EA039736B1 - Anti-pdl1 antibodies, activatable anti-pdl1 antibodies, and methods of use thereof - Google Patents

Abstract

The invention relates generally to antibodies that bind programmed death ligand 1 (PDL1), activatable antibodies that specifically bind to PDL1 and methods of making and using these anti-PDL1 antibodies and anti-PDL1 activatable antibodies in a variety of therapeutic, diagnostic and prophylactic indications.

Description

This application claims priority over U.S. Provisional Application No. 62/133231, filed March 2015; U.S. Provisional Application No. 62/139596, filed March 27, 2015; and U.S. Provisional Application No. 62/218883, filed September 15, 2015; the content of each of which is incorporated into the present description by reference in their entirety.

The technical field to which the invention belongs

The invention relates in general to antibodies that bind to programmed death polypeptide ligand 1 (PDL1), activated antibodies that specifically bind to PDL1, and methods for making and using these anti-PDL1 antibodies and activated anti-PDL1 antibodies in various therapeutic, diagnostic and prophylactic indications.

State of the art

Antibody-based drugs have proven effective in the treatment of some diseases, but in some cases, toxicity resulting from the wide expression of targets limits their therapeutic efficacy. In addition, antibody-based drugs have other limitations such as rapid clearance from the circulatory system after administration.

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

Therefore, in the field of antibody-based drugs, there is a continuing need for antibodies that mimic the desired characteristics of small molecule prodrugs.

The essence of the invention

The invention provides antibodies or antigen-binding fragments thereof that specifically bind to programmed death polypeptide ligand 1 (PDL1), also known as PD-L1, CD274, B7 homologue 1 and/or B7-H1. The use of the term PDL1 is intended to include any of its variants, for example, as a non-limiting example of PD-L1 and/or PDL-1, all variants are used interchangeably here.

In some embodiments, the antibody comprises an antibody, or an antigen-binding fragment thereof, that specifically binds to PDL1. In some embodiments, the antibody or antigen-binding fragment thereof that binds to PDL1 is a monoclonal antibody, domain antibody, single chain antibody, Fab fragment, F(ab')2 Fragment, scFv, scAb, dAb, heavy chain single domain antibody or a light chain single domain antibody. In some embodiments, the antibody or antigen-binding fragment thereof that binds to PDL1 is a mouse antibody, another rodent antibody, a chimeric, humanized, or fully human monoclonal antibody.

In some embodiments, the antibody is an isolated antibody, or an antigen-binding fragment (AB) thereof, that specifically binds to mammalian PDL1, wherein the AB has one or more of the following: (a) AB specifically binds to human PDL1 and murine PDL1; (b) AB specifically binds to human PDL1 and cynomolgus PDL1; (c) AB specifically binds to human PDL1, mouse PDL1, and cynomolgus PDL1; (d) AB inhibits the binding of human B7-1 and human PD1 to human PDL1 with an EC ₅₀ value below 10 nM; (d) AB inhibits binding of mouse B7-1 and mouse PD1 to mouse PDL1 with an EC ₅₀ value below 10 nM; and (f) AB inhibits the binding of Cynomolgus B7-1 and Cynomolgus PD1 to Cynomolgus PDL1 with an EC ₅₀ value below 10 nM.

In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a mammalian PDL1 with a dissociation constant less than or equal to 0.01 nM, less than or equal to 0.05 nM, less than or equal to 0.1 nM, less than or equal to 0.2 nM, less than or equal to 0.3 nM, less than or equal to 0.4 nM, less than or equal to 0.5 nM, less than or equal to 0.75 nM, and less than or equal to 1 nM.

In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to mammalian PDL1 with a dissociation constant in the range of 0.01 to 1 nM, 0.05 to 1 nM, 0.1 to 1 nM, 0.2 to 1 nM , 0.3 to 1 nM, 0.4 to 1 nM, 0.5 to 1 nM, 0.75 to 1 nM, 0.01 to 0.75 nM, 0.05 to 0.75 nM, 0.1 to 0.75 nM, 0.2 to 0.75 nM, 0.3 to 0.75 nM, 0.4 to 0.75 nM, 0.5 to 0.75 nM, 0.01 to 0.5 nM, 0.05 to 0.5 nM, 0.1 to 0.5 nM, 0.2 to 0.5 nM, 0.3 to 0.5 nM, 0.4 to 0.5 nM, 0.01 to 0.4 nM, 0.05 to 0.4 nM, 0.1 to 0.4 nM, 0.2 to 0.4 nM, 0.3 to 0.4 nM, 0.01 to 0.3 nM, 0.05 to 0.3 nM, 0.1 to 0.3 nM, 0.2 to 0.3 nM, 0.01 to 0.2 nM, 0.05 to 0.2 nM, 0.1 to 0.2 nM, 0.01 to 0.1 nM, 0.05 to 0.1 nM or from 0.01 to 0.05 nM.

In some embodiments, the mammalian PDL1 is selected from the group consisting of human PDL, mouse PDL1, rat PDL1, and cynomolgus monkey PDL1.

In some embodiments, the antibody, or antigen-binding fragment thereof, specifically binds human PDL1, mouse PDL1, and Cynomolgus PDL1 with a dissociation constant below 1 nM.

- 1 039736

In some embodiments, the mammalian PDL1 is human PDL1.

In some embodiments, the antibody, or antigen-binding fragment thereof, has one or more of the following: (a) AB binds specifically to human PDL1, mouse PDL1, and Cynomolgus PDL1; (b) AV inhibits the binding of human B7-1 and human PD1 to human PDL1; (c) AV inhibits binding of mouse B7-1 and mouse PD1 to mouse PDL1; and (d) AB inhibits the binding of Cynomolgus B7-1 and Cynomolgus PD1 to Cynomolgus PDL1.

In some embodiments, the antibody or antigen-binding fragment thereof blocks the ability of a natural ligand to bind to mammalian PDL1 with an EC ₅₀ value of less than or equal to 0.5 nM, less than or equal to 1 nM, less than or equal to 2 nM, less than or equal to 3 nM, less than or equal to 4 nM, less than or equal to 5 nM, less than or equal to 6 nM, less than or equal to 7 nM, less than or equal to 8 nM, less than or equal to 9 nm and/or less than or equal to 10 nM.

In some embodiments, the antibody or antigen-binding fragment thereof blocks the ability of a natural ligand to bind to mammalian PDL1 with an EC50 value in the range of 0.5 to 10 nM, 0.5 to 5 nM, 0.5 to 3 nM, 0.5 up to 2 nM, 0.5 to nM, 1 to 10 nM, 1 to 5 nM, 1 to 3 nM, 1 to 2 nM, 2 to 10 nM, 2 to 5 nM, 2 to 3 nM, 3 to 10 nM, 3 to 5 nM, or 5 to 10 nM. In some embodiments, the natural ligand is mammalian PD1. In some embodiments, the natural ligand is selected from the group consisting of human PD1, mouse PD1, and cynomolgus monkey PD1.

In some embodiments, the natural ligand is mammalian B7-1. In some embodiments, the natural ligand is selected from the group consisting of human B7-1, mouse B7-1, and cynomolgus monkey B7-1.

In some embodiments, the antibody comprises a heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 , 44, 46, 48, 50, 52, 54, and 56. In some embodiments, the antibody comprises a light chain amino acid sequence comprising SEQ ID NO: 12 or SEQ ID NO: 58. In some embodiments, the antibody comprises a light chain amino acid sequence, containing SEQ ID NO: 12. In some embodiments, the antibody comprises a light chain amino acid sequence containing SEQ ID NO: 58.

In some embodiments, the antibody comprises a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, and 56 and a light chain amino acid sequence comprising SEQ ID NO: 58.

In some embodiments, the antibody comprises a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, and 56 and a light chain amino acid sequence comprising SEQ ID NO: 12.

In some embodiments, the antibody comprises a heavy chain 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: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, and 56. In some embodiments, the antibody comprises a light chain amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the antibody comprises a light chain amino acid sequence, which is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to aminoca a slot sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54 and 56, and a light chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the antibody comprises a heavy chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or % is identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54 and 56, and a light chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 46 and the light chain amino acid sequence of SEQ ID NO: 58. In some embodiments, the antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 46 and the light chain amino acid sequence of SEQ ID NO: 12.

In some embodiments, the antibody comprises a heavy chain amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the antibody contains

- 2 039736 light chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95%, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the antibody comprises an amino acid a light chain sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain amino acid sequence that at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to an amino acid sequence selected from SEQ ID NO: 46 and a light chain amino acid sequence that is at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the antibody contains a heavy chain 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 SEQ ID NO: 46, and a light chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid identical the sequences of SEQ ID NO: 12.

In some embodiments, the antibody comprises a combination of a heavy chain variable region 1 complementarity determining region (VH CDR1, also referred to herein as CDRH1), a heavy chain variable region complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, a heavy chain variable region complementarity determining region 3 (VH CDR3, also referred to here as CDRH3), light chain variable region complementarity determining region 1 (VL CDR1, also referred to here as CDRL1), complementarity determining region sequence, light chain variable region chain 2 (VL CDR2, also referred to herein as CDRL2) and a light chain variable complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein at least one CDR sequence is selected from the group consisting of a VH C sequence DR1 shown in Table 16; the VH CDR2 sequence shown in Table 16; the VH CDR3 sequence shown in Table 16; the VL CDR1 sequence shown in Table 16; the VL CDR2 sequence shown in Table 16; and the VL CDR3 sequence shown in Table 16.

In some embodiments, the antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR1 sequence shown in Table 16; a VH CD2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR2 sequence shown in Table 16; a VH CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR3 sequence shown in Table 16; a VL CDR1 sequence that 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 shown in Table 16; a VL CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR2 sequence shown in Table 16; and a VL CDR3 sequence that 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 shown in Table 16.

In some embodiments, the antibody comprises a heavy chain that contains a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, where the combination is a combination of three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown on the same line in Table .16.

