In some embodiments, the activatable 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 heavy chain variable region sequences shown in Table 12 and a nucleic acid sequence encoding a light chain variable region amino acid sequence selected from the group consisting of light chain variable region sequences shown in Table 12.

[0198] In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding 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 heavy chain variable region sequences shown in Table 12. In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a light 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 light chain variable region sequences shown in Table 12. In some embodiments, the activatable antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding 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 heavy chain variable region sequences shown in Table 12, and a nucleic acid sequence encoding a light 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 light chain variable region sequences shown in Table 12.

[0199] In some embodiments, the activatable antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a combination of a variable heavy chain complementarity determination region 1 (VH CDR1, also known herein as CDRH1) sequence, a variable heavy chain complementarity determination region 2 (VH CDR2, also known herein as CDRH2) sequence, a variable heavy chain complementarity determination region 3 (VH CDR3, also known herein as CDRH3) sequence, a variable light chain complementarity determination region 1 (VL CDR1, also known herein as CDRL1) sequence, a variable light chain complementarity determination region 2 (VL CDR2, also known herein as CDRL2) sequence, and a variable light chain complementarity determination region 3 (VL CDR3, also known herein as CDRL3) sequence, wherein at least one CDR sequence is the VH CDR1 sequence shown in Table 13; the VH CDR2 sequence shown in Table 13; the VH CDR3 sequence shown in Table 13; the VL CDR1 sequence shown in Table 13; the VL The sequence is selected from the group consisting of CDR2 sequences and VL CDR3 sequences shown in Table 13.

[0200] In some embodiments, the activatable antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a combination of VH CDR1 sequence, VH CDR2 sequence, VH CDR3 sequence, VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence, where at least one CDR sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR1 sequence shown in Table 13; the 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 shown in Table 13; and the VH CDR1 sequence shown in Table 13. VH CDR3 sequences containing sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the CDR3 sequence; VL CDR1 sequences containing sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR1 sequences shown in Table 13; VL CDR2 sequences containing sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR2 sequences shown in Table 13; and VL CDR1 sequences shown in Table 13 The selection is made from a group of VL CDR3 sequences that include sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the CDR3 sequence.

[0201] In some aspects, the activatable antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a combination of the VH CDR1 sequence, VH CDR2 sequence, VH CDR3 sequence, VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence, where the combination is a combination of the six CDR sequences (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) shown in a single column in Table 13.

[0202] In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a light chain variable region, which includes a combination of VL CDR1, VL CDR2, and VL CDR3 sequences, where the combination is a combination of the three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) shown in a single column in Table 13.

[0203] In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a heavy chain variable region containing a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, where the combination is a combination of the three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in a single column in Table 13.

[0204] In some embodiments, the activatable antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a combination of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences, where each CDR sequence in the combination is 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 (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) shown in a single column of Table 13.

[0205] In some embodiments, the activatable antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a heavy chain variable region including a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, where each CDR sequence in the combination is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the corresponding CDR sequence in the combination of three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in a single column of Table 13.

[0206] In some embodiments, the activatable antibody is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding a light chain variable region including a combination of VL CDR1, VL CDR2, and VL CDR3 sequences, where each CDR sequence in the combination is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the corresponding CDR sequence in the combination of three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) shown in a single column of Table 13.

[0207] The Disclosure also provides a method for producing the activatable antibody of the Disclosure by culturing cells containing the nucleic acid molecule or vector of the Disclosure under conditions that result in the expression of the activatable antibody.

[0208] The Disclosure also provides a method for producing an activatable antibody that binds to CD71 in an activated state, comprising the steps of (a) culturing cells comprising a nucleic acid construct encoding the activatable antibody under conditions that result in the expression of the activatable antibody, including the activatable antibody of the Disclosure; and (b) recovering the activatable antibody.

[0209] In some embodiments, the activatable antibody comprises one or more polypeptides comprising a combination of sequences in a given column of Table D, or any combination of a mask sequence (MM) from Table E, a substrate sequence (CM), a light chain variable domain sequence or light chain variable domain CDR sequence, and a heavy chain variable domain sequence or heavy chain variable domain CDR sequence.

[0210] (Table D) Combinations of anti-CD71 activatable antibodies TIFF0007887228000145.tif163151TIFF0007887228000146.tif229151TIFF0007887228000147.tif22915 1TIFF0007887228000148.tif229151TIFF0007887228000149.tif229151TIFF0007887228000150.tif26151

[0211] (Table E) Components of anti-CD71 activatable antibodies TIFF0007887228000151.tif188152TIFF0007887228000152.tif229152TIFF0007887228000153.tif178152

[0212] In some embodiments, the activatable antibodies of the present disclosure comprise one or more polypeptides comprising a combination of sequences selected from Table D or Table E, wherein the polypeptide comprises a combination of a masking sequence selected from the column titled “Mask Sequence (MM)” in Table D or Table E, a substrate sequence from the column titled “Substrate Sequence (CM)” in Table D or Table E, a light chain variable domain or light chain CDR from the column titled “VL or VL CDR” or “VL CDR SEQ ID NO” in Table D or Table E, and a heavy chain variable domain or heavy chain CDR from the column titled “VH or VH CDR” or “VH CDR SEQ ID No” in Table D or Table E. For example, the activatable antibody of this disclosure may include the amino acid sequence of combination number 54, which includes a masking sequence of SEQ ID NO: 17, a substrate sequence of SEQ ID NO: 412, a light chain variable domain including VL CDR sequences of SEQ ID NO: 12, 14, and 15, and a heavy chain variable domain including VH CDR sequences of 9, 10, and 11. Therefore, activatable antibodies including at least a combination of sequences in any given column of Table D are described herein. Similarly, any combination of a mask sequence (MM) from Table E, a substrate sequence (CM), a light chain variable domain sequence or light chain variable domain CDR sequence, and a heavy chain variable domain sequence or heavy chain variable domain CDR sequence are described herein. Activatable antibodies including at least a masking sequence selected from Table D or Table E in the corresponding column, a substrate sequence, a variable heavy chain or variable heavy chain CDR, and a variable light chain or variable light chain CDR are also described herein.In some exemplary embodiments, an activatable antibody comprising at least a combination of sequences in any given column of Table D, or any combination of a mask sequence (MM) from Table E, a substrate sequence (CM), a light chain variable domain sequence or light chain variable domain CDR sequence, and a heavy chain variable domain sequence or heavy chain variable domain CDR sequence, can be combined with one or more toxins, including drastatin or its derivatives, auristatin or its derivatives, meitansinoid or its derivatives, duocalmycin or its derivatives, calicheamycin or its derivatives, or pyrrolobenzodiazepine or its derivatives. In some exemplary embodiments, an activatable antibody comprising at least a combination of sequences in any given column of Table D, or any combination of a mask sequence (MM) from Table E, a substrate sequence (CM), a light chain variable domain sequence or light chain variable domain CDR sequence, and a heavy chain variable domain sequence or heavy chain variable domain CDR sequence, can be combined with one or more toxins, including auristatin E, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), maytansinoid DM4, maytansinoid DM1, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, and / or duocalmycin.

[0213] Any combination in Table D or Table E above can be combined with a human immunoglobulin constant region to produce full human IgG including IgG1, IgG2, and IgG4, or with a variant constant region to produce human IgG with modified function such as IgG1 N297A, IgG1 N297Q, or IgG4 S228P. The combinations described in Table D or Table E are not limited to the specific combinations shown in any given column, and may therefore include any mask sequence from column 2 of Table D (or column 1 of Table E) combined with any substrate sequence from column 3 of Table D (or column 2 of Table E), any VL sequence or VL CDR sequence set from column 4 of Table D (or column 3 of Table E), and any VH sequence or VH CDR sequence set from column 5 of Table D (or column 4 of Table E). In addition to the mask sequences disclosed in column 2 of Table D or column 1 of Table E, any mask sequences disclosed herein can be used in combination. In addition to the substrate sequences disclosed in column 3 of Table D or column 2 of Table E, any CM disclosed herein can be used in combination. In addition to the light chain variable region sequences or light chain CDR sequences disclosed in column 4 of Table D or column 3 of Table E, any light chain variable region sequences or light chain CDR sequences disclosed herein can be used in combination. In addition to the heavy chain variable region sequences or heavy chain CDR sequences disclosed in column 5 of Table D or column 4 of Table E, any heavy chain variable region sequences or heavy chain CDR sequences disclosed herein can be used in combination.

[0214] In some embodiments, antibody-drug conjugates (ADCs) and activatable antibody-drug conjugates (AADCs) may contain one or more polypeptides comprising a combination of a light chain sequence or light chain variable domain sequence in a given column of Table F, a heavy chain sequence or heavy chain variable domain sequence, a linker, and a toxin, or any combination of a light chain sequence or light chain variable domain sequence in Table F, a heavy chain sequence or heavy chain variable domain sequence, a linker, and a toxin.

[0215] (Table F) Combinations of anti-CD71 ADCs and anti-CD71 activatable ADCs TIFF0007887228000154.tif43151TIFF0007887228000155.tif227151TIFF0007887228000156.tif227151TIFF00078872280 00157.tif227151TIFF0007887228000158.tif227151TIFF0007887228000159.tif227151TIFF0007887228000160.tif209151

[0216] An antibody-drug conjugate (ADC) or an activatable antibody-drug conjugate (AADC) of the present disclosure may comprise one or more polypeptides comprising a combination of amino acid sequences, linkers, and toxins listed in a given column of Table F. Therefore, an activatable antibody-drug conjugate (ADC) or an activatable antibody-drug conjugate (AADC) of the present disclosure comprising a combination of amino acid sequences, linkers, and toxins listed in a given column or provided as a specific combination is described herein. For example, an activatable antibody-drug conjugate of the present disclosure may comprise an amino acid sequence of combination number 45, which comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 325, a light chain comprising the amino acid sequence of SEQ ID NO: 333, and an spdb-DM4 linker-toxin. In another example of an AADC disclosed and described herein, the activatable antibody-drug conjugate of this disclosure may include an amino acid sequence of combination number 33, which includes a heavy chain containing an amino acid sequence of SEQ ID NO: 325, a light chain containing an amino acid sequence of SEQ ID NO: 331, and a vc-MMAE linker-toxin.

[0217] Any combination in Table F, which lists heavy chain and light chain variable regions, can be combined with a human immunoglobulin constant region to produce full human IgG, including IgG1, IgG2, and IgG4, or with a variant constant region to produce human IgG with modified function, such as IgG1 N297A, IgG1 N297Q, or IgG4 S228P. The combinations described in Table F are not limited to the specific combinations shown in any given column and therefore may include any heavy chain or heavy chain variable region sequence from column 2 of Table F, combined with any light chain or light chain variable region sequence from column 3 of Table F, combined with any linker from column 4, and 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 sequences disclosed herein may 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 sequences disclosed herein may be used in combination. In addition to the linkers listed in column 4, any linkers disclosed herein may be used in combination. In addition to the toxins listed in column 5, any toxins disclosed herein may be used in combination.

[0218] In some embodiments, the serum half-life of the activatable antibody is longer than the serum half-life of the corresponding antibody. For example, the pK of the activatable antibody is longer than the pK of the corresponding antibody. In some embodiments, the serum half-life of the activatable antibody is similar to that of the corresponding antibody. In some embodiments, the serum half-life of the activatable antibody is at least 15 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 12 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 11 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 10 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 9 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 8 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 7 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 5 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 2 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 24 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 20 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 18 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 16 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 14 hours when administered to an organism.In some embodiments, the serum half-life of the activatable antibody is at least 12 hours when administered to a living organism. In some embodiments, the serum half-life of the activatable antibody is at least 10 hours when administered to a living organism. In some embodiments, the serum half-life of the activatable antibody is at least 8 hours when administered to a living organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 hours when administered to a living organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 hours when administered to a living organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 hours when administered to a living organism.

[0219] In some embodiments, activatable anti-CD71 antibodies and / or conjugated activatable anti-CD71 antibodies are monospecific. In some embodiments, activatable anti-CD71 antibodies and / or conjugated activatable anti-CD71 antibodies are multispecific, for example, bispecific or trifunctional, as a non-limiting example. In some embodiments, activatable anti-CD71 antibodies and / or conjugated activatable anti-CD71 antibodies are formulated as part of a pro-bispecific T cell engager (BITE) molecule. In some embodiments, activatable anti-CD71 antibodies and / or conjugated activatable anti-CD71 antibodies are formulated as part of T cells modified by prochimeric antigen receptors (CARs) or other manipulated receptors.

[0220] In some embodiments, an activatable antibody or its antigen-binding fragment is incorporated into a multispecific activatable antibody or its antigen-binding fragment, and at least one arm of the multispecific activatable antibody specifically binds to CD71. In some embodiments, an activatable antibody or its antigen-binding fragment is incorporated into a bispecific antibody or its antigen-binding fragment, and at least one arm of the bispecific activatable antibody specifically binds to CD71.

[0221] In some embodiments, the anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies described herein are used in conjunction with one or more additional agents or combinations of additional agents. Suitable additional agents include, for example, current pharmaceutical and / or surgical therapies for intended uses such as cancer. For example, anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies can be used in conjunction with additional chemotherapeutic agents or antineoplastic agents.

[0222] In some embodiments, further factors of action are chemotherapeutic agents selected from the group consisting of, for example, docetaxel, paclitaxel, Abraxane (i.e., paclitaxel conjugated to albumin), doxorubicin, oxaliplatin, carboplatin, cisplatin, irinotecan, and gemcitabine.

[0223] In some embodiments, further agents are checkpoint inhibitors, kinase inhibitors, agents that target inhibitors present in the tumor microenvironment, and / or T cell agonists or NK agonists. In some embodiments, further agents are radiotherapy, alone or in combination with another further agent, such as a chemotherapeutic agent or an antineoplastic agent. In some embodiments, further agents are vaccines, oncoviruses, and / or DC activators, such as, in non-limiting examples, Toll-like receptor (TLR) agonists and / or α-CD40. In some embodiments, further agents are tumor-targeting antibodies designed to kill tumors via ADCCs or via direct conjugation to toxins (e.g., antibody-drug conjugates (ADCs)).

[0224] In some embodiments, the checkpoint inhibitor is an inhibitor of a target selected from the group consisting of CTLA-4, LAG-3, PD1, CD71, 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 IDO inhibitors, α-CSF1R inhibitors, α-CCR4 inhibitors, TGF-β, myeloid-derived suppressor cells, or regulatory T cells. In some embodiments, the agonist is selected from the group consisting of Ox40, GITR, CD137, ICOS, CD27, and HVEM.

[0225] 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 PD1 inhibitor. In some embodiments, the inhibitor is a CD71 inhibitor. In some embodiments, the inhibitor is a TIGIT inhibitor. In some embodiments, the inhibitor is a TIM-3 inhibitor. 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 α-CSF1R inhibitor. In some embodiments, the inhibitor is an α-CCR4 inhibitor. In some embodiments, the inhibitor is TGF-β. In some embodiments, the inhibitor is myeloid-derived suppressor cells. In some embodiments, the inhibitor is regulatory T cells.

[0226] 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 ICOS. In some embodiments, the agonist is CD27. In some embodiments, the agonist is HVEM.