In some embodiments, the antibody comprises a light chain that contains a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where the combination is a combination of three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) shown on the same line in Table .16.

In some embodiments, the antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where the combination is a combination of six CDR sequences (VH CDR1, VH CDR2, VH CDR3, VL CDR1 , VL CDR2 and VL CDR3) shown on the same line in Table 16.

In some embodiments, the antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where each CDR sequence in the combination contains a sequence that 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 one

- 3 039736 line in the table. 16.

In some embodiments, the antibody comprises a heavy chain that contains a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, where each CDR sequence in the combination contains a sequence that 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 three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in one line in Table 16.

In some embodiments, the antibody comprises a light chain that contains a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where each CDR sequence in the combination contains a sequence that 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 three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) shown in one line in Table 16.

In some embodiments, the antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VL CDR1 sequence containing RASQSISSYLN (SEQ ID NO: 209); a VL CDR2 sequence containing AASSLQS (SEQ ID NO: 215); a VL CDR3 sequence containing DNGYPST (SEQ ID NO: 228); a VH CDR1 sequence containing SYAMS (SEQ ID NO: 212); a VH CDR2 sequence containing SSIWRNGIVTVYADS (SEQ ID NO: 246); and a VH CDR3 sequence containing WSAAFDY (SEQ ID NO: 235).

In some embodiments, the antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR2 sequence contains SSIWRNGIVTVYADS (SEQ ID NO: 246). In some embodiments, the antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR3 sequence contains WSAAFDY (SEQ ID NO: 235). In some embodiments, the antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR2 sequence contains SSIWRNGIVTVYADS (SEQ ID NO: 246) and the VH CDR3 sequence contains WSAAFDY (SEQ ID NO: 235).

In some embodiments, the antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises a VL CDR1 containing RASQSISSYLN (SEQ ID NO: 209) ; a VL CDR2 sequence containing AASSLQS (SEQ ID NO: 215); a VL CDR3 sequence containing DNGYPST (SEQ ID NO: 228); a VH CDR1 sequence containing SYAMS (SEQ ID NO: 212); a VH CDR2 sequence containing SSIWRNGIVTVYADS (SEQ ID NO: 246); and a VH CDR3 sequence containing WSAAFDY (SEQ ID NO: 235).

In some embodiments, the antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, and 56. In some embodiments, the antibody is encoded by a nucleic acid sequence that contains a nucleic acid sequence encoding a heavy chain amino acid sequence selected from a group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, and 56. In some embodiments, the antibody is encoded by a nucleic acid sequence that contains a nucleic acid sequence encoding a light chain amino acid sequence containing SEQ ID NO: 12 or SEQ ID NO: 58. In some embodiments, the antibody is encoded by a nucleic acid sequence that contains a nucleic acid sequence encoding a light chain amino acid sequence comprising SEQ ID NO: 12. In some embodiments, an antibody is encoded by a nucleic acid sequence that contains a nucleic acid sequence encoding a light chain amino acid sequence comprising SEQ ID NO: 58. In some embodiments, the implementation the antibody is encoded by a nucleic acid sequence that contains a nucleic acid sequence encoding a heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 and 56, and a nucleic acid sequence that contains a nucleic acid sequence encoding a light chain amino acid sequence comprising SEQ ID NO: 58. In some embodiments, implementation An antibody is encoded by a nucleic acid sequence that contains a nucleic acid sequence encoding an amino acid sequence

- 4 039736 heavy chain selected from the group consisting of SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 and 56, and a nucleic acid sequence that contains a nucleic acid sequence encoding a light chain amino acid sequence containing SEQ ID NO: 12. In some embodiments, the antibody is encoded by a nucleic acid sequence that contains a nucleic acid sequence, a heavy chain amino acid coding sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54 and 56, and a nucleic acid sequence that contains a nucleic acid sequence encoding a light chain amino acid sequence comprising SEQ ID NOs: 58. In some embodiments, an antibody is encoded by a nucleic acid sequence that contains a nucleic acid sequence encoding an amino acid sequence. a heavy chain value selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54 and 56, and a nucleic acid sequence that contains a nucleic acid sequence encoding a light chain amino acid sequence containing SEQ ID NO: 12.

In some embodiments, an antibody is encoded by a nucleic acid sequence that contains a nucleic acid sequence encoding a heavy chain amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid sequence identical. selected from the group consisting of SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 , 54 and 56. In some embodiments, the antibody is encoded by a nucleic acid sequence that contains a nucleic acid sequence encoding a heavy chain 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 NOs: 46, 48, 50, 52, 54, and 56. In some embodiments, the antibody is encoded by a nucleic acid sequence that contains the nucleic acid sequence oic acid encoding a light chain amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the antibody is encoded by the sequence a nucleic acid that contains a nucleic acid sequence encoding a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identical SEQ ID NO: 12. In some embodiments, the antibody is encoded by a nucleic acid sequence that contains a nucleic acid sequence encoding a heavy chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97 , 98 or 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 , 44, 46, 48, 50, 52, 54 and 56, and a nucleic acid sequence that contains a nucleic acid sequence encoding a light chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96 , 97, 98, or 99% amino acid identical to the sequence of SEQ ID NO: 58. In some embodiments, the antibody is encoded by a nucleic acid sequence that contains a nucleic acid sequence encoding a heavy chain 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 NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 , 38, 40, 42, 44, 46, 48, 50, 52, 54 and 56, and a nucleic acid sequence that contains a nucleic acid sequence encoding a light chain amino acid sequence that is at least 90, 91, 92, 93 , 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody is encoded by a nucleic acid sequence that contains a nucleic acid sequence encoding a heavy chain 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 NOs: 46, 48, 50, 52, 54, and 56 and a nucleic acid sequence that contains a nucleic acid sequence encoding a light chain amino acid sequence , which is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the antibody is encoded by a nucleic acid sequence that contains the nucleic acid sequence , encoding a heavy chain amino acid sequence that is at least 90, 91, 92, 9 3, 94, 95, 96, 97, 98, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, and 56, and a nucleic acid sequence that contains the nucleic acid sequence , encoding a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the antibody is encoded by a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, 17, 21, 23, 25, 27, 29, 31, 33,

- 5 039736

35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55. In some embodiments, the antibody is encoded by a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 51, 53 and 55. In some embodiments, the antibody is encoded by the light chain nucleic acid sequence of SEQ ID NO: 57. In some embodiments, the antibody is encoded by the light chain nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the antibody is encoded by the heavy chain nucleic acid sequence a chain selected from the group consisting of SeQ ID NOs: 13, 15, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53 and 55, and a light chain nucleic acid sequence comprising SEQ ID NO: 57. In some embodiments, the antibody is encoded by a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 13, 15, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 4 1, 43, 45, 47, 49, 51, 53, and 55, and a light chain nucleic acid sequence comprising SEQ ID NO: 11. In some embodiments, an antibody is encoded by a nucleic acid sequence that contains a heavy chain nucleic acid sequence encoding an amino acid a heavy chain sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 51, 53, and 55; and a light chain nucleic acid sequence comprising SEQ ID NO: 57. In some embodiments, the antibody is encoded by a nucleic acid sequence, which contains a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 4 9, 51, 53 and 55, and a light chain nucleic acid sequence containing SEQ ID NOs: 11.

In some embodiments, the 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 the heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55. In some embodiments, the antibody is encoded a nucleic acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 51, 53, and 55. In some embodiments, an 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 the SEQ light chain nucleic acid sequence. ID NO: 57. In some embodiments, the antibody is encoded by the nucleic acid sequence which is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the light chain nucleic chain sequence of SEQ ID NO: 11. In some embodiments, the antibody is encoded by a nucleic acid sequence that at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53 and 55, and a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to a light chain nucleic acid sequence comprising SEQ ID NO: 57. In some embodiments, an 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 the heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53 and 55, and a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the light chain nucleic acid sequence containing SEQ ID NO: 11. In some embodiments, the antibody is encoded by a nucleic acid sequence that contains a nucleic acid sequence that is at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identical to a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 51, 53, and 55, and the nucleic acid sequence which is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to a light chain nucleic acid sequence comprising SEQ ID NO: 57. In some embodiments, an antibody is encoded by a nucleic acid sequence, which contains a nucleic acid sequence that is at least e is 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 51, 53, and 55, and a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chain nucleic acid sequence comprising SEQ ID NO: 11.

In some embodiments, the antibody or antigen-binding fragment thereof is included in a multispecific antibody or antigen-binding fragment, wherein at least one arm of the multispecific antibody specifically binds to PDL1. In some embodiments, the antibody or antigen-binding fragment thereof is incorporated into a bispecific antibody or antigen-binding fragment thereof, wherein at least one arm of the bispecific antibody specifically binds to PDL1.

In some embodiments, at least one arm of a multispecific antibody, such as a bispecific antibody, contains a heavy chain amino acid sequence,

- 6 039736 selected from the group consisting of SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 , 52, 54, and 56. In some embodiments, at least one arm of a multispecific antibody, such as a bispecific antibody, comprises a light chain amino acid sequence comprising SEQ ID NO: 12 or SEQ ID NO: 58. In some embodiments, at least one a multispecific antibody arm, such as a bispecific antibody, comprises a light chain amino acid sequence comprising SEQ ID NO: 12. In some embodiments, at least one arm of a multispecific antibody, such as a bispecific antibody, comprises a light chain amino acid sequence comprising SEQ ID NO: 58.

In some embodiments, at least one arm of a multispecific antibody, such as a bispecific antibody, comprises a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 46, 48, 50, 52, 54, and 56, and an amino acid a light chain sequence containing SEQ ID NO: 58.

In some embodiments, at least one arm of a multispecific antibody, such as a bispecific antibody, comprises a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 46, 48, 50, 52, 54, and 56, and an amino acid a light chain sequence containing SEQ ID NO: 12.

In some embodiments, at least one arm of a multispecific antibody, such as a bispecific antibody, comprises a heavy chain amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid sequence identical to selected from the group consisting of SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 , 54, and 56. In some embodiments, at least one arm of a multispecific antibody, such as a bispecific antibody, comprises a light chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, at least one arm of a polyspecific antibody, such as a bispecific antibody, comprises a light chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% is identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, at least one arm of a multispecific antibody, such as a bispecific antibody, comprises a heavy chain 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 NOs: 46, 48, 50, 52, 54, and 56, and a light chain amino acid sequence that is at least 90, 91, 92, 93 , 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, at least one arm of a multispecific antibody, e.g. , 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54 and 56, and the amino acid after a light chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of SEQ ID NO: 12.

In some embodiments, at least one arm of a multispecific antibody, e.g., a bispecific antibody, comprises the heavy chain amino acid sequence of SEQ ID NO: 46 and the light chain amino acid sequence of SEQ ID NO: 58. In some embodiments, at least one arm of the multispecific antibody, e.g. a bispecific antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 46 and the light chain amino acid sequence of SEQ ID NO: 12.

In some embodiments, at least one arm of a multispecific antibody, such as a bispecific antibody, comprises a heavy chain amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid of SEQ ID NO: 46. In some embodiments, at least one arm of a multispecific antibody, such as a bispecific antibody, comprises a light chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, at least one arm of a polyspecific antibody, such as a bispecific antibody, comprises a light chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95 , 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, at least one arm of the polyspecific an antibody, such as a bispecific antibody, comprises a heavy chain 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 SEQ ID NO: 46, and a light chain amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, at least one arm of a polyspecific antibody , for example a bispecific antibody, includes a heavy chain amino acid sequence that is at least 90, 91, 92, 93,

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94, 95, 96, 97, 98, or 99% identical to an amino acid sequence selected from SEQ ID NO: 46 and a light chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96,

97, 98 or 99% identical to the amino acid sequence of SEQ ID NO: 12.

In some embodiments, at least one arm of a polyspecific antibody, e.g., a bispecific antibody, comprises a combination of a heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a heavy chain complementarity determining region 2 (VH CDR2, also referred to here as CDRH2), complementarity determining region sequence 3 (VH CDR3, also referred to here as CDRH3), light chain complementarity determining region sequence 1 (VL CDR1, also referred to here as CDRL1), complementarity determining region sequence, light chain 2 (VL CDR2, also referred to here as CDRL2) and a light chain complementarity determining region 3 sequence (VL CDR3, also referred to here as CDRL3), wherein at least one CDR sequence is selected from the group consisting of the VH CDR1 sequence shown in table 16; the VH CDR2 sequence shown in Table 16; the VH CDR3 sequence shown in Table 16; the VL CDR1 sequence shown in Table 16; the VL CDR2 sequence shown in Table 16; and the VL CDR3 sequence shown in Table 16.

In some embodiments, at least one arm of a multispecific antibody, such as a bispecific antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected. from the group consisting of a VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR1 sequence shown in Table 16; a VH CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR2 sequence shown in Table 16; a VH CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR3 sequence shown in Table 16; a VL CDR1 sequence that 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 shown in Table 16; a VL CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR2 sequence shown in Table 16; and a VL CDR3 sequence that 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 shown in Table 16.

In some embodiments, at least one arm of a multispecific antibody, such as a bispecific antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where the combination is a combination of six CDR sequences ( VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3) shown in one line in Table 16.

In some embodiments, at least one arm of a multispecific antibody, such as a bispecific antibody, comprises a heavy chain that contains a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, where the combination is a combination of three heavy chain CDR sequences (VH CDR1, VH CDR2 , VH CDR3) shown in one row in Table 16.

In some embodiments, at least one arm of a polyspecific antibody, such as a bispecific antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where each CDR sequence in combination contains the sequence which 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 six CDR sequences (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3) shown on the same line in Table 16.

In some embodiments, at least one arm of a multispecific antibody, such as a bispecific antibody, comprises a heavy chain that contains a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, where each CDR sequence in the combination contains a sequence that 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 three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in one row in Table 16.

In some embodiments, at least one arm of a multispecific antibody, such as a bispecific antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a

- 8 039736

VL CDR2 and VL CDR3 sequences, wherein at least one CDR sequence is selected from the group consisting of a VL CDR1 sequence containing RASQSISSYLN (SEQ ID NO: 209); a VL CDR2 sequence containing AASSLQS (SEQ ID NO: 215); a VL CDR3 sequence containing DNGYPST (SEQ ID NO: 228); sequence VH CDR1 containing SYAMS (SEQ ID NO: 212); a VH CDR2 sequence containing SSIWRNGIVTVYADS (SEQ ID NO: 246); and a VH CDR3 sequence containing WSAAFDY (SEQ ID NO: 235).

In some embodiments, at least one arm of a polyspecific antibody, such as a bispecific antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR2 sequence contains SSIWRNGIVTVYADS (SEQ ID NO: 246). In some embodiments, at least one arm of a polyspecific antibody, e.g., a bispecific antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR3 sequence contains WSAAFDY ( SEQ ID NO: 235). In some embodiments, at least one arm of a polyspecific antibody, such as a bispecific antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR2 sequence contains SSIWRNGIVTVYADS (SEQ ID NO: 246) and the VH CDR3 sequence contains WSAAFDY (SEQ ID NO: 235).

In some embodiments, at least one arm of a polyspecific antibody, such as a bispecific antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises a VL CDR1 sequence comprising RASQSISSYLN (SEQ ID NO: 209); a VL CDR2 sequence containing AASSLQS (SEQ ID NO: 215); a VL CDR3 sequence containing DNGYPST (SEQ ID NO: 228); a VH CDR1 sequence containing SYAMS (SEQ ID NO: 212) ; a VH CDR2 sequence containing SSIWRNGIVTVYADS (SEQ ID NO: 246); and a VH CDR3 sequence containing WSAAFDY (SEQ ID NO: 235).

The invention also provides activated antibodies that include an antibody or antigen-binding fragment thereof that specifically binds to PDL1, associated with a masking fragment (MM) such that the MM compound reduces the ability of the antibody or antigen-binding fragment to bind to PDL1. In some embodiments, the MM is linked through a sequence that includes a substrate for a protease, such as a protease that is active in diseased tissue and/or a protease that co-locates with PDL1 in the area to be treated in the subject. The activated anti-PDL1 antibodies provided herein, also referred to herein as activated anti-PDL1 antibodies or activated anti-PDL1 antibodies, are stable in the circulatory system, activated at the intended sites of treatment and/or diagnosis, but not in normal, e.g., healthy tissue or other tissue that is not intended for treatment and/or diagnosis, and when activated, exhibit binding to PDL1 that is at least comparable to that of the corresponding unmodified antibody.

In some embodiments, an activated antibody that, when activated, specifically binds to mammalian PDL1 includes an antibody, or an antigen-binding fragment (AB) thereof, that specifically binds to mammalian PDL1; a masking fragment (MM) that inhibits AB binding to mammalian PDL1 when the antibody to be activated is in an uncleaved state; and a cleavable fragment (CM) associated with AB, where CM is a polypeptide that functions as a substrate for a protease.

In some embodiments, an activated antibody is provided that, when activated, has one or more of the following characteristics: (a) specifically binds to mammalian PDL1; and (b) specifically blocks natural PDL1 ligand from binding to mammalian PDL1, wherein the antibody to be activated comprises: an antibody or antigen-binding fragment (AB) thereof that specifically binds to mammalian PDL1; a masking fragment (MM) that inhibits AB binding to mammalian PDL1 when the antibody to be activated is in an uncleaved state; and a cleavable fragment (CM) associated with AB, where CM is a polypeptide that functions as a substrate for a protease.

In some embodiments, the activated antibody in the uncleaved state specifically binds to a mammalian PDL1 with a dissociation constant less than or equal to 1 nM, less than or equal to 2 nM, less than or equal to 3 nM, less than or equal to 4 nM, less than or equal to 5 nM, less than or equal to equal to 10 nM, less than or equal to 15 nM, less than or equal to 20 nM and/or less than or equal to 25 nM.

In some embodiments, the non-cleaved activated antibody specifically binds to a mammalian PDL1 with a dissociation constant greater than or equal to 1 nM, greater than or equal to 2 nM, greater than or equal to 3 nM, greater than or equal to 4 nM, greater than or equal to 5 nM, greater than

- 9 039736 or equal to 10 nM, greater than or equal to 15 nM, greater than or equal to 20 nM and/or greater than or equal to 25 nM.

In some embodiments, the activated antibody in the uncleaved state specifically binds to mammalian PDL1 with a dissociation constant in the range of 1 to 2 nM, 1 to 5 nM, 1 to 10 nM, 1 to 15 nM, 1 to 20 nM, 1 to 25 nM, 2 to 5 nM, 2 to 10 nM, 2 to 15 nM, 2 to 20 nM, 2 to 25 nM, 5 to 10 nM, 5 to 15 nM, 5 to 20 nM, 5 to 25 nM, 10 to 15 nM, 10 to 20 nM, 10 to 25 nM, 15 to 20 nM, 15 to 25 nM, or 20 to 25 nM.

In some embodiments, the activated antibody in the activated state specifically binds to a mammalian PDL1 with a dissociation constant less than or equal to 0.01 nM, less than or equal to 0.05 nM, less than or equal to 0.1 nM, less than or equal to 0.2 nM, less or equal to 0.3 nM, less than or equal to 0.4 nM, less than or equal to 0.5 nM, less than or equal to 0.75 nM, and less than or equal to 1 nM.