[0227] In some embodiments, anti-CD71 antibodies, conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies are administered during and / or after treatment in combination with one or more additional agents, such as chemotherapeutic agents, anti-inflammatory agents, and / or immunosuppressants. In some embodiments, anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies and additional agents are formulated in a single therapeutic composition, and anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies and additional agents are administered simultaneously. Alternatively, the anti-CD71 antibody, conjugated anti-CD71 antibody, activatable anti-CD71 antibody, and / or conjugated activatable anti-CD71 antibody and further activators may be separated from each other, for example, each being formulated in a separate therapeutic composition, and the anti-CD71 antibody, conjugated anti-CD71 antibody, activatable anti-CD71 antibody, and / or conjugated activatable anti-CD71 antibody and further activators may be administered simultaneously, or the anti-CD71 antibody, conjugated anti-CD71 antibody, activatable anti-CD71 antibody, and / or conjugated activatable anti-CD71 antibody and further activators may be administered at different times during the treatment regimen. For example, anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies may be administered before the administration of further activators, or anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies may be administered after the administration of further activators, or anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies may be administered alternately with further activators.As described herein, anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies, along with additional activators, are administered in single or multiple doses.

[0228] In some embodiments, anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies, along with further activators, are administered simultaneously. For example, anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies, along with further activators, may be formulated in a single composition or administered as two or more separate compositions. In some embodiments, anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies, along with further activators, may be administered sequentially, or anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies, along with further activators, may be administered at different times during a treatment regimen.

[0229] In some embodiments, during and / or after treatment, anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies are administered in combination with one or more further activators, for example, chemotherapeutic agents, anti-inflammatory agents, and / or immunosuppressants, such as alkylating agents, antimetabolites, antimicrotubule agents, topoisomerase inhibitors, cytotoxic antibiotics, and / or any other nucleic acid damaging agents, as non-limiting examples. In some embodiments, the further activator is a taxane, e.g., paclitaxel (e.g., Abraxane®). In some embodiments, the further activator is an antimetabolite, e.g., gemcitabine. In some embodiments, the further activator is an alkylating agent, e.g., platinum-based chemotherapy such as carboplatin or cisplatin. In some embodiments, the further activator is a targeted activator, e.g., a kinase inhibitor, e.g., sorafenib or erlotinib. In some embodiments, a further agent is a targeted agent, e.g., another antibody, e.g., a monoclonal antibody (e.g., bevacizumab), a bispecific antibody, or a multispecific antibody. In some embodiments, a further agent is a proteosome inhibitor, e.g., bortezomib or carfilzomib. In some embodiments, a further agent is an immunomodulator, e.g., lenolidomin or IL-2. In some embodiments, a further agent is radiation. In some embodiments, a further agent is an agent considered standard of treatment by those skilled in the art. In some embodiments, a further agent is chemotherapy well known to those skilled in the art.

[0230] In some embodiments, further activators are another antibody or its antigen-binding fragment, another conjugated antibody or its antigen-binding fragment, another activatable antibody or its antigen-binding fragment, and / or another conjugated activatable antibody or its antigen-binding fragment. In some embodiments, further activators are another antibody or its antigen-binding fragment, another conjugated antibody or its antigen-binding fragment, another activatable antibody or its antigen-binding fragment, and / or another conjugated activatable antibody or its antigen-binding fragment, targeting the same target as the first antibody or its antigen-binding fragment, for example, CD71. In some embodiments, further agents are another antibody or its antigen-binding fragment, another conjugated antibody or its antigen-binding fragment, another activatable antibody or its antigen-binding fragment, and / or another conjugated activatable antibody or its antigen-binding fragment, targeting a different target from the first antibody or its antigen-binding fragment, the first conjugated antibody or its antigen-binding fragment, the activatable antibody or its antigen-binding fragment, and / or another conjugated activatable antibody or its antigen-binding fragment.

[0231] As a non-limiting example, the AB of the antibody or antigen-binding fragment and / or activatable antibody may be a binding partner to any of the targets listed in Table 1.

[0232] (Table 1) Exemplary Targets TIFF0007887228000161.tif160160TIFF0007887228000162.tif132162

[0233] As a non-limiting example, the AB of the antibody or antigen-binding fragment and / or activatable antibody may be one of the antibodies listed in Table 2, or derived from such antibodies.

[0234] (Table 2) Exemplary sources of Ab TIFF0007887228000163.tif67150TIFF0007887228000164.tif205150

[0235] In some embodiments, the further antibody or its antigen-binding fragment, conjugated antibody or its antigen-binding fragment, activatable antibody or its antigen-binding fragment, and / or conjugated activatable antibody or its antigen-binding fragment may be a monoclonal antibody, a domain antibody, a single-chain antibody, a Fab fragment, an F(ab')2 fragment, scFv, scAb, dAb, a single-domain heavy-chain antibody, or a single-domain light-chain antibody. In some embodiments, the further antibody or its antigen-binding fragment, conjugated antibody or its antigen-binding fragment, activatable antibody or its antigen-binding fragment, and / or conjugated activatable antibody or its antigen-binding fragment may be a mouse antibody, another rodent antibody, a chimeric antibody, a humanized antibody, or a fully human monoclonal antibody.

[0236] The Disclosure also provides a method for producing an anti-CD71 antibody and / or an activatable anti-CD71 antibody polypeptide by culturing cells under conditions that result in the expression of the polypeptide, wherein the cells comprise an isolated nucleic acid molecule encoding the antibody and / or activatable antibody as described herein, and / or a vector comprising the isolated nucleic acid sequence thereof. The Disclosure also provides a method for producing an antibody and / or an activatable antibody by culturing cells under conditions that result in the expression of the antibody and / or an activatable antibody, wherein the cells comprise an isolated nucleic acid molecule encoding the antibody and / or activatable antibody as described herein, and / or a vector comprising the isolated nucleic acid sequence thereof.

[0237] The present invention also provides a method for producing an activatable antibody that binds to CD71 in an activated state, comprising the steps of (a) culturing cells comprising a nucleic acid construct encoding an activatable antibody under conditions that result in the expression of an activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM), and an antibody or antigen-binding fragment (AB) that specifically binds to CD71, (i) the CM is a polypeptide that functions as a substrate for a protease, and (ii) the CM is positioned on the activatable antibody such that the MM interferes with the specific binding of AB to CD71 when the activatable antibody is in an uncleaved state, and the MM neither interferes with nor competes with the specific binding of AB to CD71 when the activatable antibody is in a cleaved state, and (b) recovering the activatable antibody. Suitable AB, MM, and / or CM include any AB, MM, and / or CM disclosed herein.

[0238] In some embodiments, the activatable antibody, in its uncleaved state, has a structural arrangement such as MM-CM-AB or AB-CM-MM from the N-terminus to the C-terminus. In some embodiments, the activatable antibody contains a linked peptide between MM and CM. In some embodiments, the activatable antibody contains a linked peptide between CM and AB. In some embodiments, the activatable antibody comprises a first linked peptide (LP1) and a second linked peptide (LP2), and, in its uncleaved state, has a structural arrangement such as MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM from the N-terminus to the C-terminus. In some embodiments, the two linked peptides do not need to be identical to each other. In some embodiments, the activatable antibody, in its uncleaved state, has a structural arrangement such as spacer-MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM-spacer from the N-terminus to the C-terminus.

[0239] In some embodiments, at least one of LP1 or LP2 is The amino acid sequence is selected from the group consisting of TIFF0007887228000165.tif4131, and n is at least an integer of 1.

[0240] In some embodiments, at least one of LP1 or LP2 is It contains an amino acid sequence selected from the group consisting of TIFF0007887228000166.tif18145.

[0241] In some aspects, LP1 is an amino acid sequence Includes TIFF0007887228000167.tif18139.

[0242] In some embodiments, LP2 is an amino acid sequence Includes TIFF0007887228000168.tif11141.

[0243] The present invention provides a method for preventing, slowing the progression of, treating, alleviating the symptoms of, or otherwise relieving a CD71-mediated disease in a subject by administering a therapeutically effective amount of the anti-CD71 antibody, conjugated anti-CD71 antibody, activatable anti-CD71 antibody, and / or conjugated activatable anti-CD71 antibody described herein to a subject in need.

[0244] The present invention also provides a method for preventing cancer, slowing the progression of cancer, treating cancer, alleviating cancer symptoms, or otherwise improving cancer in a subject by administering a therapeutically effective amount of the anti-CD71 antibody, conjugated anti-CD71 antibody, activatable anti-CD71 antibody, and / or conjugated activatable anti-CD71 antibody described herein to the subject in need. CD71 is known to be expressed in a variety of cancers, including, but not limited to, adenocarcinoma, cholangiocarcinoma (biliary tract cancer), bladder cancer, breast cancer (e.g., triple-negative and Her2-negative breast cancer), carcinoid cancer; cervical cancer; cholangiocarcinoma; colorectal cancer; endometrial cancer; glioma; head and neck cancer (e.g., squamous cell carcinoma of the head and neck); leukemia; liver cancer; lung cancer (e.g., NSCLC, SCLC); lymphoma; melanoma; oropharyngeal cancer; ovarian cancer; pancreatic cancer; prostate cancer (e.g., metastatic castration-resistant prostate cancer); kidney cancer; skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; and urothelial carcinoma.

[0245] In some embodiments, cancer is associated with CD71-expressing tumors. In some embodiments, cancer is caused by CD71-expressing tumors.

[0246] The anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies used in these methods and in any embodiment of use may be administered at any stage of the disease. For example, such anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies may be administered to patients with cancer at any stage, from early-stage cancer to metastatic cancer. The terms "subject" and "patient" are used interchangeably herein.

[0247] In some embodiments, the subject is a mammal, e.g., human, non-human primate, companion animal (e.g., cat, dog, horse), livestock, work 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 care of a veterinarian.

[0248] Anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies, as well as therapeutic formulations thereof, are administered to subjects suffering from or susceptible to diseases or disorders associated with abnormal CD71 expression and / or activity. Subjects suffering from or susceptible to diseases or disorders associated with abnormal CD71 expression and / or activity are identified using any variety of methods known in the art. For example, subjects suffering from cancer or other neoplastic conditions are identified using any variety of clinical and / or laboratory tests, e.g., physical examinations and blood, urine, and / or fecal analyses, to assess their health status. For example, subjects suffering from inflammation and / or inflammatory disorders are identified using any variety of clinical and / or laboratory tests, e.g., physical examinations and / or fluid analyses, e.g., blood, urine, and / or fecal analyses, to assess their health status.

[0249] The administration of anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies to patients with diseases or disorders associated with abnormal CD71 expression and / or activity is considered successful if any of the various experimental or clinical objectives are achieved. For example, the administration of anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies to patients with diseases or disorders associated with abnormal CD71 expression and / or activity is considered successful if one or more symptoms associated with the disease or disorder are alleviated, reduced, inhibited, or do not progress to a further, i.e., worsened state. Administration of anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies to patients with diseases or disorders associated with abnormal CD71 expression and / or activity is considered successful if the disease or disorder enters remission or does not progress to a further, i.e., worsening state.

[0250] In some embodiments, anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies and / or conjugated activatable anti-CD71 antibodies and their therapeutic formulations are administered to subjects suffering from or highly susceptible to a disease or disorder, such as subjects suffering from cancer or other neoplasms, where the affected cells of the subject express CD71. In some embodiments, the affected cells are associated with abnormal CD71 expression and / or activity. In some embodiments, the affected cells are associated with normal CD71 expression and / or activity. Subjects suffering from or highly susceptible to a disease or disorder in which the affected cells of the subject express CD71 are identified using any of the various methods known in the art. For example, subjects suffering from cancer or other neoplasms are identified using any of the various clinical and / or laboratory tests, such as a physical examination to assess health status and blood analysis, urine analysis and / or fecal analysis. For example, subjects suffering from inflammation and / or inflammatory disorders are identified using a physical examination and / or fluid analysis to assess their health status, or any of the various clinical and / or laboratory tests such as blood analysis, urine analysis, and / or fecal analysis.

[0251] In some embodiments, anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies and / or conjugated activatable anti-CD71 antibodies and their therapeutic formulations are administered to subjects suffering from or highly susceptible to diseases or disorders associated with the presence, growth, proliferation, metastasis, and / or activity of CD71-expressing cells, such as subjects suffering from cancer or other neoplasms. In some embodiments, the cells are associated with abnormal CD71 expression and / or activity. In some embodiments, the cells are associated with normal CD71 expression and / or activity. Subjects suffering from or highly susceptible to diseases or disorders associated with CD71-expressing cells are identified using any of the various methods known in the art. For example, subjects suffering from cancer or other neoplasms are identified using any of the various clinical and / or laboratory tests, such as physical examinations to assess health status and blood, urine, and / or fecal analysis. For example, subjects suffering from inflammation and / or inflammatory disorders are identified using a physical examination and / or fluid analysis to assess their health status, or any of the various clinical and / or laboratory tests such as blood analysis, urine analysis, and / or fecal analysis.

[0252] The administration of anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies to patients suffering from a disease or disorder associated with CD71-expressing cells is considered successful if any of the various laboratory or clinical objectives are achieved. For example, the administration of anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies to patients suffering from a disease or disorder associated with CD71-expressing cells is considered successful if one or more symptoms associated with the disease or disorder are alleviated, reduced, inhibited, or do not progress to a further, i.e., worse condition. Administration of anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies to patients suffering from a disease or disorder associated with CD71-expressing cells is considered successful if the disease or disorder enters remission or does not progress to a further, i.e., worsening state.

[0253] In some embodiments, anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies are administered during and / or after treatment in combination with one or more additional agents, such as chemotherapeutic agents, anti-inflammatory agents, and / or immunosuppressants. In some embodiments, anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies and additional agents are administered simultaneously. For example, anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies and additional agents may be formulated in a single composition or administered as two or more separate compositions. In some embodiments, an anti-CD71 antibody, a conjugated anti-CD71 antibody, an activatable anti-CD71 antibody, and / or a conjugated activatable anti-CD71 antibody, along with further activators, are administered sequentially.

[0254] The Disclosure also provides a method for treating, alleviating, or slowing the progression of a disorder or disease in which affected cells express CD71, comprising the step of administering a therapeutically effective amount of the antibody or conjugated antibody of the Disclosure or a pharmaceutical composition containing the antibody or conjugated antibody of the Disclosure to a subject in need. In some embodiments, the disorder or disease is cancer.

[0255] The Disclosure also provides a method for treating, alleviating, or slowing the progression of a disorder or disease associated with CD71-expressing cells, comprising the step of administering a therapeutically effective amount of the antibody or conjugated antibody of the Disclosure or a pharmaceutical composition containing the antibody or conjugated antibody of the Disclosure to a subject in need. In some embodiments, the disorder or disease associated with CD71-expressing cells is cancer. In some aspects, the cancer is adenocarcinoma, cholangiocarcinoma (biliary tract cancer), bladder cancer, bone cancer, breast cancer, triple-negative breast cancer, Her2-negative breast cancer, carcinoid cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, colon cancer, endometrial cancer, glioma, head and neck cancer, head and neck squamous cell carcinoma, leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, melanoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, metastatic castration-resistant prostate cancer, kidney cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, genitourinary cancer, or urothelial carcinoma. In some aspects, the natural ligand is transferrin. In some aspects, the expression and / or activity of mammalian CD71 is abnormal. In some aspects, the method includes a step of administering a further active agent. In some aspects, the further active agent is a therapeutic agent.

[0256] The Disclosure also provides a method for inhibiting or reducing the growth, proliferation, or translocation of mammalian CD71-expressing cells, comprising the step of administering a therapeutically effective amount of the antibody or conjugated antibody of the Disclosure or a pharmaceutical composition comprising the antibody or conjugated antibody of the Disclosure to a subject in need. In some embodiments, the natural ligand is transferrin. In some embodiments, the expression and / or activity of mammalian CD71 is abnormal. In some embodiments, the method comprises the step of administering a further active agent. In some embodiments, the further active agent is a therapeutic agent.

[0257] The Disclosure also provides a method for inhibiting, blocking, or preventing the binding of a native ligand to mammalian CD71, comprising the step of administering a therapeutically effective amount of the antibody or conjugated antibody of the Disclosure or a pharmaceutical composition comprising the antibody or conjugated antibody of the Disclosure to a subject in need. In some embodiments, the native ligand is transferrin. In some embodiments, the expression and / or activity of mammalian CD71 is abnormal. In some embodiments, the method comprises the step of administering a further active agent. In some embodiments, the further active agent is a therapeutic agent.

[0258] The Disclosure also provides a method for treating, alleviating, or slowing the progression of a disorder or disease in which affected cells express CD71, comprising the step of administering a therapeutically effective amount of the activatable antibody of the Disclosure, a conjugated activatable antibody of the Disclosure, or a pharmaceutical composition comprising the activatable antibody of the Disclosure, or a pharmaceutical composition comprising the conjugated activatable antibody of the Disclosure, to a subject in need. In some embodiments, the disorder or disease is cancer.

[0259] The Disclosure also provides a method for treating, alleviating, or slowing the progression of a disorder or disease associated with CD71-expressing cells, comprising the step of administering a therapeutically effective amount of the activatable antibody of the Disclosure, a conjugated activatable antibody of the Disclosure, or a pharmaceutical composition comprising the activatable antibody of the Disclosure, or a pharmaceutical composition comprising the conjugated activatable antibody of the Disclosure, to a subject in need. In some embodiments, the disorder or disease associated with CD71-expressing cells is cancer. In some aspects, the cancer is adenocarcinoma, cholangiocarcinoma (biliary tract cancer), bladder cancer, bone cancer, breast cancer, triple-negative breast cancer, Her2-negative breast cancer, carcinoid cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, colon cancer, endometrial cancer, glioma, head and neck cancer, head and neck squamous cell carcinoma, leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, melanoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, metastatic castration-resistant prostate cancer, kidney cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, genitourinary cancer, or urothelial carcinoma. In some aspects, the natural ligand is transferrin. In some aspects, the expression and / or activity of mammalian CD71 is abnormal. In some aspects, the method includes a step of administering a further active agent. In some aspects, the further active agent is a therapeutic agent.

[0260] The Disclosure also provides a method for inhibiting or reducing the growth, proliferation, or translocation of mammalian CD71-expressing cells, comprising the step of administering a therapeutically effective amount of the activatable antibody of the Disclosure, a conjugated activatable antibody of the Disclosure, or a pharmaceutical composition comprising the activatable antibody of the Disclosure, or a pharmaceutical composition comprising the conjugated activatable antibody of the Disclosure, to a subject in need. In some embodiments, the natural ligand is transferrin. In some embodiments, the expression and / or activity of mammalian CD71 is abnormal. In some embodiments, the method comprises the step of administering a further active agent. In some embodiments, the further active agent is a therapeutic agent.

[0261] The Disclosure also provides a method for inhibiting, blocking, or preventing the binding of a native ligand to mammalian CD71, comprising the step of administering a therapeutically effective amount of the activatable antibody of the Disclosure, or a conjugated activatable antibody of the Disclosure, or a pharmaceutical composition comprising the activatable antibody of the Disclosure, or a pharmaceutical composition comprising the conjugated activatable antibody of the Disclosure, to a subject in need. In some embodiments, the native ligand is transferrin. In some embodiments, the expression and / or activity of mammalian CD71 is abnormal. In some embodiments, the method comprises the step of administering a further active agent. In some embodiments, the further active agent is a therapeutic agent.

[0262] The present invention also provides methods and kits for using activatable anti-CD71 antibodies and / or conjugated activatable anti-CD71 antibodies in various diagnostic and / or prophylactic applications. For example, the present invention provides a method for (i) contacting a subject or sample with an anti-CD71 activatable antibody, wherein the anti-CD71 activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) to be cleaved by a cleavage agent, and an antigen-binding domain or fragment thereof (AB) that specifically binds to a target of interest, wherein the anti-CD71 activatable antibody, in an uncleaved and unactivated state, has a structural arrangement such as MM-CM-AB or AB-CM-MM from the N-terminus to the C-terminus, (a) MM is a peptide that inhibits the binding of AB to CD71, and MM does not have the amino acid sequence of AB's natural binding partner, and is not a modified version of AB's natural binding partner, and (b) when AB is uncleaved and unactivated, MM inhibits the specificity of AB to CD71 The method and kit provide for detecting the presence or absence of a cleavage agent and a target of interest in a subject or sample, comprising the steps of (i) interfering with target binding and, if AB is cleaved and activated, MM does not interfere with or compete for the specific binding of AB to CD71, and (ii) measuring the level of activated anti-CD71 activatable antibody in the subject or sample, wherein the presence of a detectable level of activated anti-CD71 activatable antibody in the subject or sample indicates the presence of the cleavage agent and CD71 in the subject or sample, and the absence of a detectable level of activated anti-CD71 activatable antibody in the subject or sample indicates the absence of the cleavage agent, the absence of CD71, or the absence of both the cleavage agent and CD71 in the subject or sample.

[0263] In some embodiments, the activatable anti-CD71 antibody is an activatable anti-CD71 antibody conjugated with a therapeutic agent. In some embodiments, the activatable anti-CD71 antibody is not conjugated with a therapeutic agent. In some embodiments, the activatable anti-CD71 antibody includes a detectable label. In some embodiments, the detectable label is located on AB. In some embodiments, the level of the activatable anti-CD71 antibody in a subject or sample is measured using a secondary reagent that specifically binds to the activated antibody, and the reagent includes a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

[0264] In some embodiments of these methods and kits, the activatable anti-CD71 antibody includes a detectable label. In some embodiments of these methods and kits, the detectable label includes an imaging agent, a contrasting 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 includes a radioisotope. In some embodiments of these methods and kits, the radioisotope is indium or technetium. In some embodiments of these methods and kits, the contrasting agent includes iodine, gadolinium, or iron oxide. In some embodiments of these methods and kits, the enzyme includes horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments of these methods and kits, the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or europium derivatives. In some embodiments of these methods and kits, the luminescence label comprises N-methylacrydium derivatives. In some embodiments of these methods, the label comprises Alexa Fluor® label, e.g., Alex 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.

[0265] 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, e.g., a non-human primate, a companion animal (e.g., a cat, a dog, a horse), a livestock, a work animal, or a zoo animal. In some embodiments, the subject is a rodent.

[0266] 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.

[0267] In some embodiments of the methods and kits described herein, the methods are used to identify, or otherwise refine, a patient population suitable for treatment with the anti-CD71 activatable antibody of the Disclosure, and subsequently treat subjects in need of treatment by administering the activatable anti-CD71 antibody and / or a conjugated activatable anti-CD71 antibody. For example, a patient who tests positive for both the target (e.g., CD71) and the protease that cleaves the substrate at the cleavable portion (CM) of the anti-CD71 activatable antibody being tested in these methods is identified as a candidate suitable for treatment with such anti-CD71 activatable antibody, including such CM, and is then administered a therapeutically effective dose of the tested activatable anti-CD71 antibody and / or a conjugated activatable anti-CD71 antibody. Similarly, a patient who tests negative for either or both the target (e.g., CD71) and the protease that cleaves the substrate at the CM of the activatable antibody being tested using these methods may be identified as a candidate suitable for another form of therapy. In some embodiments, such patients may be tested with other anti-CD71 activatable antibodies until a suitable anti-CD71 activatable antibody for treatment (e.g., an anti-CD71 activatable antibody containing a CM cleaved by the patient at the disease site) is identified. In some embodiments, the patient is then administered a therapeutically effective dose of the activatable anti-CD71 antibody and / or conjugated activatable anti-CD71 antibody that tests positive in the patient. Suitable ABs, MMs, and / or CMs include any ABs, MMs, and / or CMs disclosed herein.

[0268] The pharmaceutical compositions according to the present invention may contain the antibody and carrier of the present invention. These pharmaceutical compositions may be included in a kit, such as a diagnostic kit.