In some embodiments, the activated antibody in the activated state specifically binds to mammalian PDL1 with a dissociation constant in the range of 0.01 to 1 nM, 0.05 to 1 nM, 0.1 to 1 nM, 0.2 pM to 1 nM, 0.3 to 1 nM, 0.4 to 1 nM, 0.5 to 1 nM, 0.75 to 1 nM, 0.01 to 0.75 nM, 0.05 to 0 .75 nM, 0.1 to 0.75 nM, 0.2 to 0.75 nM, 0.3 nmol to 0.75 nM, 0.4 to 0.75 nM, 0.5 to 0.75 nM, 0.01 to 0.5 nM, 0.05 to 0.5 nM, 0.1 to 0.5 nM, 0.2 to 0.5 nM, 0.3 to 0.5 nM, 0.4 to 0.5 nM, 0.01 to 0.4 nM, 0.05 to 0.4 nM, 0.1 to 0.4 nM, 0.2 to 0 .4 nM, 0.3 to 0.4 nM, 0.01 to 0.3 nM, 0.05 to 0.3 nM, 0.1 to 0.3 nM, 0.2 to 0 .3 nM, 0.01 to 0.2 nM, 0.05 to 0.2 nM, 0.1 to 0.2 nM, 0.01 to 0.1 nM, 0.05 to 0 .1 nM or 0.01 to 0.05 nM.

In some embodiments, AV specifically binds to mammalian PDL1 with a dissociation constant less than or equal to 0.01 nM, less than or equal to 0.05 nM, less than or equal to 0.1 nM, less than or equal to 0.2 nM, less than or equal to 0 .3 nM, less than or equal to 0.4 nM, less than or equal to 0.5 nM, less than or equal to 0.75 nM, and less than or equal to 1 nM.

In some embodiments, AV specifically binds to mammalian PDL1 with a dissociation constant in the range of 0.01 to 1 nM, 0.05 to 1 nM, 0.1 to 1 nM, 0.2 to 1 nM, 0, 3 to 1 nM, 0.4 to 1 nM, 0.5 to 1 nM, 0.75 to 1 nM, 0.01 to 0.75 nM, 0.05 to 0.75 nM, from 0.1 to 0.75 nM, 0.2 nmol to 0.75 nM, 0.3 to 0.75 nM, 0.4 to 0.75 nM, 0.5 to 0.75 nM, 0.01 to 0.5 nM, 0.05 to 0.5 nM, 0.1 to 0.5 nM, 0.2 to 0.5 nM, 0.3 to 0.5 nM, 0.4 to 0.5 nM, 0.01 pM to 0.4 nM, 0.05 to 0.4 nM, 0.1 to 0.4 nM, 0.2 to 0.4 nM, 0.3 to 0.4 nM, 0.01 to 0.3 nM, 0.05 to 0.3 nM, 0.1 to 0.3 nM, 0.2 to 0.3 nM, 0.01 to 0.2 nM, 0.05 to 0.2 nM, 0.1 to 0.2 nM, 0.01 to 0.1 nM, 0.05 to 0.1 nM, or from 0.01 to 0.05 nM.

In some embodiments, the mammalian PDL1 is selected from the group consisting of human PDL, mouse PDL1, rat PDL1, and cynomolgus monkey PDL1. In some embodiments, the AV specifically binds to human PDL, mouse PDL1, or cynomolgus monkey PDL1 with a dissociation constant below 1 nM. In some embodiments, the mammalian PDL1 is human PDL1.

In some embodiments, the AV has one or more of the following characteristics: (a) the AV specifically binds to human PDL1 and murine PDL1; (b) AB binds specifically to human PDL1 and cynomolgus PDL1; and (c) AB specifically binds to human PDL1, mouse PDL1, and Cynomolgus monkey PDL1.

In some embodiments, the AV has one or more of the following characteristics: (a) the AV specifically binds to human PDL, mouse PDL1, or Cynomolgus PDL1; (b) AV inhibits the binding of human B7-1 and human PD1 to human PDL1; (c) AV inhibits binding of mouse B7-1 and mouse PD1 to mouse PDL1; and (d) AB inhibits the binding of Cynomolgus B7-1 and Cynomolgus PD1 to Cynomolgus PDL1.

In some embodiments, the AB blocks the ability of a natural ligand to bind to a mammalian PDL1 with an EC ₅₀ value of less than or equal to 0.5 nM, less than or equal to 1 nM, less than or equal to 2 nM, less than or equal to 3 nM, less than or equal to 4 nM, less than or equal to 5 nM, less than or equal to 6 nM, less than or equal to 7 nM, less than or equal to 8 nM, less than or equal to 9 nM and/or less than or equal to 10 nM.

In some embodiments, the AB blocks the ability of a natural ligand to bind to mammalian PDL1 with an EC ₅₀ value of 0.5 to 10 nM, 0.5 to 5 nM, 0.5 to 3 nM, 0.5 to 2 nM, 0 .5 to 1 nM, 1 to 10 nM, 1 to 5 nM, 1 to 3 nM, 1 to nM, 2 to 10 nM, 2 to 5 nM, 2 to 3 nM, 3 to 10 nM, 3 to 5 nM, or 5 to 10 nM.

In some embodiments, the natural ligand is mammalian PD1. In some embodiments, the natural ligand is selected from the group consisting of human PD1, mouse PD1, or cynomolgus monkey PD1.

In some embodiments, the natural ligand is mammalian B7-1. In some embodiments, the natural ligand is selected from the group consisting of human B7-1, mouse B7-1, and cynomolgus monkey B7-1.

In some embodiments, the activated antibody has one or more of the following characteristics: (a) AB induces type 1 diabetes in non-obese diabetic (NOD) mice; and (b) an activated antibody in a non-cleaved state inhibits the induction of type 1 diabetes in a non-obese NOD diabetic mouse.

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In some embodiments, the activated antibody inhibits the induction of type 1 diabetes in a NOD mouse following administration of the activated antibody at a single dose of 0.1 to 3 mg/kg, 0.5 to 3 mg/kg, 1 to 3 mg/kg, from 2 to 3 mg/kg, 0.1 to 2 mg/kg, 0.5 to 2 mg/kg, 1 to 2 mg/kg, 0.1 to 1 mg/kg, 0.5 to 1 mg/kg or 0.1 to 0.5 mg/kg.

In some embodiments, the activated antibody induces type 1 diabetes in the subject after administration of the activated antibody at a single dose of 1 mg/kg. In some embodiments, the subject is a NOD mouse.

In some embodiments, the activated antibody induces type 1 diabetes in a subject upon administration of the activated antibody at a single dose less than or equal to 3 mg/kg, less than or equal to 2 mg/kg, less than or equal to 1 mg/kg, less than or equal to 0.5 mg/kg and/or less than or equal to 0.1 mg/kg.

In some embodiments, the activated antibody induces type 1 diabetes in a subject upon administration of the activated antibody at a single dose in the range selected from the group consisting of 0.1 to 3 mg/kg, 0.5 to 3 mg/kg, 1 to 3 mg/kg, 2 to 3 mg/kg, 0.1 to 2 mg/kg, 0.5 to 2 mg/kg, 1 to 2 mg/kg, 0.1 to 1 mg/kg , 0.5 to 1 mg/kg and 0.1 to 0.5 mg/kg. In some embodiments, the subject is a NOD mouse.

In some embodiments, the activated antibody has one or more of the following characteristics: (a) the activated antibody in the uncleaved state does not induce type 1 diabetes in more than 50% of the population of non-obese diabetic (NOD) mice, and AB induces type 1 diabetes in more than 50% of the NOD mouse population.

In some embodiments, the activated antibody does not induce type 1 diabetes in more than 50% of a population of NOD mice after administration to each mouse in the population at a single dose less than or equal to 3 mg/kg, less than or equal to 2 mg/kg, less than or equal to 1 mg /kg, less than or equal to 0.5 mg/kg and/or less than or equal to 0.1 mg/kg.

In some embodiments, the activated antibody does not induce type 1 diabetes in more than 50% of the population of NOD mice after administration of each mouse in the population of the singly activated antibody at a dose of 0.1 to 3 mg/kg, 0.5 to 3 mg/kg, 1 to 3 mg/kg, 2 to 3 mg/kg, 0.1 to 2 mg/kg, 0.5 to 2 mg/kg, 1 to 2 mg/kg, 0.1 to 1 mg/kg, 0.5 to 1 mg/kg, or 0.1 to 0.5 mg/kg.

In some embodiments, the activated antibody has one or more of the following characteristics: (a) the activated antibody, in its uncleaved state, does not induce type 1 diabetes in more than 50% of a non-obese (NOD) diabetic mouse population when administered at a single dose of 1 mg/kg; and (b) AB induces type 1 diabetes in more than 50% of the NOD mouse population when administered at a single dose of 1 mg/kg.

In some embodiments, the AV induces type 1 diabetes in more than 50% of a population of NOD mice after each mouse in the population is administered a single dose of the AV at a dose less than or equal to 3 mg/kg, less than or equal to 2 mg/kg, less than or equal to 1 mg/kg. kg, less than or equal to 0.5 mg/kg and/or less than or equal to 0.1 mg/kg.

In some embodiments, the AV induces type 1 diabetes in more than 50% of a population of NOD mice after each mouse in the population is administered a single dose of the AV at a dose of 0.1 to 3 mg/kg, 0.5 to 3 mg/kg, 1 to 3 mg/kg, 2 to 3 mg/kg, 0.1 to 2 mg/kg, 0.5 to 2 mg/kg, 1 to 2 mg/kg, 0.1 to 1 mg/kg , 0.5 to 1 mg/kg or 0.1 to 0.5 mg/kg.

In some embodiments, the NOD mouse is a female NOD/subsp. ShiLtJ mouse.

In some embodiments, the activated antibody inhibits the induction of type 1 diabetes in a NOD mouse at least 3-fold higher than AB.

In some embodiments, the activated antibody has a margin of safety that is at least three times the margin of safety compared to AB.

In some embodiments, the non-cleaved activated antibody binds to a lower percentage of the peripheral blood CD4+ CD8+ T lymphocyte population than does AB.

In some embodiments, the activated antibody does not bind to more than 50% of the peripheral blood CD4+ CD8+ T lymphocyte population following administration of the single activated antibody at a dose less than or equal to 5 mg/kg, less than or equal to 4 mg/kg, less than or equal to 3 mg/kg. kg, less than or equal to 2 mg/kg, less than or equal to 1 mg/kg, less than or equal to 0.5 mg/kg and/or less than or equal to 0.1 mg/kg.

In some embodiments, the activated antibody does not bind to more than 50% of the population of CD4+ CD8+ T lymphocytes in peripheral blood after administration of a single activated antibody at a dose of 0.1 to 5 mg/kg, 0.5 to 5 mg/kg, 1 up to 5 mg/kg, from 2 to 5 mg/kg, from 3 to 5 mg/kg, from 0.1 to 3 mg/kg, from 0.5 to 3 mg/kg, from 1 to 3 mg/kg, 2 to 3 mg/kg, 0.1 to 2 mg/kg, 0.5 to 2 mg/kg, 1 to 2 mg/kg, 0.1 to 1 mg/kg, 0.5 to 1 mg/kg or 0.1 to 0.5 mg/kg.

In some embodiments, the AV binds to more than 50% of the peripheral blood CD4+ CD8+ T-lymphocyte population following single administration of the AV at a dose of 0.1 to 5 mg/kg, 0.5 to 5 mg/kg, 1 to 5 mg /kg, 2 to 5 mg/kg, 3 to 5 mg/kg, 0.1 to 3 mg/kg, 0.5 to 3 mg/kg, 1 to 3 mg/kg, 2 to 3 mg/kg, 0.1 to 2 mg/kg, 0.5 to 2 mg/kg, 1 to 2 mg/kg, 0.1 to 1 mg/kg, or 0.1 to 0, 5 mg/kg.

In some embodiments, CD4+ CD8+ peripheral blood T lymphocytes are

- 11 039736 mice. In some embodiments, mouse peripheral blood CD4+ CD8+ T lymphocytes are derived from a tumor-bearing mouse.

In some embodiments, the percentage of the peripheral blood CD4+ CD8+ T lymphocyte population to which AB binds is below 60% when the activated antibody is administered at a dose of 1 to 5 mg/kg. In some embodiments, the percentage of the peripheral blood CD4+ CD8+ T lymphocyte population to which AB binds is below 50% when the activated antibody is administered at a dose of 1 to 5 mg/kg. In some embodiments, the percentage of the peripheral blood CD4+ CD8+ T lymphocyte population to which AB binds is between 30% and 60% when the activated antibody is administered at a dose of 1 to 5 mg/kg. In some embodiments, the percentage of the peripheral blood CD4+ CD8+ T lymphocyte population to which AB binds is 30 to 50% when the activated antibody is administered at a dose of 1 to 5 mg/kg.

The invention also relates to methods for treating, preventing and/or delaying the onset or slowing progression, or alleviating a symptom associated with aberrant expression and/or activity of PDL1 in a subject, using activated antibodies that bind to PDL1, in particular, activated antibodies that bind to and neutralize or otherwise inhibit at least one biological activity mediated by PDL1 and/or PDL1 signaling pathway.

Activated antibodies in the activated state bind to PDL1 and include (i) an antibody or antigen-binding fragment (AB) thereof that specifically binds to PDL1; (ii) a masking fragment (MM) that, when the activated antibody is in an uncleaved state, inhibits AB binding to PDL1; and (c) a cleavable fragment (CM) associated with AB, where CM is a polypeptide that functions as a substrate for the protease.

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

In some embodiments, the activated antibody comprises a linker peptide between MM and CM.

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

In some embodiments, the activated antibody comprises a first linker peptide (LP1) and a second linker peptide (LP2), and where the activated antibody in the uncleaved state has the following structural organization from the N-terminus to the C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CMLP1-MM. In some embodiments, the two linker peptides need not be identical to each other.

In some embodiments, at least one of LP1 or LP2 contains an amino acid sequence selected from the group consisting of (GS) _n , (GGS) _n , (GSGGS) _n (SEQ ID NO: 191) and (GGGS) _n (SEQ ID NO: 192), where n is an integer, at least one.

In some embodiments, at least one of LP1 or LP2 contains an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 193), GGSGG (SEQ ID NO: 194), GSGSG (SEQ ID NO: 195), GSGGG (SEQ ID NO: 196), GGGSG (SEQ ID NO: 197) and GSSSG (SEQ ID NO: 198).

In some embodiments, LP1 contains the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 199), GSSGGSGGSGG (SEQ ID NO: 200), GSSGGSGGSGGS (SEQ ID NO: 201), GSSGGGGGGGGGGGS (SEQ ID NO: 202), GSSGGSGGSG (SEQ ID NO: 202), 203) or GSSGGSGGSGS (SEQ ID NO: 204).

In some embodiments, LP2 contains the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 205), GSSGT (SEQ ID NO: 206), or GSSG (SEQ ID NO: 207).

In some embodiments, the AB has a dissociation constant of about 100 nM or less for binding to PDL1.

In some embodiments, the activated antibody comprises an antibody, or an antigen-binding fragment thereof, that specifically binds to PDL1. In some embodiments, the antibody or antigen-binding fragment thereof that binds to PDL1 is a monoclonal antibody, domain antibody, single chain antibody, Fab fragment, F(ab')2 fragment, scFv, scAb, dAb, heavy chain single domain antibody or a light chain single domain antibody. In some embodiments, the antibody or antigen-binding fragment thereof that binds to PDL1 is a mouse antibody, another rodent antibody, a chimeric, humanized, or fully human monoclonal antibody.

In some embodiments, the activated antibody comprises a heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, and 56. In some embodiments, the activated antibody comprises a light chain amino acid sequence comprising SEQ ID NO: 12 or SEQ ID NO: 58. In some embodiments, the activated antibody comprises the amino acid sequence light chain comprising SEQ ID NO: 12. In some embodiments, the activated antibody comprises a light chain amino acid sequence comprising

SEQ ID NO: 58.

In some embodiments, the activated antibody comprises a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, and 56 and a light chain amino acid sequence comprising SEQ ID NO: 58 .

In some embodiments, the activated antibody comprises a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, and 56 and a light chain amino acid sequence comprising SEQ ID NO: 12 .

In some embodiments, the activated antibody comprises a heavy chain 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 : 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, and 56. In some embodiments, an activated the antibody comprises a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the activated antibody comprises the amino acid sequence a light chain that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the activated antibody comprises a heavy chain amino acid sequence that at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, and 56, and a light chain amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the activated antibody comprises a heavy chain amino acid sequence that is at least 90 , 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54 and 56 and the light chain amino acid sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the AV activated anti-PDL1 antibody comprises a heavy chain amino acid sequence selected from the group consisting of the heavy chain sequences shown in Table 15. In some embodiments, the AV-activated anti-PDL1 antibody comprises a light chain amino acid sequence selected from the group consisting of the light chain sequences shown in Table 15. In some embodiments, the AV activated anti-PDL1 antibody comprises a heavy chain amino acid sequence selected from the group consisting of the heavy chain sequences shown in Table 15 and a light chain amino acid sequence selected from the group consisting of the light chain sequences shown in Table 15. in Table 15.

In some embodiments, the AV activated anti-PDL1 antibody contains a heavy chain 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 the heavy chain sequences shown in Table 15. In some embodiments, the AV activated anti-PDL1 antibody comprises a light chain 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 the light chain sequences shown in Table 15. In some embodiments, the AV activated anti-PDL1 antibody contains a heavy chain 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 the heavy chain sequences shown in Table 15 and a light chain 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 the light chain sequences shown in Table 15.

In some embodiments, the activated antibody comprises a combination of a heavy chain variable region complementarity determining region 1 sequence (VH CDR1, also referred to herein as CDRH1), a heavy chain variable complementarity determining region 2 sequence (VH CDR2, also referred to herein as CDRH2), complementarity determining region sequence, heavy chain variable region 3 (VH CDR3, also referred to here as CDRH3), complementarity determining region sequence, variable light chain 1 (VL CDR1, also referred to here as CDRL1), complementarity determining region sequence, variable light chain region 2 (VL CDR2, also referred to herein as CDRL2) and a complementarity determining region variable light chain 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein at least one CDR sequence is selected from the group consisting of the following the ovality of VH CDR1 shown in Table 16; the VH CDR2 sequence shown in Table 16; the VH CDR3 sequence shown in Table 16; the VL CDR1 sequence shown in Table 16; VL sequences

CDR2 shown in Table 16; and the VL CDR3 sequence shown in Table 16.

In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence that is includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR1 sequence shown in Table 16; a VH CD2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR2 sequence shown in Table 16; a VH CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR3 sequence shown in Table 16; a VL CDR1 sequence that 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 shown in Table 16; a VL CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR2 sequence shown in Table 16; and a VL CDR3 sequence that 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 shown in Table 16.

In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where the combination is a combination of six CDR sequences (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3), shown in one line in the table. 16.

In some embodiments, the activated antibody comprises a heavy chain that contains a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, where the combination is a combination of three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown on the same line in table 16.

In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein each CDR sequence in the combination contains a sequence that 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 one line in the table. 16.

In some embodiments, the activated antibody comprises a heavy chain that contains a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, where each CDR sequence in the combination contains a sequence that 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 three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in one row in Table 16.

In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VL CDR1 sequence containing RASQSISSYLN (SEQ ID NO: 209); a VL CDR2 sequence containing AASSLQS (SEQ ID NO: 215); a VL CDR3 sequence containing DNGYPST (SEQ ID NO: 228); sequence VH CDR1 containing SYAMS (SEQ ID NO: 212); a VH CDR2 sequence containing SSIWRNGIVTVYADS (SEQ ID NO: 246); and a VH CDR3 sequence containing WSAAFDY (SEQ ID NO: 235).

In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR2 sequence contains SSIWRNGIVTVYADS (SEQ ID NO: 246). In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR3 sequence includes WSAAFDY (SEQ ID NO: 235). In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR2 sequence includes SSIWRNGIVTVYADS (SEQ ID NO: 246) and the VH CDR3 sequence includes WSAAFDY (SEQ ID NO: 235).