[0269] In some embodiments, the pharmaceutical composition comprises an antibody of the Disclosure, an activatable antibody of the Disclosure, a conjugated antibody of the Disclosure, and / or a conjugated activatable antibody of the Disclosure, as well as a carrier. In some embodiments, the pharmaceutical composition comprises a further active agent. In some embodiments, the further active agent is a therapeutic agent. [Invention 1001] An isolated antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB specifically binds to human CD71 and cynomolgus monkey CD71. [Invention 1002] The antibody or its antigen-binding fragment VH CDR1 sequence TIFF0007887228000169.tif4128; VH CDR2 sequence TIFF0007887228000170.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000171.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) An isolated antibody of the present invention 1001, including [the specified substance]. [Invention 1003] An isolated antibody according to Invention 1001 or Invention 1002, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8. [Invention 1004] An isolated antibody or its antigen-binding fragment that binds to the same epitope as any of the isolated antibodies of the present invention 1001 to 1003 on human CD71 and / or cynomolgus monkey CD71. [Invention 1005] An isolated antibody or its antigen-binding fragment that cross-competes with any of the isolated antibodies of the present invention 1001 to 1004 for binding to human CD71 and / or cynomolgus monkey CD71. [Invention 1006] An antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB specifically binds to human CD71 and cynomolgus monkey CD71; A masking moiety (MM) that inhibits the binding of AB to CD71 when the activatable antibody is in an uncleaved state; and A cleavable portion (CM) coupled to AB, which is a polypeptide that functions as a substrate for a protease, the CM An activatable antibody that, in its activated state, binds to CD71. [Invention 1007] The activatable antibody of the present invention 1006, wherein MM has a dissociation constant for binding to AB that is greater than the dissociation constant of AB to CD71. [Invention 1008] An activatable antibody according to Invention 1006 or Invention 1007, wherein when the activatable antibody is in a cleaved state, MM does not interfere with or compete with AB for binding to CD71. [Invention 1009] An activatable antibody according to any of the present invention 1006 to 1008, wherein MM is a polypeptide with a length of 40 amino acids or less. [Invention 1010] An activatable antibody according to any of the present invention 1006 to 1009, wherein the polypeptide sequence of MM is different from the polypeptide sequence of human CD71. [Invention 1011] An activatable antibody according to any of the Invention 1006-1010, wherein the identity of the polypeptide sequence of MM to any natural binding partner of AB is 50% or less. [Invention 1012] An activatable antibody according to any of the Invention 1006 to 1011, wherein MM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 16-295 and 297-314. [Invention 1013] An activatable antibody according to any of the Invention 1006-1012, wherein MM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 17, and 297-314. [Invention 1014] CM is an activatable antibody according to any of the present invention 1006 to 1013, wherein CM is a substrate of an active protease in the affected tissue. [Invention 1015] An activatable antibody according to any of the present invention 1006 to 1014, wherein CM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 356-423, 680-698, 713, 714, and 789-808. [Invention 1016] An activatable antibody according to any of the present invention 1006 to 1015, wherein CM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 406-423, 680-698, 713, 714, and 807-808. [Invention 1017] The antigen-binding fragment is selected from the group consisting of Fab fragment, F(ab')2 fragment, scFv, scAb, dAb, single-domain heavy chain antibody, and single-domain light chain antibody, and is an antibody according to any of Invention 1001 to 1005 or an activatable antibody according to any of Invention 1006 to 1016. [Invention 1018] An antibody according to any of Invention 1001 to 1005 or an activatable antibody according to any of Invention 1006 to 1017, wherein AB specifically binds to human CD71. [Invention 1019] AB VH CDR1 sequence TIFF0007887228000172.tif4128; VH CDR2 sequence TIFF0007887228000173.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000174.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) An activatable antibody according to any of the inventions 1006 to 1018, including the above. [Invention 1020] An activatable antibody according to any of the inventions 1006 to 1019, wherein AB comprises a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8 and 809 to 908. [Invention 1021] An activatable antibody according to any of the present invention 1006 to 1020, wherein AB is linked to CM. [Invention 1022] An activatable antibody according to any of the invention 1006 to 1021, wherein AB is directly linked to CM. [Invention 1023] An activatable antibody according to any of the present invention 1006 to 1021, wherein AB is linked to CM via a linked peptide. [Invention 1024] An activatable antibody according to any of the invention 1006 to 1023, wherein the activatable antibody, in its uncleaved state, has a structural arrangement such as MM-CM-AB or AB-CM-MM from the N-terminus to the C-terminus, with MM linked to CM. [Invention 1025] An activatable antibody according to any of the present invention 1006 to 1024, comprising a linked peptide between MM and CM. [Invention 1026] An activatable antibody according to any of the present invention 1006 to 1024, comprising a linked peptide between CM and AB. [Invention 1027] An activatable antibody according to any of the invention 1006 to 1026, comprising a first linked peptide (LP1) and a second linked peptide (LP2), and having a structural arrangement such as MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM from the N-terminus to the C-terminus in the uncleaved state. [Invention 1028] The activatable antibody of the present invention 1027, wherein the two linked peptides do not need to be identical to each other. [Invention 1029] An activatable antibody according to Invention 1027 or Invention 1028, wherein each of LP1 and LP2 is a peptide approximately 1 to 20 amino acids long. [Invention 1030] An activatable antibody according to any of the invention 1006 to 1029, comprising a heavy chain sequence with SEQ ID NO: 325 or 699 and a light chain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 327, 329, 331, 333, 335, 337, 650, 652, 654, 656, 658, 660, 670-673, 701-712, 721-788, 809-836, and 841-908. [Invention 1031] The activatable antibody comprises a combination of amino acid sequences, and the combination of amino acid sequences is selected from a single column in Table D. Now, regarding the given combination... (a) The heavy chain of AB contains the amino acid sequence of the VH CDR sequence corresponding to a given combination in a single column listed in Table D, (b) The light chain of AB contains the amino acid sequence of the VL CDR sequence corresponding to a given combination in a single column listed in Table D, (c) MM includes the amino acid sequence of the mask sequence (MM) corresponding to a given combination in a single column listed in Table D, and (d) The CM contains the amino acid sequence of the substrate sequence (CM) corresponding to a given combination in a single column listed in Table D. An activatable antibody according to any of the present invention items 1006 to 1030. [Invention 1032] The activatable antibody comprises a combination of amino acid sequences, where, given a given combination of amino acid sequences, (a) The heavy chain of AB contains an amino acid sequence of a VH sequence or VH CDR sequence selected from the group consisting of VH sequences or VH CDR sequences listed in the corresponding column of Table E, (b) The light chain of AB contains an amino acid sequence of a VL sequence or VL CDR sequence selected from the group consisting of VL sequences or VL CDR sequences listed in the corresponding column of Table E, (c) MM includes the amino acid sequence of a mask sequence (MM) selected from the group consisting of MM sequences listed in the corresponding column of Table E, and (d) The CM contains the amino acid sequence of a substrate sequence (CM) selected from the group consisting of CM sequences listed in the corresponding column of Table E. An activatable antibody according to any of the present invention items 1006 to 1030. [Invention 1033] An activatable antibody comprising an antibody that specifically binds to mammalian CD71 or its antigen-binding fragment (AB), MM, and CM, SEQ ID NO: Heavy chain sequence with 325 or 699; Light chain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 327, 329, 331, 333, 335, 337, 650, 652, 654, 656, 658, 660, 670-673, 701-712, 721-788, 809-836, and 841-908. said activatable antibody. [Invention 1034] Antibodies or their antigen-binding fragments (ABs) that specifically bind to mammalian CD71; SEQ ID NO: MM containing amino acid sequences selected from the groups consisting of 16-295 and 297-314; and SEQ ID NO: CM containing an amino acid sequence selected from the group consisting of 356-423, 680-698, 713, 714, and 789-808. An activatable antibody containing the above. [Invention 1035] An activatable antibody according to Invention 1034, wherein MM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 17, and 297-314, and CM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 406-423, 680-698, 713, 714, and 807-808. [Invention 1036] AB VH CDR1 sequence TIFF0007887228000175.tif4128; VH CDR2 sequence TIFF0007887228000176.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000177.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) An activatable antibody according to invention 1034 or 1035, comprising the above. [Invention 1037] An activatable antibody according to any of the invention 1034 to 1036, wherein AB comprises a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8 and 809 to 908. [Invention 1038] An antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB specifically binds to the same epitope as any of the isolated antibodies of the present invention 1001 to 1005 on human CD71 and / or cynomolgus monkey CD71; A masking moiety (MM) that inhibits the binding of AB to CD71 when the activatable antibody is in an uncleaved state; and A cleavable portion (CM) coupled to AB, which is a polypeptide that functions as a substrate for a protease, the CM An activatable anti-CD71 antibody, including... [Invention 1039] An antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB specifically cross-competes with any of the isolated antibodies of the present invention 1001 to 1005 for binding to human CD71 and / or cynomolgus monkey CD71; A masking moiety (MM) that inhibits the binding of AB to CD71 when the activatable antibody is in an uncleaved state; and A cleavable portion (CM) coupled to AB, which is a polypeptide that functions as a substrate for a protease, the CM An activatable anti-CD71 antibody, including... [Invention 1040] A conjugated antibody or a conjugated activatable antibody comprising an antibody according to any of Invention 1001 to 1005 conjugated to an active substance, or an activatable antibody according to any of Invention 1006 to 1039 conjugated to an active substance. [Invention 1041] A conjugated antibody or a conjugated activatable antibody according to the present invention 1040, wherein the active substance is a toxin or a fragment thereof. [Invention 1042] A conjugated antibody or a conjugated activatable antibody according to Invention 1040 or Invention 1041, wherein the active substance is a microtubule inhibitor. [Invention 1043] A conjugated antibody or a conjugated activatable antibody according to Invention 1040 or Invention 1041, wherein the active substance is a nucleic acid damaging active substance. [Invention 1044] A conjugated antibody or conjugated activatable antibody according to Invention 1040 or Invention 1041, wherein the active substance is selected from the group consisting of drastatin or its derivatives, auristatin or its derivatives, meitansinoid or its derivatives, duocalmycin or its derivatives, calicheamycin or its derivatives, and pyrrolobenzodiazepine or its derivatives. [Invention 1045] A conjugated antibody or a conjugated activatable antibody according to any of invention 1040-1042 and 1044, wherein the active substance is auristatin E or a derivative thereof. [Invention 1046] A conjugated antibody or a conjugated activatable antibody according to any of invention 1040-1042 and 1044, wherein the active substance is monomethyl auristatin E (MMAE). [Invention 1047] A conjugated antibody or a conjugated activatable antibody according to any of invention 1040-1042 and 1044, wherein the active substance is monomethyl auristatin D (MMAD). [Invention 1048] A conjugated antibody or a conjugated activatable antibody according to any of the present invention 1040-1042 and 1044, wherein the active substance is a meitansinoid selected from the group consisting of DM1 and DM4. [Invention 1049] A conjugated antibody or a conjugated activatable antibody according to any of invention 1040-1042 and 1044, wherein the active substance is the meitansinoid DM4. [Invention 1050] A conjugated antibody or a conjugated activatable antibody according to any of Invention 1040, 1041, 1043, and 1044, wherein the active substance is duocalmycin. [Invention 1051] A conjugated antibody or a conjugated activatable antibody according to any of the present invention 1040 to 1050, wherein the active substance is conjugated to AB via a linker. [Invention 1052] A conjugated antibody or conjugated activatable antibody according to any of the invention 1040 to 1051, wherein the linker that conjugates the active substance to AB includes an SPDB portion, a vc portion, or a PEG2-vc portion. [Invention 1053] A conjugated antibody or conjugated activatable antibody according to any of Invention 1040 to 1052, wherein the linker and AB conjugated toxin comprises an SPDB-DM4 moiety, a vc-MMAD moiety, a vc-MMAE moiety, a vc-duocalmycin moiety, or a PEG2-vc-MMAD moiety. [Invention 1054] A conjugated antibody or a conjugated activatable antibody according to the present invention 1051, wherein the linker is a cleavable linker. [Invention 1055] A conjugated antibody or a conjugated activatable antibody according to the present invention 1051, wherein the linker is a non-cleavable linker. [Invention 1056] A conjugated antibody or a conjugated activatable antibody according to any of the present invention 1040 to 1055, wherein the active substance is a detectable portion. [Invention 1057] A conjugated antibody or a conjugated activatable antibody according to the present invention 1056, wherein the detectable portion is a diagnostic active substance. [Invention 1058] An antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB specifically binds to human CD71 and cynomolgus monkey CD71; A masking moiety (MM) that inhibits the binding of AB to CD71 when the activatable antibody is in an uncleaved state; A cleavable moiety (CM) coupled to AB, which is a polypeptide that functions as a substrate for a protease; and Active substance conjugated to AB A conjugated, activatable antibody that, in its activated state, binds to CD71. [Invention 1059] A conjugated activatable antibody according to Invention 1058, wherein the active substance is selected from the group consisting of drastatin or its derivatives, auristatin or its derivatives, meitansinoid or its derivatives, duocalmycin or its derivatives, calicheamycin or its derivatives, and pyrrolobenzodiazepine or its derivatives. [Invention 1060] A conjugated, activatable antibody according to Invention 1058 or 1059, wherein the active substance is selected from the group consisting of auristatin E, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), meitansinoid DM4, meitansinoid DM1, duocalmycin, pyrrolobenzodiazepine, and pyrrolobenzodiazepine dimer. [Invention 1061] A conjugated activatable antibody according to any of the present invention 1058 to 1060, wherein the active substance is conjugated to AB via a linker. [Invention 1062] A conjugated antibody or conjugated activatable antibody according to any of Invention 1058 to 1061, wherein the linker that conjugates the active substance to AB includes an SPDB portion, a vc portion, or a PEG2-vc portion. [Invention 1063] A conjugated antibody or conjugated activatable antibody according to any of Invention 1058 to 1062, wherein the linker and AB conjugated toxin comprises an SPDB-DM4 moiety, a vc-MMAD moiety, a vc-MMAE moiety, a vc-duocalmycin moiety, or a PEG2-vc-MMAD moiety. [Invention 1064] The antibody or its antigen-binding fragment VH CDR1 sequence TIFF0007887228000178.tif4128; VH CDR2 sequence TIFF0007887228000179.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000180.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) A conjugated, activatable antibody according to any of the inventions 1058 to 1063, including the present invention. [Invention 1065] A conjugated, activatable antibody according to any of the present invention 1058 to 1064, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8 and 809 to 908. [Invention 1066] A conjugated, activatable antibody according to any of the Invention 1058-1065, wherein MM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 16-295 and 297-314. [Invention 1067] A conjugated, activatable antibody according to any of the Invention 1058-1066, wherein MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 17, and 297-314. [Invention 1068] A conjugated, activatable antibody according to any of the Invention 1058-1067, wherein CM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 356-423, 680-698, 713, 714, and 789-808. [Invention 1069] A conjugated, activatable antibody according to any of the Invention 1058-1068, wherein CM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 406-423, 680-698, 713, 714, and 807-808. [Invention 1070] The activatable antibody comprises a combination of amino acid sequences, and the combination of amino acid sequences is selected from a single column in Table D. Now, regarding the given combination... (a) The heavy chain of AB contains the amino acid sequence of the VH CDR sequence corresponding to a given combination in a single column listed in Table D, (b) The light chain of AB contains the amino acid sequence of the VL CDR sequence corresponding to a given combination in a single column listed in Table D, (c) MM includes the amino acid sequence of the mask sequence (MM) corresponding to a given combination in a single column listed in Table D, and (d) The CM contains the amino acid sequence of the substrate sequence (CM) corresponding to a given combination in a single column listed in Table D. A conjugated, activatable antibody according to any of invention 1058 to 1069. [Invention 1071] The activatable antibody comprises a combination of amino acid sequences, where, given a given combination of amino acid sequences, (a) The heavy chain of AB contains an amino acid sequence of a VH sequence or VH CDR sequence selected from the group consisting of VH sequences or VH CDR sequences listed in the corresponding column of Table E, (b) The light chain of AB contains an amino acid sequence of a VL sequence or VL CDR sequence selected from the group consisting of VL sequences or VL CDR sequences listed in the corresponding column of Table E, (c) MM includes the amino acid sequence of a mask sequence (MM) selected from the group consisting of MM sequences listed in the corresponding column of Table E, and (d) The CM contains the amino acid sequence of a substrate sequence (CM) selected from the group consisting of CM sequences listed in the corresponding column of Table E. A conjugated, activatable antibody according to any of invention 1058 to 1070. [Invention 1072] SEQ ID NO: Heavy chain containing amino acid sequence 325 or 699; and Light chain containing amino acid sequences selected from the group consisting of SEQ ID NO: 327, 329, 331, 333, 335, 337, 650, 652, 654, 656, 658, 660, 670-673, 701-712, 721-788, 809-836, and 841-908. A conjugated activatable antibody according to any of the inventions 1058 to 1071, including the present invention. [Invention 1073] Conjugated antibodies including the following: (a) An antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB is (i) VH CDR1 sequence TIFF0007887228000181.tif4128; VH CDR2 sequence TIFF0007887228000182.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000183.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) or (ii) Heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5, and light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8 The antibody or its antigen-binding fragment (AB) includes; (b) A conjugated active substance to AB, selected from the group consisting of auristatin E, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), meitansinoid DM4, meitansinoid DM1, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, and duocalmycin. [Invention 1074] An antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB specifically binds to human CD71 and cynomolgus monkey CD71; A masking moiety (MM) that inhibits the binding of AB to CD71 when the activatable antibody is in an uncleaved state; A cleavable moiety (CM) coupled to AB, which is a polypeptide that functions as a substrate for a protease; and Active substance conjugated to AB A conjugated activatable antibody that includes, in its activated state, binds to CD71, AB is as follows: (i) VH CDR1 sequence TIFF0007887228000184.tif4128; VH CDR2 sequence TIFF0007887228000185.tif4128; VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11) VL CDR1 sequence TIFF0007887228000186.tif4128; VL CDR2 sequence STSNLAS (SEQ ID NO: 14) ; and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) or (ii) A heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to 5, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to 8, or (iii) A heavy chain containing an amino acid sequence of SEQ ID NO: 325 or 699, and a light chain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 327, 329, 331, 333, 335, 337, 650, 652, 654, 656, 658, 660, 670-673, 701-712, 721-788, 809-836, and 841-908. Includes; and The active substance is selected from the group consisting of auristatin E, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), meitansinoid DM4, meitansinoid DM1, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, and duocalmycin. Said activatable antibody. [Invention 1075] A conjugated, activatable antibody according to Invention 1074, wherein MM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 16-295 and 297-314. [Invention 1076] A conjugated activatable antibody according to Invention 1074 or Invention 1075, wherein MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 17, and 297-314. [Invention 1077] A conjugated, activatable antibody according to any of the Invention 1074-1076, wherein CM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 356-423, 680-698, 713, 714, and 789-808. [Invention 1078] A conjugated, activatable antibody according to any of the Invention 1074-1077, wherein CM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 406-423, 680-698, 713, 714, and 807-808. [Invention 1079] A conjugated activatable antibody according to any of invention 1074 to 1078, wherein the active substance is conjugated to AB via a linker, and the linker conjugating the active substance to AB includes an SPDB portion, a vc portion, or a PEG2-vc portion. [Invention 1080] A conjugated antibody or conjugated activatable antibody according to any of Invention 1074 to 1079, wherein the linker and AB conjugated toxin comprises an SPDB-DM4 moiety, a vc-MMAD moiety, a vc-MMAE moiety, a vc-duocalmycin moiety, or a PEG2-vc-MMAD moiety. [Invention 1081] In the activated state, it is an antibody or its antigen-binding fragment (AB) that binds to CD71; and Toxins conjugated to AB via the linker A conjugated activatable antibody or a conjugated antibody containing, The conjugated activatable antibody or conjugated antibody comprises an amino acid sequence, a linker, and a toxin selected from a single column of Table F, wherein for a given combination, (a) AB comprises a heavy chain containing an amino acid sequence of a heavy chain sequence or a heavy chain variable domain sequence corresponding to a given combination in a single column listed in Table F, (b) AB comprises a light chain containing an amino acid sequence of a light chain sequence or a light chain variable domain sequence corresponding to a given combination in a single column listed in Table F, and (c) The linker and toxin include a linker and toxin corresponding to a given combination in a single column listed in Table F, The conjugated activatable antibody or the conjugated antibody. [Invention 1082] A pharmaceutical composition comprising an antibody according to any of Invention 1001 to 1005, an activatable antibody according to any of Invention 1006 to 1039, or a conjugated antibody or conjugated activatable antibody according to any of Invention 1040 to 1081, and a carrier. [Invention 1083] A pharmaceutical composition of the present invention 1082, comprising further active substances. [Invention 1084] A pharmaceutical composition of the present invention 1083, wherein the further active substance is a therapeutic active substance. [Invention 1085] An isolated nucleic acid molecule encoding an isolated antibody according to any of Invention 1001 to 1005 or an activatable antibody according to any of Invention 1006 to 1081. [Invention 1086] A vector comprising an isolated nucleic acid molecule according to the present invention 1085. [Invention 1087] A method for producing an antibody or an activatable antibody by culturing cells containing a nucleic acid molecule of the present invention 1085 or a vector of the present invention 1086 under conditions that result in the expression of an antibody or an activatable antibody. [Invention 1088] A method for producing an activatable antibody that binds to CD71 in its activated state, (a) A step of culturing cells containing a nucleic acid construct encoding an activatable antibody under conditions that result in the expression of an activatable antibody, including any of the activatable antibodies described in 1006 to 1039 of the present invention; and (b) Step of recovering the activatable antibody The method, including the method described above. [Invention 1089] A method for treating, alleviating, or slowing the progression of a disorder or disease in which affected cells express CD71, comprising the step of administering a therapeutically effective amount of an antibody according to any of Invention 1001-1005, an activatable antibody according to any of Invention 1006-1039, a conjugated antibody or conjugated activatable antibody according to any of Invention 1040-1081, or a pharmaceutical composition according to any of Invention 1082-1084 to a subject in need. [Invention 1090] The method of the present invention 1089, wherein the disorder or disease is cancer. [Invention 1091] A method for treating, alleviating, or slowing the progression of a disorder or disease associated with CD71-expressing cells, comprising the step of administering a therapeutically effective amount of an antibody according to any of Invention 1001-1005, an activatable antibody according to any of Invention 1006-1039, a conjugated antibody or conjugated activatable antibody according to any of Invention 1040-1081, or a pharmaceutical composition according to any of Invention 1082-1084 to a subject in need. [Invention 1092] The method of the present invention 1091, wherein the disorder or disease associated with cells expressing CD71 is cancer. [Invention 1093] The method of Invention 1090 or Invention 1092, wherein the cancer is adenocarcinoma, bile duct (biliary tract) cancer, bladder cancer, bone cancer, breast cancer, triple-negative breast cancer, Her2-negative breast cancer, carcinoid cancer, cervical cancer, bile duct cancer, colorectal cancer, colon cancer, endometrial cancer, glioma, head and neck cancer, head and neck squamous cell carcinoma, leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, melanoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, metastatic castration-resistant prostate cancer, kidney cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, genitourinary cancer, or urothelial carcinoma. [Invention 1094] A method for inhibiting or reducing the growth, proliferation, or metastasis of mammalian CD71-expressing cells, comprising the step of administering a therapeutically effective amount of an antibody according to any of Invention 1001-1005, an activatable antibody according to any of Invention 1006-1039, a conjugated antibody or conjugated activatable antibody according to any of Invention 1040-1081, or a pharmaceutical composition according to any of Invention 1082-1084 to a subject in need. [Invention 1095] A method for inhibiting, blocking, or preventing the binding of a natural ligand to mammalian CD71, comprising the step of administering a therapeutically effective amount of an antibody according to any of Invention 1001-1005, an activatable antibody according to any of Invention 1006-1039, a conjugated antibody or conjugated activatable antibody according to any of Invention 1040-1081, or a pharmaceutical composition according to any of Invention 1082-1084 to a subject in need. [Invention 1096] The method of the present invention 1095, wherein the natural ligand is transferrin. [Invention 1097] Any method according to items 1094 to 1096 of the present invention, wherein the expression and / or activity of mammalian CD71 is abnormal. [Invention 1098] A method according to any one of the present invention 1089 to 1097, further comprising the step of administering a further active substance. [Invention 1099] The method of the present invention 1098, wherein the further active substance is a therapeutic active substance. [Brief explanation of the drawing]