- 14 039736

In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises a VL CDR1 containing RASQSISSYLN (SEQ ID NO: 209) ; a VL CDR2 sequence containing AASSLQS (SEQ ID NO: 215); a VL CDR3 sequence containing DNGYPST (SEQ ID NO: 228); a VH CDR1 sequence containing SYAMS (SEQ ID NO: 212); a VH CDR2 sequence containing SSIWRNGIVTVYADS (SEQ ID NO: 246); and a VH CDR3 sequence containing WSAAFDY (SEQ ID NO: 235).

In some embodiments, the activated antibody comprises a combination of a heavy chain variable region complementarity determining region 1 sequence (VH CDR1, also referred to herein as CDRH1), a heavy chain variable complementarity determining region sequence 2 (VH CDR2, also referred to herein as CDRH2), heavy chain variable region complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3), complementarity determining region light chain variable region 1 sequence (VL CDR1, also referred to here as CDRL1), complementarity determining region sequence, variable region light chain 2 (VL CDR2, also referred to herein as CDRL2), and a light chain variable region complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein at least one CDR sequence is selected from the group consisting of the sequence the VH CDR1 values shown in Table 17; the VH CDR2 sequence shown in Table 17; the VH CDR3 sequence shown in Table 17; the VL CDR1 sequence shown in Table 17; the VL CDR2 sequence shown in Table 17; and the VL CDR3 sequence shown in Table 17.

In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence that is includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR1 sequence shown in Table 17; a CD2 VH sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR2 sequence shown in Table 17; a VH CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR3 sequence shown in Table 17; a VL CDR1 sequence that 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 shown in Table 17; a VL CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR2 sequence shown in Table 17; and a VL CDR3 sequence that 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 shown in Table 17.

In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination is the combination shown in Table 17.

In some embodiments, the activated antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein each CDR sequence in the combination contains a sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the corresponding CDR sequence in the combination shown in Table 17.

In some embodiments, the activated antibody comprises an amino acid sequence selected from the group consisting of SeQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103,

105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147,

149, 151, 153, 155, 157, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961,963,

965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005,

1144-1191, 1200 and 1201. In some embodiments, the implementation of the activated antibody contains an amino acid sequence selected from the group consisting of SEQ ID NOS: 46, 48, 50, 52, 54 and 56. In some embodiments, the implementation of the activated antibody contains an amino acid sequence, selected from the group consisting of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105,

107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,

151, 153, 155, 157, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965,

967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1144-

1191, 1200 and 1201, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54 and 56.

- 15 039736

In some embodiments, the activated antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1144-119 1200 and 1201. In some embodiments, the activated antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, and 56. In some embodiments, the activated antibody comprises an amino acid sequence selected from the group, consisting of SEQ ID NOs: 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1144-1191, 1200 and 1201, and an amino acid sequence selected from the group consisting of SEQ ID NO: 46, 48, 50, 52, 54 and 56.

In some embodiments, the activated antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961 963 965 967 969 971 973 975 977 979 981 983 985 987 989 991 993 995 997 999 1001 1003 1005 1145 1149 1151 1153 1155 1157 1159 1161 1163 1165 1167 1169 1171 1173 1175 1177 1179 1181 1183 1185 1187 1199 the activated antibody contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, and 56. In some embodiments, the activated antibody contains an amino acid sequence selected from the group consisting of SEQ ID NO: 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 100 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189, 1191 and 1201 and 1201 and 1201 , and an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54 and 56.

In some embodiments, the activated antibody comprises 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 SEQ ID NO: 83 , 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133 , 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 931, 933, 935, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961 , 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 11441001 and 1201, and 1201 In some embodiments, the activated antibody comprises 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 SEQ ID NO: 46, 48, 50, 52, 54, and 56. In some embodiments, the activated antibody comprises am a foreign 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 NOs: 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141 143 145 147 149 931 933 935 937 939 941 943 945 947 949 951 953 955 957 959 961 963 965 967 969 971, 973, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1144-1191, 1200 and 1201, and 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 SEQ ID NOS: 46, 48, 50, 52, 54, and 56.

In some embodiments, the activated antibody comprises 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 SEQ ID NO: 931 , 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981 , 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1144-1191, 1200, and 1201. 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, and 56. In some embodiments, an activated antibody 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 groups py, consisting of SEQ ID NO: 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973 975 977 979 981 983 985 987 989 991 993 995 997 999 1001 1003 1005 1144-1191 1200 and 1201 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54 and 56.

In some embodiments, the activated antibody comprises 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 SEQ ID NO: 931 , 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981 , 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 116.3 , 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189, 1191, 1200 and 1201. B

- 16 039736 in some embodiments, the activated antibody comprises 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 SEQ ID NO : 46, 48, 50, 52, 54, and 56. In some embodiments, the activated antibody comprises an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid a sequence selected from the group consisting of SEQ ID NOs: 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969 971 973 975 977 979 981 983 985 987 989 991 993 995 997 999 1001 1003 1005 1145 1147 1149 1151 115 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189, 1191 and 1201, and an amino acid sequence that is at least 90, 91 , 92, 93, 94, 95, 96, 97, 9 8 or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54 and 56.

In some embodiments, the activated antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 931, 933, 935, 937, 939, 941, 943, 945, 947, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1144-1191, 1200 and 1201, and the heavy chain amino acid sequence of SEQ ID NO: 46.

In some embodiments, the activated antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1144-119 1200 and 1201, and the heavy chain amino acid sequence of SEQ ID NO: 46.

In some embodiments, the activated antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961 963 965 967 969 971 973 975 977 979 981 983 985 987 989 991 993 995 997 999 1001 1003 1005 1145 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1199 and the heavy amino acid sequence chains SEQ ID NO: 46.

In some embodiments, the activated antibody comprises 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 SEQ ID NO: 83 , 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133 , 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 931, 933, 935, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961 , 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 11441001 and 1201, and 1201 , and an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the heavy chain amino acid sequence of SEQ ID NO: 46.

In some embodiments, the activated antibody comprises 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 SEQ ID NO: 931 , 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981 , 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1144-1191, 1200 and 1201, and an amino acid sequence that is at least 90, 91, 92, 93, 94%, 95%, 96%, 97%, 98% or 99% identical to the heavy chain amino acid sequence of SEQ ID NO: 46.

In some embodiments, the activated antibody comprises 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 SEQ ID NO: 931 , 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981 , 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 116.3 , 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189, 1191 and 1201, and an amino acid sequence that is at least 98 or 99% identical to the heavy chain amino acid sequence of SEQ ID NO: 46.

In some embodiments, the activated antibody comprises the amino acid sequence of SEQ ID NO: 428. In some embodiments, the activated antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 430, 432, 434, and 1202. In some embodiments, the activated antibody comprises an amino acid sequence of SEQ ID NO: 428; and an amino acid sequence selected from the group consisting of SEQ ID NO: 430, 432 and 434.

In some embodiments, the implementation of the activated antibody contains the amino acid sequence of SEQ ID NO: 1008. In some embodiments, the implementation of the activated antibody contains an amino acid sequence selected from the group consisting of SEQ ID NO: 430, 432, 434 and 1202. In some embodiments, the implementation of the activated antibody contains the amino acid sequence of SEQ ID NO: 1008 and an amino acid sequence selected from the group

- 17 039736 consisting of SEQ ID NOs: 430, 432, 434 and 1202.

In some embodiments, the activated antibody comprises an amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 428. In some embodiments, the activated antibody 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 SEQ ID NOs: 430, 432, 434, and 1202 In some embodiments, the activated antibody comprises an amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 428 and 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 SEQ ID NOs: 430, 432, 434 and 1202.

In some embodiments, the activated antibody comprises an amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 1008. In some embodiments, the activated antibody 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 SEQ ID NOs: 430, 432, 434, and 1202 In some embodiments, the activated antibody comprises an amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 1008 and 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 SEQ ID NOs: 430, 432, 434 and 1202.

In some embodiments, the activated antibody comprises a masking fragment (MM) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 59-81, 208, and 426; a cleavable fragment (CM) containing an amino acid sequence selected from the group consisting of SEQ ID nO: 341, 352, 359, 364, 372, 377, 379, 383, 394, 437, 883-893, 901-920 and 921; and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises a VL CDR1 sequence containing RASQSISSYLN (SEQ ID NO: 209); a VL CDR2 sequence containing AASSLQS (SEQ ID NO: 215); a VL CDR3 sequence containing DNGYPST (SEQ ID NO: 228); a VH CDR1 sequence containing SYAMS (SEQ ID NO: 212) ; a VH CDR2 sequence containing SSIWRNGIVTVYADS (SEQ ID NO: 246); and a VH CDR3 sequence containing WSAAFDY (SEQ ID NO: 235).

In some embodiments, the activated antibody comprises a masking fragment (MM) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 60, 62, 63, 66-68, 71, 75, and 76; a cleavable fragment (CM) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 341, 364, 377, 394, 437, 883-893, 901-911 and 920; and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises a VL CDR1 sequence containing RASQSISSYLN (SEQ ID NO: 209); a VL CDR2 sequence containing AASSLQS (SEQ ID NO: 215); a VL CDR3 sequence containing DNGYPST (SEQ ID NO: 228); a VH CDR1 sequence containing SYAMS (SEQ ID NO: 212) ; a VH CDR2 sequence containing SSIWRNGIVTVYADS (SEQ ID NO: 246); and a VH CDR3 sequence containing WSAAFDY (SeQ ID NO: 235).

In some embodiments, the activated antibody comprises a masking fragment (MM) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 59-81, 208, and 426; a cleavable fragment (CM) containing an amino acid sequence selected from the group consisting of SEQ ID nO: 341, 352, 359, 364, 372, 377, 379, 383, 394, 437, 883-893, 901-920 and 921; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46.

In some embodiments, the activated antibody comprises a masking fragment (MM) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 60, 62, 63, 66-68, 71, 75, and 76; a cleavable fragment (CM) containing an amino acid sequence selected from the group consisting of SeQ ID NOs: 341, 364, 377, 394, 437, 883-893, 901-911 and 920; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46.

In some embodiments, the activated antibody comprises a masking fragment (MM) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 59-81, 208, and 426; a cleavable fragment (CM) containing an amino acid sequence selected from the group consisting of sEq ID nO: 341, 352, 359, 364, 372, 377, 379, 383, 394, 437, 883-893, 901-920 and 921; and a light chain variable region containing the amino acid sequence

- 18 039736

SEQ ID NO: 12, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 46.

In some embodiments, the activated antibody comprises a masking fragment (MM) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 60, 62, 63, 66-68, 71, 75, and 76; a cleavable fragment (CM) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 341, 364, 377, 394, 437, 883-893, 901-11 and 920; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence that contains a nucleic acid sequence encoding a heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30 , 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, and 56. In some embodiments, the antibody to be activated is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, and 56. In some embodiments, the activated antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a light chain amino acid sequence comprising SEQ ID NO: 58 In some embodiments, the activated antibody is encoded by the nucleic acid sequence s, which includes a nucleic acid sequence encoding a light chain amino acid sequence containing SEQ ID NO: 12. In some embodiments, the activated antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 and 56, and the sequence a nucleic acid sequence that includes a nucleic acid sequence encoding a light chain amino acid sequence comprising SEQ ID NO: 58. In some embodiments, the antibody to be activated is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a heavy chain amino acid sequence selected from the group consisting of from SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 3 4, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 and 56, and a nucleic acid sequence that includes a nucleic acid sequence encoding a light chain amino acid sequence containing SEQ ID NO: 12. In some embodiments, the activated antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a heavy chain amino acid sequence from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, and 56, and a nucleic acid sequence that includes a nucleic acid sequence acid encoding a light chain amino acid sequence comprising SEQ ID NO: 58. In some embodiments, the activated antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 46, 48, 50, 52, 54 and 56, and n a nucleic acid sequence comprising a nucleic acid sequence encoding a light chain amino acid sequence comprising SEQ ID NO: 12.

In some embodiments, the activated antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a heavy chain amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid identical. a sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, and 56. In some embodiments, the activated antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a heavy chain amino acid sequence, a 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 NOs: 46, 48, 50, 52, 54, and 56. In some embodiments, the activated antibody is encoded by a nucleic acid sequence , which includes a nucleic acid sequence encoding a light chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, In one embodiment, the activated antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a light chain amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the activated antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a heavy chain amino acid sequence that is at least 90

- 19 039736

91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 , 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 and 56, and a nucleic acid sequence that includes a nucleic acid sequence encoding a light chain amino acid sequence that is at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the activated antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a heavy chain amino acid sequence that at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 and 56, and in sequence a nucleic acid that includes a nucleic acid sequence encoding a light chain amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 12. B in some embodiments, the activated antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a heavy chain amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid sequence identical selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54 and 56, and a nucleic acid sequence that includes a nucleic acid sequence encoding a light chain amino acid sequence that is at least 90, 91, 92 , 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the activated antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence encoding a heavy chain 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 a group consisting of SEQ ID NOs: 46, 48, 50, 52, 54 and 56, and a nucleic acid sequence that includes a nucleic acid sequence encoding a light chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the activated antibody is encoded by a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51, 53, and 55. In some embodiments, the activated antibody is encoded by a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOS: 45, 47, 49, 51, 53, and 55. In some embodiments, the activated antibody is encoded by the light chain nucleic acid sequence of SEQ ID NO: 57. In some embodiments, the activated antibody is encoded by the light chain nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the activated antibody is encoded by the heavy chain nucleic acid sequence selected from the group consisting of SEQ IDNO: 13, 15, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53 and 55, and the nucleicino sequence a light chain acid containing SEQ ID NO: 57. In some embodiments, the activated antibody is encoded by a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 13, 15, 17, 21, 23, 25, 27, 29 , 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55, and a light chain nucleic acid sequence comprising SEQ ID NO: 11. In some embodiments, the activated antibody is encoded by a nucleic acid sequence an acid that includes a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 51, 53, and 55 and a light chain nucleic acid sequence comprising SEQ ID NO: 57. In some embodiments, an activated an antibody is encoded by a nucleic acid sequence that includes a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 51, 53 and 55 and a light chain nucleic acid sequence chain containing SEQ ID NO: 11.

In some embodiments, the activated 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 heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55. In some embodiments, an activated the 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 heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 45, 47, 49, 51, 53, and 55. In some embodiments, the activated antibody encodes a nucleic acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the nucleic acid sequence. light chain SEQ ID NO: 57. In some embodiments, the activated antibody is encoded after a nucleic acid identity that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the light chain nucleic acid sequence of SEQ ID NO: 11.

- 20 039736 leic acid, which is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 13, 15, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53 and 55, and a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to a light chain nucleic acid sequence comprising SEQ ID NO: 57. In some embodiments, an activated 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 heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, 17, 21, 23, 25, 27 , 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53 and 55, and a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95 , 96, 97, 98 or 99% identical to the next of a light chain nucleic acid comprising SEQ ID NO: 11. In some embodiments, the activated antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97 , 98 or 99% identical to a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 51, 53 and 55, and a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the light chain nucleic acid sequence containing SEQ ID NO: 57. In some embodiments, the activated antibody is encoded by a nucleic acid sequence that includes a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 51, 53 and 55 and a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the light chain nucleic acid sequence comprising SEQ ID NO: 11.

In some embodiments, the MM has a dissociation constant for binding to AB that is higher than the dissociation constant for binding AB to PDL1.

In some embodiments, the implementation of the MM has a dissociation constant for binding to AB, which is not higher than the dissociation constant for binding AB to PDL1.

In some embodiments, the MM has a dissociation constant for binding to AB that is lower than the dissociation constant for binding AB to PDL1.

In some embodiments, the implementation of the MM has a dissociation constant for binding to AB, which is approximately equal to the dissociation constant for binding AB to PDL1.

In some embodiments, MM does not interfere with or compete with AB for binding to PDL1 when the antibody to be activated is in a cleaved state.

In some embodiments, the implementation of the MM is a polypeptide from 2 to 40 amino acids in length. In some embodiments, the MM is a polypeptide up to 40 amino acids in length.

In some embodiments, the MM polypeptide sequence is different from the PDL1 sequence. In some embodiments, the MM polypeptide sequence is no more than 50% identical to any natural AB binding partner. In some embodiments, the MM polypeptide sequence differs from the PDL1 sequence and is no more than 40%, 30%, 25%, 20%, 15% or 10% identical to any natural AB binding partner.

In some embodiments, the MM contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 59-81, 208, and 426. In some embodiments, the MM contains 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 SEQ ID NOs: 59-81, 208 and 426.

In some embodiments, the binding of MM to AB reduces the ability of AB to bind to PDL1 such that the dissociation constant (Kd) of AB when combined with MM relative to PDL1 is at least twice as high as the Kd of AB when not bound to MM in relation to PDL1.

In some embodiments, binding of MM to AB reduces the ability of AB to bind to PDL1 such that the dissociation constant (Kd) of AB when combined with MM relative to PDL1 is at least five times higher than the Kd of AB when not bound to MM in relation to PDL1.

In some embodiments, binding of MM to AB reduces the ability of AB to bind to PDL1 such that the dissociation constant (Kd) of AB when combined with MM relative to PDL1 is at least 10-fold higher than the Kd of AB when not bound to MM in relation to PDL1.

In some embodiments, the binding of MM to AB reduces the ability of AB to bind to PDL1 such that the dissociation constant (Kd) of AB when combined with MM relative to PDL1 is at least 20 times higher than the Kd of AB when not bound to MM in relation to PDL1.

In some embodiments, binding of MM to AB reduces the ability of AB to bind to PDL1 such that the dissociation constant (Kd) of AB when combined with MM relative to PDL1 is at least 40 times higher than the Kd of AB when not bound to MM in relation to PDL1.

In some embodiments, the binding of MM to AB reduces the ability of AB to bind to PDL1 such that the dissociation constant (Kd) of AB when combined with MM relative to PDL1

- 21 039736 at least 100 times higher than the Kd of AB when it is not associated with MM in relation to PDL1.

In some embodiments, the binding of MM to AB reduces the ability of AB to bind to PDL1 such that the dissociation constant (Kd) of AB when combined with MM relative to

PDL1 is at least 1000 times higher than the Kd of AB when not associated with MM relative to PDL1.

In some embodiments, binding of MM to AB reduces the ability of AB to bind to PDL1 such that the dissociation constant (Kd) of AB when combined with MM relative to PDL1 is at least 10,000 times higher than the Kd of AB when not bound to MM in relation to PDL1.

In some embodiments, in the presence of PDL1, MM reduces the ability of AB to bind to PDL1 by at least 90% when CM is not cleaved compared to when SM is cleaved, as determined in an in vitro assay using a target displacement test such as , for example, the assay described in PCT International Application Publication WO 2010/081173, the contents of which are incorporated herein by reference in its entirety.

In some embodiments, the protease that cleaves SM is active, eg, upregulated in diseased tissue, and the protease cleaves SM in the activated antibody when the activated antibody is exposed to the protease.

In some embodiments, the protease co-locates to tissue with PDL1 and the protease cleaves the CM in the activated antibody when the activated antibody is exposed to the protease.

In some embodiments, the protease is present at relatively higher levels in close proximity to the tissue containing the target, in the area to be treated or diagnosed, than in the tissue of the areas not to be treated (e.g., healthy tissue), and the protease cleaves SM in an activated antibody when the activated antibody is exposed to the protease.

In some embodiments, the implementation of the CM is located in the activated antibody such that when the activated antibody is in the uncleaved state, the binding of the activated antibody to PDL1 is reduced with a dissociation constant that is at least twice the dissociation constant for binding of unmodified AB to PDL1, while in the cleaved state (ie when the antibody to be activated is in the cleaved state) AB binds to PDL1.