[0270] [Figure 1] This graph shows the ability of the anti-CD71 antibody of this disclosure to bind to human CD71. [Figure 2-1] This graph shows the ability of various activatable anti-CD71 antibodies of this disclosure to bind to human CD71. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 3]Figure 3A: A graph showing the ability of the non-conjugate and conjugate anti-CD71 activatable antibodies of this disclosure to bind to human CD71 when the activatable antibody is intact or proteolytically activated. Figure 3B: A graph showing the in vitro cytotoxicity of the conjugate anti-CD71 activatable antibody and the activated conjugate anti-CD71 activatable antibody of this disclosure. [Figure 4] This graph shows the efficacy of the conjugated, activatable anti-CD71 antibody of this disclosure in NCI-H292 xenograft tumors. [Figure 5] This graph shows the efficacy of the conjugated, activatable anti-CD71 antibody of this disclosure in xenograft tumors of breast cancer HCC1806. [Figure 6A] Figure 6A: A graph showing that the anti-CD71 antibody of this disclosure binds to primary kidney epithelial cells of cynomolgus monkeys. [Figure 6B] Figure 6B: This graph shows that the anti-CD71 antibody of this disclosure binds to cynomolgus monkey CD71 but not to mouse CD71. [Figure 6C] Figure 6C: A graph showing that the mouse and humanized anti-CD71 antibodies of this disclosure bind to human BxPC3 cell lines. [Figure 7] This graph shows that a single dose of the activatable anti-CD71 antibody of this disclosure demonstrates an extended half-life compared to the CD71 parent antibody in cynomolgus monkeys. [Figure 8] This graph shows the pharmacokinetics of the conjugated, activatable anti-CD71 antibody of this disclosure when administered to cynomolgus monkeys. [Figure 9] This graph shows the tolerability of the conjugated, activatable anti-CD71 antibody of this disclosure when administered to cynomolgus monkeys. [Figure 10] This graph shows an exemplary assay method for the binding of recombinant CD71 to its ligand transferrin. [Figure 11A] Figure 11A: A graph showing the competitive binding assay method for CD71 between the anti-CD71 antibody and transferrin of this disclosure. [Figure 11B] Figure 11B: A graph showing a binding assay method for transferrin binding to CD71, demonstrating the inhibition of binding by the anti-CD71 antibody of this disclosure. [Figure 12A] This paper presents exemplary immunohistochemical (IHC) assays for determining CD71 expression levels in various primary and metastatic cancer tissue types. [Figure 12B] This paper presents exemplary immunohistochemical (IHC) assays for determining CD71 expression levels in various primary and metastatic cancer tissue types. [Figure 12C] This paper presents exemplary immunohistochemical (IHC) assays for determining CD71 expression levels in various primary and metastatic cancer tissue types. [Figure 13A] This disclosure presents an exemplary efficacy study of the activatable anti-CD71 conjugate antibody (AADC) using non-Hodgkin lymphoma tumor xenografts in mice. [Figure 13B] This disclosure presents an exemplary efficacy study of the activatable anti-CD71 conjugate antibody (AADC) using non-Hodgkin lymphoma tumor xenografts in mice. [Figure 13C] This disclosure presents an exemplary efficacy study of the activatable anti-CD71 conjugate antibody (AADC) using non-Hodgkin lymphoma tumor xenografts in mice. [Figure 14A] This disclosure presents an exemplary tolerability study of the anti-CD71 conjugate-activatable antibody (AADC) in cynomolgus monkeys. [Figure 14B] This disclosure presents an exemplary tolerability study of the anti-CD71 conjugate-activatable antibody (AADC) in cynomolgus monkeys. [Figure 14C] This disclosure presents an exemplary tolerability study of the anti-CD71 conjugate-activatable antibody (AADC) in cynomolgus monkeys. [Figure 14D] This disclosure presents an exemplary tolerability study of the anti-CD71 conjugate-activatable antibody (AADC) in cynomolgus monkeys. [Figure 14E] This disclosure presents an exemplary tolerability study of the anti-CD71 conjugate-activatable antibody (AADC) in cynomolgus monkeys. [Figure 14F] This disclosure presents an exemplary tolerability study of the anti-CD71 conjugate-activatable antibody (AADC) in cynomolgus monkeys. [Figure 14G] This disclosure presents an exemplary tolerability study of the anti-CD71 conjugate-activatable antibody (AADC) in cynomolgus monkeys. [Figure 15] This disclosure presents exemplary CD71 expression levels in various cell lines using an anti-CD71 antibody, as well as cytotoxic studies in the same cell lines using the anti-CD71 antibody of this disclosure in combination with a drug-conjugated secondary antibody. [Figure 16] Figures 16A–16D illustrate exemplary studies of the ability of various anti-CD71 activatable antibodies of this disclosure to bind to human CD71 on various human-derived cell lines. [Figure 17A] Exemplary studies of the cytotoxicity of the anti-CD71 antibody drug conjugate of this disclosure against various cell lines are presented. [Figure 17B] Exemplary studies of the cytotoxicity of the anti-CD71 antibody drug conjugate of this disclosure against various cell lines are presented. [Figure 17C] Exemplary studies of the cytotoxicity of the anti-CD71 antibody drug conjugate of this disclosure against various cell lines are presented. [Figure 17D] Exemplary studies of the cytotoxicity of the anti-CD71 antibody drug conjugate of this disclosure against various cell lines are presented. [Figure 18] This disclosure presents exemplary studies of the cytotoxicity of various activatable anti-CD71 antibody drug conjugates against non-Hodgkin lymphoma-derived Raji cell lines. [Figure 19] This disclosure presents exemplary studies of the ability of various anti-CD71 activatable antibodies to bind to human CD71. [Figure 20A]This disclosure presents exemplary studies of the efficacy of the anti-CD71 antibody-drug conjugates (ADCs) and anti-CD71 activatable antibody-drug conjugates (AADCs) in patient-derived xenograft tumor models. [Figure 20B] This disclosure presents exemplary studies of the efficacy of the anti-CD71 antibody-drug conjugates (ADCs) and anti-CD71 activatable antibody-drug conjugates (AADCs) in patient-derived xenograft tumor models. [Figure 21A] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 antibody drug conjugates against various colorectal cancer-derived cell lines. [Figure 21B] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 antibody drug conjugates against various colorectal cancer-derived cell lines. [Figure 21C] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 antibody drug conjugates against various colorectal cancer-derived cell lines. [Figure 21D] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 antibody drug conjugates against various colorectal cancer-derived cell lines. [Figure 21E] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 antibody drug conjugates against various colorectal cancer-derived cell lines. [Figure 21F] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 antibody drug conjugates against various colorectal cancer-derived cell lines. [Figure 21G] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 antibody drug conjugates against various colorectal cancer-derived cell lines. [Figure 21H] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 antibody drug conjugates against various colorectal cancer-derived cell lines. [Figure 22] Figures 22A and 22B illustrate exemplary studies of the cytotoxicity of various anti-CD71 antibody-drug conjugates and activatable drug conjugates of this disclosure against HT29 colorectal cancer-derived cell lines. [Figure 23]Figures 23A and 23B show exemplary efficacy studies of the activatable anti-CD71 conjugate antibody (AADC) of this disclosure using non-Hodgkin lymphoma tumor xenografts in mice. [Figure 24] Figures 24A and 24B show exemplary efficacy studies of the activatable anti-CD71 conjugate antibody (AADC) of this disclosure using non-small cell lung cancer (NCI-H292) tumor xenografts in mice. [Figure 25] This disclosure presents an exemplary efficacy study of the activatable anti-CD71 conjugate antibody (AADC) using xenografts of pancreatic cancer (BxPC3) tumors in mice. [Figure 26] Figures 26A and 26B illustrate exemplary efficacy studies of the activatable anti-CD71 conjugate antibody (AADC) of this disclosure using patient-derived tumor xenografts (non-Hodgkin lymphoma) in mice. [Figure 27] Figures 27A and 27B show exemplary efficacy studies of the activatable anti-CD71 conjugate antibody (AADC) of this disclosure using patient-derived tumor xenografts in mice. [Figure 28] This disclosure presents exemplary studies of the ability of various anti-CD71 antibodies and activatable antibodies to bind to human CD71. [Figure 29] Figures 29A–29C illustrate exemplary studies of the ability of various anti-CD71 activatable antibodies and activatable antibody-drug conjugates of this disclosure to bind to human CD71 on various human-derived cell lines. [Figure 30A] This disclosure presents exemplary studies of the ability of various anti-CD71 activatable antibodies to bind to human CD71 in vitro and on various human-derived cell lines. [Figure 30B] This disclosure presents exemplary studies of the ability of various anti-CD71 activatable antibodies to bind to human CD71 in vitro and on various human-derived cell lines. [Figure 30C] This disclosure presents exemplary studies of the ability of various anti-CD71 activatable antibodies to bind to human CD71 in vitro and on various human-derived cell lines. [Figure 30D] This disclosure presents exemplary studies of the ability of various anti-CD71 activatable antibodies to bind to human CD71 in vitro and on various human-derived cell lines. [Figure 31A] Figures 31A–31J illustrate exemplary studies of the cytotoxicity of various anti-CD71 activatable drug conjugates of this disclosure against various cancer-derived cell lines. [Figure 31B] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 activatable drug conjugates against various cancer-derived cell lines. [Figure 31C] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 activatable drug conjugates against various cancer-derived cell lines. [Figure 31D] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 activatable drug conjugates against various cancer-derived cell lines. [Figure 31E] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 activatable drug conjugates against various cancer-derived cell lines. [Figure 31F] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 activatable drug conjugates against various cancer-derived cell lines. [Figure 31G] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 activatable drug conjugates against various cancer-derived cell lines. [Figure 31H] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 activatable drug conjugates against various cancer-derived cell lines. [Figure 31I] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 activatable drug conjugates against various cancer-derived cell lines. [Figure 31J] This disclosure presents exemplary studies of the cytotoxicity of various anti-CD71 activatable drug conjugates against various cancer-derived cell lines. [Figure 32]Figures 32A and 32B illustrate exemplary studies of the ability of the anti-CD71 antibodies and antibody-drug conjugates of this disclosure to bind to human CD71 on various human-derived small cell lung cancer cell lines. [Figure 33] Figures 33A and 33B illustrate exemplary studies of the antiproliferative effects of the anti-CD71 antibody of this disclosure against various cancer-derived cell lines. [Figure 34] This paper presents an exemplary study of CD71 expression levels in multiple patient-derived metastatic cancer samples. [Figure 35] This paper presents an exemplary study of the in vivo binding of the anti-CD71 antibody of this disclosure to tumors in a mouse model of pancreatic cancer. [Figure 36] This paper presents an exemplary study of the in vivo binding of the anti-CD71 antibody of this disclosure to tumors in a mouse model of metastatic breast cancer. [Figure 37] This disclosure presents an exemplary study of in-situ binding of the anti-CD71 antibody in a lung cancer xenograft model. [Modes for carrying out the invention]

[0271] Detailed description of the invention This disclosure provides monoclonal antibodies (mAbs) and activatable monoclonal antibodies that specifically bind to CD71, also known as transferrin receptor protein 1 (TfR1). In some embodiments, the monoclonal antibodies and activatable monoclonal antibodies are internally translocated by CD71-containing cells. The use of the term “CD71” is intended to encompass any variant thereof, such as CD-71 and / or CD71, as non-limiting examples, and all variants are used interchangeably herein.

[0272] CD71 is a transmembrane glycoprotein that primarily binds to transferrin. CD71 is essential for cellular homeostasis. It is continuously recycled through ligand-mediated endocytosis, where transferrin is the primary ligand. CD71 is also known to be ubiquitously expressed on dividing cells.

[0273] Abnormal expression and / or activity of CD71 and CD71-related signaling are linked to the pathogenesis of many diseases and disorders, including cancer. CD71 is overexpressed in many cancers, including both solid tumors and hematological malignancies. CD71 has widespread cell surface expression. In malignant cells, CD71 mediates higher iron uptake necessary for cell division. CD71 is also associated with poor prognosis in leukemia.

[0274] CD71 is a desirable target because it is widely found in the signs of multiple cancers.

[0275] This disclosure provides anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies that are useful in methods for treating, preventing, slowing the progression of, improving and / or alleviating symptoms of diseases or disorders associated with abnormal CD71 expression and / or activity. For example, activatable anti-CD71 antibodies are used in methods for treating, preventing, slowing the progression of, improving and / or alleviating symptoms of cancer or other neoplastic conditions.

[0276] This disclosure provides anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies useful in methods for treating, preventing, slowing the progression of, improving and / or alleviating symptoms of diseases or disorders associated with cells expressing CD71. In some embodiments, the cells are associated with abnormal CD71 expression and / or activity. In some embodiments, the cells are associated with normal CD71 expression and / or activity. For example, activatable anti-CD71 antibodies are used in methods for treating, preventing, slowing the progression of, improving and / or alleviating symptoms of cancer or other neoplastic conditions.

[0277] This disclosure provides anti-CD71 antibodies, conjugated anti-CD71 antibodies, activatable anti-CD71 antibodies, and / or conjugated activatable anti-CD71 antibodies useful in methods for treating, preventing, slowing the progression of, improving and / or alleviating symptoms of diseases or disorders in which affected cells express CD71. In some embodiments, affected cells are associated with abnormal CD71 expression and / or activity. In some embodiments, affected cells are associated with normal CD71 expression and / or activity. For example, activatable anti-CD71 antibodies are used in methods for treating, preventing, slowing the progression of, improving and / or alleviating symptoms of cancer or other neoplastic conditions.

[0278] An activatable anti-CD71 antibody and / or a conjugated activatable anti-CD71 antibody comprises an antibody or antigen-binding fragment that specifically binds to CD71, coupled to a masking moiety (MM) such that the coupling of the MM reduces the ability of the antibody or antigen-binding fragment to bind to CD71. In some embodiments, the MM is coupled via a sequence containing a substrate for a protease, e.g., a protease that co-localizes with CD71 at the treatment site of interest.

[0279] Exemplary activatable anti-CD71 antibodies of the present invention include, for example, activatable antibodies comprising heavy chains and light chains that are or derived from the heavy chain variable sequences and light chain variable sequences shown below (CDR sequences are shown in bold and underlined). TIFF0007887228000187.tif190147

[0280] Examples of activatable anti-CD71 antibodies of the present invention include, for example, an activatable antibody comprising a combination of the following sequences: variable heavy chain complementarity determination region 1 (VH CDR1, also known herein as CDRH1) sequence, variable heavy chain complementarity determination region 2 (VH CDR2, also known herein as CDRH2) sequence, variable heavy chain complementarity determination region 3 (VH CDR3, also known herein as CDRH3) sequence, variable light chain complementarity determination region 1 (VL CDR1, also known herein as CDRL1) sequence, variable light chain complementarity determination region 2 (VL CDR2, also known herein as CDRL2) sequence, and variable light chain complementarity determination region 3 (VL CDR3, also known herein as CDRL3) sequence, wherein at least one CDR sequence is an amino acid sequence VH CDR1 sequence containing TIFF0007887228000188.tif4128; amino acid sequence VH CDR2 sequence containing TIFF0007887228000189.tif4128; amino acid sequence ENWDPGFAF (SEQ ID NO: 11) VH CDR3 sequence containing; amino acid sequence VL CDR1 sequence containing TIFF0007887228000190.tif4128; amino acid sequence STSNLAS (SEQ ID NO: 14) VL CDR2 sequence containing; and amino acid sequence QQRRNYPYT (SEQ ID NO: 15) VL CDR3 sequence including Selected from the group consisting of

[0281] In some embodiments, the activatable anti-CD71 antibody comprises a heavy chain comprising or derived from the heavy chain amino acid sequence shown in PCT Publications WO 2014 / 144060, WO 2014 / 189973, WO 2014 / 020140, U.S. Patents 8,663,598, 8,129,503, 7,736,647, 7,572,895, 4,434,156, U.S. Patent Application Publications US2014114054, US20140212423, US2013177579, US2013045206, US20130216476, US20120282176, and / or Chinese Patent CN101245107B, the contents of which are incorporated herein by reference as a whole.

[0282] In some embodiments, the activatable anti-CD71 antibody includes a heavy chain containing or derived from the heavy chain amino acid sequence shown in Table 12. In some embodiments, the activatable anti-CD71 antibody includes a light chain containing or derived from the heavy chain amino acid sequence shown in Table 12. In some embodiments, the activatable anti-CD71 antibody includes a heavy chain containing or derived from the heavy chain amino acid sequence shown in Table 12, and a light chain containing or derived from the light chain amino acid sequence shown in Table 12. In some embodiments, the activatable anti-CD71 antibody includes a combination of heavy chain variable region and light chain variable region sequences derived from the combination shown in Group A of Table 12. In some embodiments, the activatable anti-CD71 antibody includes a combination of heavy chain variable region and light chain variable region sequences derived from the sequence shown in Group B of Table 12. In some embodiments, the activatable anti-CD71 antibody includes a combination of heavy chain variable region and light chain variable region sequences derived from the sequence shown in Group C of Table 12. In some embodiments, the activatable anti-CD71 antibody includes a combination of heavy chain variable region and light chain variable region sequences derived from the sequences shown in Group D of Table 12. In some embodiments, the activatable anti-CD71 antibody includes a combination of heavy chain variable region and light chain variable region sequences shown in Group E of Table 12. In some embodiments, the activatable anti-CD71 antibody includes a combination of heavy chain variable region and light chain variable region sequences shown in Group F of Table 12. In some embodiments, the activatable anti-CD71 antibody includes a combination of heavy chain variable region and light chain variable region sequences derived from the sequences shown in Group G of Table 12. In some embodiments, the activatable anti-CD71 antibody includes a combination of heavy chain variable region and light chain variable region sequences derived from the sequences shown in Group H of Table 12. In some embodiments, the activatable anti-CD71 antibody includes a combination of heavy chain variable region and light chain variable region sequences shown in Group I of Table 12. In some embodiments, the activatable anti-CD71 antibody includes a heavy chain variable region sequence shown in Group J of Table 12.In some embodiments, the activatable anti-CD71 antibody comprises a heavy chain variable region sequence shown in group J of Table 12, or a combination of heavy chain variable region and light chain variable region sequences shown in group K of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain variable region and light chain variable region sequences derived from the sequences shown in group L of Table 12.

[0283] In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence complementarity-determining region (CDR) sequences derived from the heavy chain sequences shown in Group A of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of light chain sequence CDRs derived from the light chain sequences shown in Group A of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group A of Table 12 and light chain sequence CDRs derived from the heavy chain sequences shown in Group A of Table 12.

[0284] In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group B of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of light chain sequence CDRs derived from the light chain sequences shown in Group B of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group B of Table 12 and light chain sequence CDRs derived from the heavy chain sequences shown in Group B of Table 12.

[0285] In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group C of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of light chain sequence CDRs derived from the light chain sequences shown in Group C of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group C of Table 12 and light chain sequence CDRs derived from the heavy chain sequences shown in Group C of Table 12.

[0286] In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group D of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of light chain sequence CDRs derived from the light chain sequences shown in Group D of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group D of Table 12 and light chain sequence CDRs derived from the heavy chain sequences shown in Group D of Table 12.

[0287] In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group E of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of light chain sequence CDRs derived from the light chain sequences shown in Group E of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group E of Table 12 and light chain sequence CDRs derived from the heavy chain sequences shown in Group E of Table 12.

[0288] In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group F of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of light chain sequence CDRs derived from the light chain sequences shown in Group F of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group F of Table 12 and light chain sequence CDRs derived from the heavy chain sequences shown in Group F of Table 12.

[0289] In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group G of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of light chain sequence CDRs derived from the light chain sequences shown in Group G of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group G of Table 12 and light chain sequence CDRs derived from the heavy chain sequences shown in Group G of Table 12.

[0290] In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group H of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of light chain sequence CDRs derived from the light chain sequences shown in Group H of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group H of Table 12 and light chain sequence CDRs derived from the heavy chain sequences shown in Group H of Table 12.

[0291] In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group I of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of light chain sequence CDRs derived from the light chain sequences shown in Group I of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group I of Table 12 and light chain sequence CDRs derived from the heavy chain sequences shown in Group I of Table 12.

[0292] In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group J of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of light chain sequence CDRs derived from the light chain sequences shown in Group J of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group J of Table 12 and light chain sequence CDRs derived from the heavy chain sequences shown in Group J of Table 12.

[0293] In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group K of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of light chain sequence CDRs derived from the light chain sequences shown in Group K of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group K of Table 12 and light chain sequence CDRs derived from the heavy chain sequences shown in Group K of Table 12.

[0294] In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group L of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of light chain sequence CDRs derived from the light chain sequences shown in Group L of Table 12. In some embodiments, the activatable anti-CD71 antibody comprises a combination of heavy chain sequence CDRs derived from the heavy chain sequences shown in Group L of Table 12 and light chain sequence CDRs derived from the heavy chain sequences shown in Group L of Table 12.

[0295] (Table 12) Variable heavy chain region (VH) and variable light chain region (VL) sequences of activatable antibodies that bind to CD71 TIFF0007887228000191.tif185158TIFF0007887228000192.tif220158TIFF0007887228000193.tif218158TIFF0007887228000194.tif209158TIFF0007887228000195.tif218158TIFF0007887228000196.tif227158TIFF0007887228000197.tif116158(VH / VL = VH concatenated to VL)

[0296] In some embodiments, the activatable anti-CD71 antibody includes the CDR sequence shown in Table 13, a combination of VL CDR sequences (VL CDR1, VL CDR2, VL CDR3) selected from the group consisting of combinations shown in a single column of Table 13, a combination of VH CDR sequences (VH CDR1, VH CDR2, VH CDR3) selected from the group consisting of combinations shown in Table 13, or a combination of VL CDR and VH CDR sequences (VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, VH CDR3) selected from the group consisting of combinations shown in Table 13.

[0297] (Table 13) CDR sequences of antibodies that bind to CD71 and antibodies that can be activated TIFF0007887228000198.tif24158TIFF0007887228000199.tif225158TIFF0007887228000200.tif227158TIFF0007887228000201.tif212158

[0298] In some embodiments, the activatable anti-CD71 antibody is referred to as BA120, as disclosed in U.S. Patent No. 7,736,647, and is a hybridoma deposited on June 14, 2005, under the number CNCM I-3449 with the Collection Nationale de Cultures de Microorganismes (CNCM) (Pasteur Institute, Paris, France, 25, Rue du Docteur Roux, F-75724, Paris, Cedex 15), a hybridoma disclosed in U.S. Patent No. 7,572,895, and is a hybridoma deposited with the ATCC under PTA-6055, PCT Publication WO 2014 / 020140 and WO This includes, or is derived from, antibodies manufactured, secreted, or otherwise produced by hybridomas such as the hybridoma disclosed in 2005 / 111082 and deposited with CNCM on 10 May 2001 under number 1-2665, the hybridoma disclosed in U.S. Patent No. 4,434,156 and deposited with ATCC under HB-8094, and the hybridoma disclosed in U.S. Patent No. 5,648,469 and deposited with ATCC under HB-11011 and HB-11010.

[0299] The anti-CD71 antibodies and ABs in the activatable antibodies of this disclosure specifically bind to CD71 targets, such as mammalian CD71 and / or human CD71. Anti-CD71 antibodies and ABs that bind to the same CD71 epitopes as the antibodies and / or activated activatable antibodies described herein are also included in this disclosure. Anti-CD71 antibodies and ABs that compete with the anti-CD71 antibodies and / or activated anti-CD71 activatable antibodies described herein for binding to CD71 targets, such as human CD71, are also included in this disclosure. Anti-CD71 antibodies and ABs that cross-compete with the anti-CD71 antibodies and / or activated anti-CD71 activatable antibodies described herein for binding to CD71 targets, such as human CD71, are also included in this disclosure.

[0300] The activatable anti-CD71 antibodies provided herein include a masking moiety. In some embodiments, the masking moiety is an amino acid sequence that is coupled to or otherwise added to the anti-CD71 antibody, and the masking moiety is positioned within the activatable anti-CD71 antibody construct such that it reduces the anti-CD71 antibody's ability to specifically bind to CD71. Preferred masking moieties are identified using any of a variety of known techniques. For example, peptide masking moieties are identified using the method described in PCT Publication WO 2009 / 025846 by Daugherty et al., the contents of which are incorporated herein by reference in their entirety.

[0301] The activatable anti-CD71 antibodies provided herein contain a cleavable moiety. In some embodiments, the cleavable moiety 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 techniques. For example, peptide substrates are identified using the methods described in U.S. Patent No. 7,666,817 by Daugherty et al.; U.S. Patent No. 8,563,269 by Stagliano et al.; and PCT Publication WO 2014 / 026136 by La Porte et al., the contents of each of these are incorporated herein by reference in whole. (See also Boulware et al. "Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics." Biotechnol Bioeng. 106.3 (2010): 339-46.)

[0302] Exemplary substrates include, but are not limited to, those that can be cleaved by one or more of the enzymes or proteases listed in Table 4.

[0303] (Table 4) Exemplary proteases and / or enzymes TIFF0007887228000202.tif201160

[0304] The activatable anti-CD71 antibodies described herein overcome the limitations of antibody therapies, particularly those known to be toxic to at least some extent in vivo. Target-mediated toxicity is a major limitation in the development of therapeutic antibodies. The activatable anti-CD71 antibodies provided herein are designed to address the toxicity associated with the inhibition of targets in normal tissues by conventional therapeutic antibodies. These activatable anti-CD71 antibodies remain masked until they are proteolytically activated at the site of disease. Starting from an anti-CD71 antibody as the parent therapeutic antibody, the activatable anti-CD71 antibodies of the present invention were prepared by coupling the antibody to an inhibitory mask through a linker incorporating a protease substrate.

[0305] When AB is modified with MM and in the presence of a target, the specific binding of AB to that target is reduced or inhibited compared to the specific binding of unmodified AB or parent AB to the target.

[0306] K of AB modified with MM against the target d This is the K of AB or parent AB that is not modified by MM against the target. dAt least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 times or greater than that, or 5-10, 10-100, 10-10,000, 10-100,000, 10-10,000,000, 10-10,000,000 1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-10,000,000, 10,000-100,000, 10,000-100,000, 10,000-10,000,000, 10,000-10,000,000, or 100,000-10,000,000 times larger. Conversely, the binding affinity of MM-modified AB to the target is at least 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 times greater than or equal to the binding affinity of unmodified AB or parent AB to the target, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,0 00, 10~1,000,000, 10~10,000,000, 100~1,000, 100~10,000, 100~100,000, 100~1,000,000, 100~10,000,000, 1,000~10,000, 1,000~100,000, 1,000~10,000,000, 10,000~100,000, 10,000~10,000,000, 10,000~10,000,000, 100,000~1,000,000, or 100,000~10,000,000 times lower.

[0307] The dissociation constant (K) of MM with respect to AB d ) is generally the K of AB against the target.d Larger than . K of MM relative to AB d , is the K of AB against the target d It can be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 times, or even 10,000,000 times greater than AB. Conversely, the binding affinity of MM to AB is generally lower than the binding affinity of AB to the target. The binding affinity of MM to AB is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 times, or even 10,000,000 times lower than the binding affinity of AB to the target.

[0308] In some embodiments, the dissociation constant (Kd) of MM with respect to AB is the K of AB with respect to the target. d This is almost equivalent. In some embodiments, the dissociation constant (Kd) of MM with respect to AB is less than or equal to the dissociation constant of AB with respect to the target.

[0309] In some embodiments, the dissociation constant (Kd) of MM with respect to AB is smaller than the dissociation constant of AB with respect to the target.

[0310] In some embodiments, the dissociation constant (Kd) of MM with respect to AB is greater than the dissociation constant of AB with respect to the target.

[0311] In some embodiments, MM has a Kd of binding to AB that is less than or equal to the Kd of binding of AB to the target.

[0312] In some embodiments, MM has a Kd of binding to AB that is greater than or equal to the Kd of binding of AB to the target.

[0313] In some embodiments, MM has a Kd for binding to AB that is approximately equivalent to the Kd for binding of AB to the target.

[0314] In some embodiments, MM has a Kd for binding to AB that is smaller than the Kd for binding of AB to the target.

[0315] In some embodiments, MM has a Kd greater for binding to AB than for binding to the target.

[0316] In some embodiments, MM has a Kd for binding to AB that is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 times greater than the Kd for binding to AB to the target. In some embodiments, MM has a Kd for binding to AB that is 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1,000, or 100-1,000 times greater than the Kd for binding to AB to the target.

[0317] In some embodiments, MM has a lower affinity for binding to AB than the binding affinity of AB to the target.

[0318] In some embodiments, MM has an affinity for binding to AB that is less than or equal to the binding affinity of AB to the target.

[0319] In some embodiments, MM has an affinity for binding to AB that is approximately equivalent to the binding affinity of AB to the target.

[0320] In some embodiments, MM has an affinity for binding to AB that is greater than or equal to the binding affinity of AB to the target.

[0321] In some embodiments, MM has a higher affinity for binding to AB than the binding affinity of AB to the target.

[0322] In some embodiments, MM has an affinity for binding to AB that is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 times lower than the binding affinity of AB to the target. In some embodiments, MM has an affinity for binding to AB that is 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1,000, or 100-1,000 times lower than the binding affinity of AB to the target. In some embodiments, MM has an affinity for binding to AB that is 2-20 times lower than the binding affinity of AB to the target. In some embodiments, MM that is not covalently linked to AB and is present in equimolar concentrations with AB does not inhibit the binding of AB to the target.

[0323] When AB is modified with MM and in the presence of a target, the specific binding of AB to that target is reduced or inhibited compared to the specific binding of unmodified AB or parent AB to the target. Compared to the binding of MM-modified AB to a target or the binding of parental AB, the ability of MM-modified AB to a target can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and even 100% over a period of 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, as measured in vivo or in vitro assays.

[0324] MM inhibits the binding of AB to the target. MM binds to the antigen-binding domain of AB and inhibits the binding of AB to the target. MM can sterically inhibit the binding of AB to the target. MM can allosterically inhibit the binding of AB to its target. In these embodiments, when AB is modified or coupled to MM and in the presence of the target, when measured in vivo or in vitro assays, the binding is measured 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. For longer periods, there is no or virtually no binding of AB to the target, or the binding of AB to the target is 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% or less compared to the binding of AB, parent AB, or AB not coupled to MM to the target.

[0325] When AB is coupled to or modified by MM, MM “masks,” reduces, or otherwise inhibits the specific binding of AB to its target. When AB is coupled to or modified by MM, such coupling or modification can result in a structural change that reduces or inhibits AB’s ability to specifically bind to its target.

[0326] AB coupled to or modified by MM can be represented by the following formula (in order from the amino (N) terminal region to the carboxyl (C) terminal region): (MM)-(AB) (AB)-(MM) (MM)-L-(AB) (AB)-L-(MM) In the formula, MM is a masking portion, AB is an antibody or a fragment of that antibody, and L is a linker. In many embodiments, it may be desirable to insert one or more linkers, such as flexible linkers, into the composition to provide mobility.

[0327] In certain embodiments, MM is not a natural binding partner of AB. In some embodiments, MM has no homology or substantially no homology to any natural binding partner of AB. In some embodiments, the similarity of MM to any natural binding partner of AB is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% or less. In some embodiments, the identity of MM to any natural binding partner of AB is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% or less. In some embodiments, the identity of MM to any natural binding partner of AB is 25% or less. In some embodiments, the identity of MM to all natural binding partners of AB is 50% or less. In some embodiments, the identity of MM to all natural binding partners of AB is 20% or less. In some embodiments, the identity of MM to all natural binding partners of AB is 10% or less.

[0328] In some embodiments, the activatable antibody comprises an antibody (AB) modified by a masking moiety (MM) and also containing one or more cleavable moieties (CMs). Such an activatable antibody exhibits activatable / switchable binding to the target of the AB. Generally, the activatable antibody comprises an antibody or antibody fragment (AB) modified by or coupled to a masking moiety (MM) and a modifiable or cleavable moiety (CM). In some embodiments, the CM contains an amino acid sequence that acts as a substrate for at least one protease.

[0329] The elements of the activatable antibody are positioned such that AB binds to the target in a cleaved (or relatively active) state and in the presence of the target, while the activatable antibody is in an uncleaved (or relatively inactive) state and in the presence of the target, the specific binding of AB to that target is reduced or inhibited. The specific binding of AB to that target may be reduced due to the inhibition or masking of AB's ability to specifically bind to that target by the MM.

[0330] K of AB modified with MM and CM against the target d This refers to the K of AB or parent AB that is not modified with MM and CM against the target. dAt least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 times or more, or 5-10, 10-100, 10-10,000, 10-100,000, 10-10,000,000, 10-10,000,000, 10- 1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-10,000,000, 10,000-100,000, 10,000-100,000, 10,000-10,000,000, 10,000-10,000,000, or 100,000-10,000,000 times larger. Conversely, the binding affinity of MM and CM-modified AB to the target is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 times or greater than the binding affinity of MM and CM-modified AB or parent AB to the target, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,0 00, 10~1,000,000, 10~10,000,000, 100~1,000, 100~10,000, 100~100,000, 100~1,000,000, 100~10,000,000, 1,000~10,000, 1,000~100,000, 1,000~10,000,000, 10,000~100,000, 10,000~10,000,000, 10,000~10,000,000, 100,000~1,000,000, or 100,000~10,000,000 times lower.

[0331] When AB is modified with MM and CM and is present 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 AB to its target is reduced or inhibited compared to the specific binding of AB or parent AB that is not modified with MM and CM to the target. Compared to the binding of parental AB or AB not modified with MM and CM to the target, the ability of AB modified with MM and CM to bind to the target can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and even 100% over a period of 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, as measured in vivo or in an in vitro assay.

[0332] As used herein, the term "cleaved state" refers to the state of an activatable antibody after modification of CM by at least one protease. The term "uncleaved state" refers to the state of an activatable antibody without cleavage of CM by a protease. As discussed above, the term "activatable antibody" is used herein to refer to both its uncleaved (natural) state and its cleaved state. In some embodiments, it will be apparent to those skilled in the art that a cleaved activatable antibody may lack MM due to cleavage of CM by a protease, resulting in the release of at least MM (for example, if MM is not covalently attached to the activatable antibody by a disulfide bond between cysteine ​​residues).

[0333] Activatable or switchable means that when an activatable antibody is inhibited, masked, or uncleaved (i.e., in the first conformation), it exhibits a first level of binding to its target, and when it is uninhibited, unmasked, and / or cleaved (i.e., in the second conformation), it exhibits a second level of binding to its target, where the second level of target binding is greater than the first level of binding. Generally, the target access of AB by an activatable antibody is greater in the presence of a cleaving agent, i.e., a protease, that can cleave CM than in the absence of such a cleaving agent. Therefore, when an activatable antibody is in the uncleaved state, AB can be inhibited from target binding and masked from target binding (i.e., the first conformation is such that AB cannot bind to the target), and when it is in the cleaved state, AB is neither inhibited nor masked for target binding.

[0334] The CM and AB of the activatable antibody are selected such that AB corresponds to a binding site to a given target and CM corresponds to a substrate for a protease. In some embodiments, the protease co-localizes with the target at the treatment or diagnostic site in the subject. As used herein, co-localization means being at the same site or in relatively close proximity. In some embodiments, the protease cleaves the CM, producing an activated antibody that binds to a target located near the cleavage site. The activatable antibodies disclosed herein find particular applications when, for example, a protease capable of cleaving a site in the CM, i.e., the protease, is present at a relatively higher level in the target-containing tissue at the treatment or diagnostic site than in the tissue at the untreated site (e.g., in healthy tissue). In some embodiments, the CM of this disclosure is also cleaved by one or more other proteases. In some embodiments, these are one or more other proteases that co-localize with the target and are responsible for cleaving the CM in vivo.

[0335] In some embodiments, the activatable antibody provides a reduction in toxicity and / or adverse side effects that may otherwise be caused by the binding of AB at an untreated site, when AB is not masked or otherwise inhibited from binding to the target.

[0336] Generally, activatable antibodies can be designed by selecting the antibody-branch of interest (AB) and constructing the rest of the activatable antibody such that, when conformally bound, the masking molecule (MM) provides masking of the AB or reduction of the AB's binding to its target. Structural design criteria can be taken into consideration to provide this functional feature.

[0337] An activatable antibody is provided that exhibits a switchable phenotype with a desirable dynamic range for target binding in inhibited versus uninhibited conformations. Dynamic range generally refers to the ratio of (a) the maximum detection level of a parameter under a first set of conditions to (b) the minimum detection level of that parameter under a second set of conditions. For example, in the context of an activatable antibody, the dynamic range refers to the ratio of (a) the maximum detection level of target protein binding to the activatable antibody in the presence of at least one protease capable of cleaving the CM of the activatable antibody to (b) the minimum detection level of target protein binding to the activatable antibody in the absence of the protease. The dynamic range of an activatable antibody can be calculated as the ratio of the dissociation constant of treatment with an activatable antibody cleavage agent (e.g., enzyme) to the dissociation constant of treatment with an activatable antibody cleavage agent. The larger the dynamic range of the activatable antibody, the better the switchable phenotype of the activatable antibody. Activatable antibodies with a relatively high dynamic range (e.g., greater than 1) exhibit a more desirable switching phenotype, such that target protein binding by the activatable antibody occurs to a greater extent (e.g., predominantly) in the presence of a cleavage agent (e.g., an enzyme) capable of cleaving the CM of the activatable antibody than in the absence of the cleavage agent.

[0338] Activatable antibodies can be provided in a variety of structural configurations. Exemplary formulas of activatable antibodies are provided below. It is particularly intended that the N-terminus-C-terminus order of AB, MM, and CM may be reversed within the activatable antibody. It is also particularly intended that CM and MM may be duplicated in their amino acid sequence, for example, so that CM is contained within MM.

[0339] For example, an activatable antibody can be represented by the following formula (in order from the amino (N) terminal region to the carboxyl (C) terminal region): (MM)-(CM)-(AB) (AB)-(CM)-(MM) In the formula, MM is a masking portion, CM is a cleavable portion, and AB is an antibody or a fragment thereof. Although MM and CM are shown as separate components in the above formula, it should be noted that in all exemplary embodiments disclosed herein (including the formula), the amino acid sequences of MM and CM are intended to overlap, for example, such that CM is entirely or partially contained within MM. In addition, the above formula provides additional amino acid sequences that can be positioned at the N-terminus or C-terminus of an activatable antibody element.

[0340] In certain embodiments, MM is not a natural binding partner of AB. In some embodiments, MM has no homology or substantially no homology to any natural binding partner of AB. In some embodiments, the similarity of MM to any natural binding partner of AB is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% or less. In some embodiments, the identity of MM to any natural binding partner of AB is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% or less. In some embodiments, the identity of MM to any natural binding partner of AB is 50% or less. In some embodiments, the identity of MM to all natural binding partners of AB is 25% or less. In some embodiments, the identity of MM to all natural binding partners of AB is 20% or less. In some embodiments, the identity of MM to all natural binding partners of AB is 10% or less.

[0341] In many embodiments, it may be desirable to insert one or more linkers, such as flexible linkers, into the activatable antibody construct to provide mobility at one or more of the MM-CM junctions, CM-AB junctions, or both. For example, AB, MM, and / or CM may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, especially Gly and Ser, particularly Gly) to provide the desired mobility. In such cases, the switchable phenotype of such an activatable antibody construct may benefit from the introduction of one or more amino acids to provide a flexible linker. In addition, when the activatable antibody is provided as a conformationally bound construct, as described below, a flexible linker can be functionally inserted to facilitate the formation and maintenance of a cyclic structure in the uncleaved activatable antibody.

[0342] For example, in a particular embodiment, an activatable antibody may include one of the following formulas (each formula below represents an amino acid sequence in either the N-terminus-C-terminus direction or the C-terminus-N-terminus direction): (MM)-L1-(CM)-(AB) (MM)-(CM)-L2-(AB) (MM)-L1-(CM)-L2-(AB) In the formula, MM, CM, and AB are as defined above; L1 and L2 are independent, optionally present or absent, and are the same or different flexible linkers containing at least one mobile amino acid (e.g., Gly). In addition, the above formula provides additional amino acid sequences that can be positioned at the N-terminus or C-terminus of an activatable antibody element. Examples include, but are not limited to, target moieties (e.g., ligands for cell receptors present in target tissue) and serum half-life extension moieties (e.g., polypeptides that bind to serum proteins such as immunoglobulins (e.g., IgG) or serum albumin (e.g., human serum albumin (HAS))).

[0343] CM is approximately 0.001 to 1500 x 10 4 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 1500 × 10 4 M -1 S -1 It is specifically cleaved by at least one protease at a rate of . In some embodiments, CM is cleaved at approximately 100,000 M -1 S -1 It is specifically cut at a speed of . In some embodiments, CM is approximately 1 × 10⁻² to approximately 1 × 10⁻⁶ M -1 S -1 (That is, approximately 1 × 10⁻⁶ 2 ~Approx. 1×10 6 M -1S -1 It is specifically cut at the speed of ).

[0344] Contact between the enzyme and CM is established for specific enzymatic cleavage. CM can be cleaved if activatable antibodies, including AB coupled to MM and CM, are present in the presence of the target and sufficient enzymatic activity. Sufficient enzymatic activity can refer to the enzyme's ability to contact CM and result in cleavage. It is easily conceivable that the enzyme may be near CM but unable to cleave it due to other cellular factors or protein modifications of the enzyme.

[0345] Linkers suitable for use in the compositions described herein generally provide mobility for modified AB or activatable antibodies, thereby facilitating the inhibition of AB binding to a target. Such linkers are generally referred to as flexible linkers. Suitable linkers can be readily selected and may be any of the following: 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, and various lengths, e.g., 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and may have lengths of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0346] Exemplary flexible linkers include glycine polymer (G)n, glycine-serine polymers (e.g., (GS)n, (GSGGS)n (SEQ ID NO: 339) and (GGGS)n (SEQ ID NO: 340) (wherein n is an integer of at least 1)), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine polymers and glycine-serine polymers are relatively unstructured and can therefore act as neutral linkers between their constituents. Glycine accesses a significantly larger phi-psi space than its equal alanine and is far less restricted than residues with longer side chains (Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, This includes, but is not limited to, TIFF0007887228000203.tif18142. Those skilled in the art will recognize that the design of an activatable antibody may include a linker that is all or partially mobile, so that the linker may include one or more parts that confer a flexible linker and a less mobile structure to provide a desired activatable antibody structure.

[0347] This disclosure also provides compositions and methods comprising an activatable anti-CD71 antibody, wherein the antibody or antibody fragment (AB) specifically binds to CD71, and the AB is coupled to a masking moiety (MM) that reduces the AB's ability to bind to its target. In some embodiments, the activatable anti-CD71 antibody further comprises a cleavable moiety (CM) that is a substrate for a protease. The compositions and methods provided herein enable the addition of one or more active substances to one or more cysteine ​​residues in the AB without impairing the activity of the activatable anti-CD71 antibody (e.g., masking activity, activating activity, or binding activity). In some embodiments, the compositions and methods provided herein enable the addition of one or more active substances to one or more cysteine ​​residues in the AB without reducing or otherwise interfering with one or more disulfide bonds in the MM. The compositions and methods provided herein produce an activatable anti-CD71 antibody conjugated to one or more active substances, e.g., various therapeutic, diagnostic, and / or prophylactic active substances, for example, in some embodiments none of the active substances are conjugated to the MM of the activatable anti-CD71 antibody. The compositions and methods provided herein produce a conjugated activatable anti-CD71 antibody in which the MM retains the ability to effectively and efficiently mask the AB of the activatable antibody in an uncleaved state. The compositions and methods provided herein produce a conjugated activatable anti-CD71 antibody in which the activatable antibody is further activated, i.e., cleaved in the presence of a protease capable of cleaving the CM.

[0348] An activatable anti-CD71 antibody has at least one point of conjugation to the active substance, however, in the methods and compositions provided herein, fewer than all possible points of conjugation are available for conjugation to the active substance. In some embodiments, one or more points of conjugation are sulfur atoms involved in disulfide bonds. In some embodiments, one or more points of conjugation are sulfur atoms involved in interchain disulfide bonds. In some embodiments, one or more points of conjugation are sulfur atoms involved in interchain sulfide bonds but not in intrachain disulfide bonds. In some embodiments, one or more points of conjugation are sulfur atoms of cysteine ​​or other amino acid residues containing a sulfur atom. Such residues may be naturally present in the antibody structure or may be incorporated into the antibody by site-directed mutagenesis, chemical transformation, or accidental incorporation of non-native amino acids.

[0349] Also provided is a method for preparing a conjugate of an activatable anti-CD71 antibody having one or more interchain disulfide bonds in AB and one or more intrachain disulfide bonds in MM, and a drug that reacts with free thiols. The method generally includes the steps of partially reducing the interchain disulfide bonds in the activatable antibody with a reducing agent such as TCEP; and conjugating the drug that reacts with free thiols to the partially reduced activatable antibody. As used herein, the term partial reduction refers to a situation in which an activatable anti-CD71 antibody is brought into contact with a reducing agent and fewer than all disulfide bonds, e.g., fewer than all possible conjugation sites, are reduced. In some embodiments, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, less than 10%, or less than 5% of all possible conjugation sites are reduced.

[0350] In yet another embodiment, a method is provided for reducing an active substance, such as a drug, to conjugate it into an activatable anti-CD71 antibody, thereby resulting in selectivity in the arrangement of the active substance. The method generally includes the steps of partially reducing the activatable anti-CD71 antibody with a reducing agent such that no conjugation sites in the masking or other non-AB portions of the activatable antibody are reduced, and conjugating the active substance to an interchain thiol in AB. The conjugation site is selected so as to cause conjugation to occur at a desired site, enabling the desired arrangement of the active substance. The reducing agent is, for example, TCEP. Reduction reaction conditions, such as the ratio of the reducing agent to the activatable antibody, the length of incubation, the temperature during incubation, and the pH of the reduction reaction solution, are determined by identifying the conditions that produce a conjugated activatable antibody in which the MM retains the ability to effectively and efficiently mask the AB of the activatable antibody in its uncleaved state. The ratio of the reducing agent to the activatable anti-CD71 antibody will vary depending on the activatable antibody. In some embodiments, the ratio of the reducing agent to the activatable anti-CD71 antibody is approximately 20:1-1:1, 10:1-1:1, 9:1-1:1, 8:1-1:1, 7:1-1:1, 6:1-1:1, 5:1-1:1, 4:1-1:1, 3:1-1:1, 2:1-1:1, and 20:1-1:1. The ratio will likely be in the range of 0.5, approximately 10:1 to 1:1.5, approximately 9:1 to 1:1.5, approximately 8:1 to 1:1.5, approximately 7:1 to 1:1.5, approximately 6:1 to 1:1.5, approximately 5:1 to 1:1.5, approximately 4:1 to 1:1.5, approximately 3:1 to 1:1.5, approximately 2:1 to 1:1.5, approximately 1.5:1 to 1:1.5, or approximately 1:1 to 1:1. In some aspects, the ratio is in the range of approximately 5:1 to 1:1. In some aspects, the ratio is in the range of approximately 5:1 to 1.5:1. In some aspects, the ratio is in the range of approximately 4:1 to 1:1. In some aspects, the ratio is in the range of approximately 4:1 to 1.5:1. In some aspects, the ratio is in the range of approximately 8:1 to approximately 1:1. In some aspects, the ratio is in the range of approximately 2.5:1 to 1:1.

[0351] In some embodiments, a method is provided for selectively placing an active substance on AB by reducing the interchain disulfide bonds in the AB of an activatable anti-CD71 antibody and conjugating a thiol-containing active substance, such as a drug, to the resulting interchain thiols. The method generally includes the steps of: partially reducing AB with a reducing agent to form at least two interchain thiols without forming all possible interchain thiols in the activatable antibody; and conjugating an active substance to the interchain thiols of the partially reduced AB. For example, the AB of an activatable antibody is partially reduced at about 37°C for about 1 hour in a desired ratio of reducing agent to activatable antibody. In some embodiments, the ratio of the reducing agent to the activatable antibody may be in the range of approximately 20:1 to 1:1, approximately 10:1 to 1:1, approximately 9:1 to 1:1, approximately 8:1 to 1:1, approximately 7:1 to 1:1, approximately 6:1 to 1:1, approximately 5:1 to 1:1, approximately 4:1 to 1:1, approximately 3:1 to 1:1, approximately 2:1 to 1:1, approximately 20:1 to 1:1.5, approximately 10:1 to 1:1.5, approximately 9:1 to 1:1.5, approximately 8:1 to 1:1.5, approximately 7:1 to 1:1.5, approximately 6:1 to 1:1.5, approximately 5:1 to 1:1.5, approximately 4:1 to 1:1.5, approximately 3:1 to 1:1.5, approximately 2:1 to 1:1.5, approximately 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 approximately 5:1 to 1:1. In some embodiments, the ratio is in the range of approximately 5:1 to 1.5:1. In some embodiments, the ratio is in the range of approximately 4:1 to 1:1. In some embodiments, the ratio is in the range of approximately 4:1 to 1.5:1. In some embodiments, the ratio is in the range of approximately 8:1 to approximately 1:1. In some embodiments, the ratio is in the range of approximately 2.5:1 to 1:1.

[0352] The thiol-containing reagent may be, for example, cysteine ​​or N-acetylcysteine. The reducing agent may be, for example, TCEP. In some embodiments, the reduced activatable antibody may be purified before conjugation, for example, by column chromatography, dialysis, or diafiltration. Alternatively, the reduced antibody may not be purified after partial reduction and before conjugation.

[0353] The present invention also provides a partially reduced activatable anti-CD71 antibody in which at least one interchain disulfide bond in the activatable antibody is reduced by a reducing agent without interfering with any intrachain disulfide bonds in the activatable antibody, the activatable antibody comprising an antibody or antigen-binding fragment (AB) that specifically binds to CD71, a masking moiety (MM) that inhibits the binding of AB of the activatable antibody to the CD71 target in the uncleaved state, and a cleavable moiety (CM) coupled to AB, wherein the CM is a polypeptide that functions as a substrate for a protease. In some embodiments, MM is coupled to AB via CM. In some embodiments, one or more intrachain disulfide bonds of the activatable antibody are not interfered with by the reducing agent. In some embodiments, one or more intrachain disulfide bonds of MM in the activatable antibody are not interfered with by the reducing agent. In some embodiments, the activatable antibody in the uncleaved state has the following N-terminus-C-terminus structural configuration: MM-CM-AB or AB-CM-MM. In some embodiments, the reducing agent is TCEP.

[0354] In further embodiments, methods for reducing an activatable anti-CD71 antibody and conjugating an active substance, such as a drug, provide selectivity in the placement of the active substance by providing an activatable anti-CD71 antibody having a defined number and position of lysine and / or cysteine ​​residues. In some embodiments, the defined number of lysine and / or cysteine ​​residues is greater than or less than the number of corresponding residues in the amino acid sequence of the parent antibody or activatable antibody. In some embodiments, the defined number of lysine and / or cysteine ​​residues may consequently define the number of active substance equivalents that can be conjugated to the anti-CD71 antibody or activatable anti-CD71 antibody. In some embodiments, the defined number of lysine and / or cysteine ​​residues may consequently define the number of active substance equivalents that can be conjugated to the anti-CD71 antibody or activatable anti-CD71 antibody in a site-specific manner. In some embodiments, the modified activatable antibody is modified in a site-specific manner with one or more non-natural amino acids, and therefore, in some embodiments, restricts the conjugation of the active substance to only the sites of the non-natural amino acids. In some embodiments, an anti-CD71 antibody or activatable anti-CD71 antibody having a defined number and position of lysine and / or cysteine ​​residues can be partially reduced with a reducing agent discussed herein such that no conjugation sites in the masking portion or other non-AB portion of the activatable antibody are reduced, and the active substance is conjugated to the interchain thiol in AB.

[0355] The disclosure also provides partially reduced activatable antibodies in which at least one interchain disulfide bond in the activatable antibody is reduced by a reducing agent without interfering with any intrachain disulfide bonds in the activatable antibody, the activatable antibody comprising an antibody or antigen-binding fragment (AB) that specifically binds to a target, e.g., CD71; a masking moiety (MM) that inhibits the binding of the activatable antibody's AB to the target in the uncleaved state; and a cleavable moiety (CM) coupled to AB, wherein the CM is a polypeptide that functions as a substrate for at least one protease. In some embodiments, the MM is coupled to AB via the CM. In some embodiments, one or more intrachain disulfide bonds of the activatable antibody are not interfered with by the reducing agent. In some embodiments, one or more intrachain disulfide bonds of the MM in the activatable antibody are not interfered with by the reducing agent. In some embodiments, the activatable antibody in the uncleaved state has the following structural configuration from N-terminus to C-terminus: MM-CM-AB or AB-CM-MM. In some embodiments, the reducing agent is TCEP.

[0356] In some embodiments, the activatable antibodies described herein also include active agents conjugated to the activatable antibodies. In some embodiments, the conjugated active agent is a therapeutic active agent, such as an anti-inflammatory and / or antineoplastic active agent. In such embodiments, for example, the active agent is conjugated to the carbohydrate portion of the activatable antibody, in some embodiments where the carbohydrate portion is located outside the antigen-binding region of the antibody or antigen-binding fragment in the activatable antibody. In some embodiments, the active agent is conjugated to the sulfhydryl group of the antibody or antigen-binding fragment in the activatable antibody.

[0357] In some embodiments, the active substance is a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof) or a radioisotope (i.e., a radioconjugate).

[0358] In some embodiments, the active substance is a detectable moiety, such as a label or other marker. For example, the active substance is or comprises one or more biotinylated moieties detectable by radiolabeled amino acids, marked avidins (e.g., streptavidin containing a fluorescent marker or enzymatic activity detectable by optical or calorimetry), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzymatic labels, and / or one or more chemiluminescent active substances. In some embodiments, the detectable moiety is added by a spacer molecule.

[0359] This disclosure also relates to cytotoxic agents such as toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof) or immunoconjugates including antibodies conjugated to radioisotopes (i.e., radioconjugates). Suitable cytotoxic agents include, for example, dorastatins and their derivatives (e.g., auristatin E, AFP, MMAF, MMAE, MMAD, DMAF, DMAE). For example, the agent is monomethyl auristatin E (MMAE) or monomethyl auristatin D (MMAD). In some embodiments, the agent is an agent selected from the group listed in Table 5. In some embodiments, the agent is dorastatin. 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 monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the active substance is a maytansinoid or a maytansinoid derivative. In some embodiments, the active substance is DM1 or DM4. In some embodiments, the active substance is duocalmycin or a derivative thereof. In some embodiments, the active substance is calicheamycin or a derivative thereof. In some embodiments, the active substance is a pyrrolobenzodiazepine. In some embodiments, the active substance is a pyrrolobenzodiazepine dimer.

[0360] In some embodiments, the active substance is linked to AB using a maleimidocaproyl-valine-citrulline linker or a maleimidoPEG-valine-citrulline linker. In some embodiments, the active substance is linked to AB using a maleimidocaproyl-valine-citrulline linker. In some embodiments, the active substance is linked to AB using a maleimidoP...

Claims

1. An antibody or its antigen-binding fragment (AB) that specifically binds to human CD71 and cynomolgus monkey CD71, The AB is, The VH CDR1 sequence is GYTFTSYWMH (SEQ ID NO: 9), The VH CDR2 sequence is AIYPGNSETG (SEQ ID NO: 10), The VH CDR3 sequence is ENWDPGFAF (SEQ ID NO: 11). VL CDR1 sequences that are SASSSVYYMY (SEQ ID NO: 12) or CRASSSVYYMY (SEQ ID NO: 13), The VL CDR2 sequence is STSNLAS (SEQ ID NO: 14), and The VL CDR3 sequence is QQRRNYPYT (SEQ ID NO: 15). An antibody or its antigen-binding fragment (AB) containing such an antibody.

2. The antibody or antigen-binding fragment (AB) according to claim 1, wherein AB comprises a heavy chain variable region containing SEQ ID NO: 5 and a light chain variable region containing SEQ ID NO:

7.

3. The antibody or antigen-binding fragment (AB) according to claim 1 or 2, wherein AB comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 325 and a light chain containing the amino acid sequence of SEQ ID NO:

701.

4. The antigen-binding fragment is a Fab fragment, F(ab') 2 An antibody or its antigen-binding fragment (AB) according to any one of claims 1 to 3, selected from the group consisting of fragments, scFv, and scAb.

5. In its activated state, it is an activatable antibody that binds to mammalian CD71, and said activatable antibody An antibody or its antigen-binding fragment (AB) that specifically binds to mammalian CD71, wherein the AB specifically binds to human CD71 and cynomolgus monkey CD71, and the AB is The VH CDR1 sequence is GYTFTSYWMH (SEQ ID NO: 9), The VH CDR2 sequence is AIYPGNSETG (SEQ ID NO: 10), The VH CDR3 sequence is ENWDPGFAF (SEQ ID NO: 11). The VL CDR1 sequence is SASSSVYYMY (SEQ ID NO: 12), The VL CDR2 sequence is STSNLAS (SEQ ID NO: 14), and The VL CDR3 sequence is QQRRNYPYT (SEQ ID NO: 15). An antibody or its antigen-binding fragment (AB) containing, A masking moiety (MM) that inhibits the binding of AB to mammalian CD71 when the activatable antibody is in an uncleaved state, wherein the MM is a polypeptide with a length of 40 amino acids or less, and the MM includes the amino acid sequence of SEQ ID NO:309, A cleavable moiety (CM) coupled to AB, wherein the CM is a polypeptide that functions as a substrate for a protease, and the CM contains the amino acid sequence of SEQ ID NO:

688. Includes, An activatable antibody in which, in its uncleaved state, MM is linked to CM such that it contains a structural configuration of MM-CM-AB or AB-CM-MM from the N-terminus to the C-terminus.

6. The activatable antibody according to claim 5, wherein AB comprises a heavy chain variable region containing SEQ ID NO: 5 and a light chain variable region containing SEQ ID NO:

7.

7. The activatable antibody according to claim 5 or 6, wherein AB comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 325 and a light chain containing the amino acid sequence of SEQ ID NO:

701.

8. The antigen-binding fragment is a Fab fragment, F(ab') 2 An activatable antibody according to any one of claims 5 to 7, selected from the group consisting of a fragment, scFv, and scAb.

9. An activatable antibody according to any one of claims 5 to 8, further comprising a first linked peptide (LP1) and a second linked peptide (LP2).

10. The activatable antibody according to claim 9, wherein, in its uncleaved state, the activatable antibody has a structural arrangement of MM-LP1-CM-LP2-AB from the N-terminus to the C-terminus.

11. The activatable antibody according to claim 9 or 10, wherein each of LP1 and LP2 is a peptide having a length of 1 to 20 amino acids.

12. A conjugated activatable antibody that specifically binds to human CD71 and cynomolgus monkey CD71 in its activated state, wherein the conjugated activatable antibody An antibody or its antigen-binding fragment (AB) that specifically binds to human CD71 and cynomolgus monkey CD71, wherein the AB is VH CDR1 sequence GYTFTSYWMH (SEQ ID NO: 9), VH CDR2 sequence AIYPGNSETG (SEQ ID NO: 10), VH CDR3 sequence ENWDPGFAF (SEQ ID NO: 11), VL CDR1 sequence SASSSVYYMY (SEQ ID NO: 12) or CRASSSVYYMY (SEQ ID NO: 13), VL CDR2 sequence STSNLAS (SEQ ID NO: 14), and VL CDR3 sequence QQRRNYPYT (SEQ ID NO: 15) An antibody or its antigen-binding fragment (AB) containing, A masking moiety (MM) coupled to AB inhibits the binding of AB to CD71 when the activatable antibody is in an uncleaved state, wherein the MM includes the amino acid sequence of SEQ ID NO:309, A cleavable moiety (CM) coupled to AB, wherein the CM is a polypeptide that functions as a substrate for a protease, and the CM contains the amino acid sequence of SEQ ID NO:

688. An active substance conjugated to AB via a linker, wherein the linker and the active substance contain vc-MMAE, Includes, A conjugated activatable antibody in which MM is linked to CM such that, in the uncleaved state, the activatable antibody contains a structural configuration of MM-CM-AB or AB-CM-MM from the N-terminus to the C-terminus.

13. The conjugated activatable antibody according to claim 12, wherein the conjugated activatable antibody comprises a first linked peptide (LP1) and a second linked peptide (LP2).

14. The conjugated activatable antibody according to claim 13, wherein in the uncleaved state, the conjugated activatable antibody has a structural arrangement of MM-LP1-CM-LP2-AB from the N-terminus to the C-terminus.

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