In some embodiments, the implementation of the CM is located in the activated antibody such that when the activated antibody is in the uncleaved state, the binding of the activated antibody to PDL1 is reduced with a dissociation constant that is at least five times higher than the dissociation constant for binding of unmodified AB to PDL1, while in the cleaved state (ie when the antibody to be activated is in the cleaved state) AB binds to PDL1.

In some embodiments, the implementation of the SM is located in the activated antibody such that when the activated antibody is in the uncleaved state, the binding of the activated antibody to PDL1 is reduced with a dissociation constant that is at least 10 times higher than the dissociation constant for binding unmodified AB to PDL1, while in the cleaved state (ie when the antibody to be activated is in the cleaved state) AB binds to PDL1.

In some embodiments, the implementation of the CM is located in the activated antibody such that when the activated antibody is in the uncleaved state, the binding of the activated antibody to PDL1 is reduced with a dissociation constant that is at least 20 times higher than the dissociation constant for binding unmodified AB to PDL1, while in the cleaved state (ie when the antibody to be activated is in the cleaved state) AB binds to PDL1.

In some embodiments, the implementation of the SM is located in the activated antibody such that when the activated antibody is in the uncleaved state, the binding of the activated antibody to PDL1 is reduced with a dissociation constant that is at least 40 times higher than the dissociation constant for binding of unmodified AB to PDL1, whereas in the cleaved state, AB binds to PDL1.

In some embodiments, the implementation of the CM is located in the activated antibody such that when the activated antibody is in the uncleaved state, the binding of the activated antibody to PDL1 is reduced with a dissociation constant that is at least 50 times higher than the dissociation constant for binding of unmodified AB to PDL1, whereas in the cleaved state, AB binds to PDL1.

In some embodiments, the CM is located in the activated antibody such that when the activated antibody is in an uncleaved state, the binding of the activated antibody to PDL1 is reduced with a dissociation constant that is at least 100 times higher than the dissociation constant for binding unmodified AB to PDL1, whereas in the cleaved state, AB binds to PDL1.

In some embodiments, the implementation of the CM is located in the activated antibody in such a way that when the activated antibody is in an uncleaved state, then the binding of the activated

- 22 039736 antibody with PDL1 is reduced with a dissociation constant that is at least 200 times higher than the dissociation constant for binding unmodified AB to PDL1, while in the cleaved state AB binds to PDL1.

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

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

In some embodiments, the implementation of the SM is a substrate for at least one protease, which is believed to be upregulated in cancer. In some embodiments, the implementation of the SM is a substrate for at least one protease, which is believed to be upregulated in inflammation.

In some embodiments, the implementation of the SM is a substrate for at least one protease, which is believed to be upregulated in autoimmunity.

In some embodiments, CM is a substrate for at least one protease selected from the group consisting of matrix metalloprotease (MMP), thrombin, neutrophil elastase, cysteine protease, legumain, and a serine protease such as matriptase (MT-SP1) and urokinase ( uPA). While not wishing to be bound by theory, these proteases are believed to be upregulated in at least one of cancer, inflammation, and/or autoimmunity.

Typical substrates include, but are not limited to, substrates cleaved by one or more of the following enzymes or proteases listed in Table 12.

In some embodiments, the SM is selected for use with a specific protease, such as a protease known to co-localize with the target of an activated antibody.

In some embodiments, the implementation of the CM is selected for use with a specific protease, for example, a protease, which is known to be in close proximity to the target of the activated antibody.

In some embodiments, the SM is a substrate for at least one MMP. Examples of MMPs include the MMPs shown in Table 12. In some embodiments, SM is a substrate for a protease selected from the group consisting of MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. In some embodiments, SM is a substrate for MMP9. In some embodiments, SM is a substrate for MMP 14.

In some embodiments, the SM is a substrate that includes the sequence

TGRGPSWV (SEQ ID NO: 338); SARGPSRW (SEQ ID NO: 339); TARGPSFK (SEQ ID NO: 340); LSGRSDNH (SEQ ID NO: 341); GGWHTGRN (SEQ ID

NO: 342); HTGRSGAL (SEQ ID NO: 343); PLTGRSGG (SEQ ID NO: 344);

IAARGPAIH (SEQ ID NO: 345); RGPAFNPM (SEQ ID NO: 346); SSRGPAYL (SEQ ID NO: 347); RGPATPIM (SEQ ID NO: 348); RGPA (SEQ ID NO:

349); GGQPSGMWGW (SEQ ID NO: 350); FPRPLGITGL (SEQ ID NO: 351);

VHMPLGFLGP (SEQ ID NO: 352); SPLTGRSG (SEQ ID NO: 353); SAGFSLPA (SEQ ID NO: 354); LAPLGLQRR (SEQ ID NO: 355); SGGPLGVR (SEQ ID

NO: 356); PLGL (SEQ ID NO: 357); LSGRSGNH (SEQ ID NO: 883);

SGRSANPRG (SEQ ID NO: 884) ; LSGRSDDH (SEQ ID NO: 885) ; LSGRSDIH (SEQ ID NO: 886); LSGRSDQH (SEQ ID NO: 887); LSGRSDTH (SEQ ID

NO: 888); LSGRSDYH (SEQ ID NO: 889); LSGRSDNP (SEQ ID NO: 890);

LSGRSANP (SEQ ID NO: 891); LSGRSANI (SEQ ID NO: 892); and/or

LSGRSDNI (SEQ ID NO: 893).

In some embodiments, the implementation of the SM contains the amino acid sequence LSGRSDNH (SEQ ID NO: 341). In some embodiments, the implementation of the CM contains the amino acid sequence TGRGPSWV (SEQ ID NO: 338). In some embodiments, the implementation of the CM contains the amino acid sequence PLTGRSGG (SEQ ID NO: 344). In some embodiments, the CM contains the amino acid sequence GGQPSGMWGW (SEQ ID NO: 350). In some embodiments, the implementation of the SM contains the amino acid sequence FPRPLGITGL (SEQ ID NO: 351). In some embodiments, the implementation of the CM contains the amino acid sequence VHMPLGFLGP (SEQ ID NO: 352). In some embodiments, the implementation of the SM contains the amino acid sequence PLGL (SEQ ID NO: 357). In some embodiments, the implementation of the SM contains the amino acid sequence SARGPSRW (SEQ ID NO: 339). In some embodiments, the implementation of the CM contains the amino acid sequence TARGPSFK (SEQ ID NO: 340). In some embodiments, the implementation of the CM contains the amino acid sequence GGWHTGRN (SEQ ID NO: 342). In some embodiments, the implementation of the SM contains the amino acid sequence HTGRSGAL (SEQ ID NO: 343). In some embodiments, the implementation of the CM contains the amino acid sequence AARGPAIH (SEQ ID NO: 345). In some embodiments, the implementation of the SM contains the amino acid sequence RGPAFNPM (SEQ ID NO: 346). In some embodiments, the implementation of the CM contains the amino acid sequence SSRGPAYL (SEQ ID NO: 347). In some embodiments, the implementation of the SM contains the amino acid sequence RGPATPIM (SEQ ID NO: 348). In some embodiments, the implementation of the CM contains the amino acid sequence RGPA (SEQ ID NO: 349). In some embodiments, the implementation of the CM contains the amino acid sequence LSGRSGNH (SEQ ID NO: 883). In some embodiments, the implementation of the SM contains the amino acid sequence SGRSANPRG (SEQ ID NO: 884). In some embodiments, the implementation of the SM contains the amino acid sequence LSGRSDDH (SEQ ID NO: 885). In some embodiments, the implementation of the SM contains the amino acid sequence LSGRSDIH (SEQ ID NO: 886). In some embodiments, the implementation of the SM contains the amino acid sequence LSGRSDQH (SEQ ID NO: 887). In some embodiments, the implementation of the SM contains the amino acid sequence LSGRSDTH (SEQ ID NO: 888). In some embodiments, the implementation of the SM contains the amino acid sequence LSGRSDYH (SEQ ID NO: 889). In some embodiments, the implementation of the CM contains the amino acid sequence LSGRSDNP (SEQ ID NO: 890). In some embodiments, the implementation of the SM contains the amino acid sequence LSGRSANP (SEQ ID NO: 891). In some embodiments, the implementation of the CM contains the amino acid sequence LSGRSANI (SEQ ID NO: 892). In some embodiments, the implementation of the SM contains the amino acid sequence LSGRSDNI (SEQ ID NO: 893).

In some embodiments, the implementation of the SM is a substrate for MMP and includes the sequence

ISSGLSS (SEQ ID

NO: 358); QNQALRMA (SEQ ID NO: 359); AQNLLGMV (SEQ ID NO: 360);

STPFGMF (SEQ ID NO: 361); PVGYTSSL (SEQ ID NO: 362); DWLYWPGI (SEQ ID NO: 363), ISSGLLSS (SEQ ID NO: 364), LKAAPRWA (SEQ ID

NO: 365); GPSHLVLT (SEQ ID NO: 366); LPGGLSPW (SEQ ID NO: 367);

MGLFSEAG (SEQ ID NO: 368); SPLPLRVP (SEQ ID NO: 369); RMHLRSLG (SEQ ID NO: 370); LAAPLGLL (SEQ ID NO: 371); AVGLAPP (SEQID

NO: 372); LLAPSHRA (SEQ ID NO: 373); PAGLWLDP (SEQ ID NO: 374);

MIAPVAYR (SEQ ID NO: 894); RPSPMWAY (SEQ ID NO: 895); WATPRPMR (SEQ ID NO: 896); FRLLDWQW (SEQ ID NO: 897); ISSGL (SEQ ID NO:

898); ISSGLLS (SEQ ID NO: 899); and/or ISSGLL (SEQ ID NO: 900).

In some embodiments, the implementation of the SM contains the amino acid sequence

ISSGLSS (SEQ ID NO: 358). In some embodiments, the implementation of the CM contains the amino acid sequence QNQALRMA (SEQ ID NO: 359) . In some embodiments, the implementation of the CM contains the amino acid sequence AQNLLGMV (SEQ ID NO: 360). In some embodiments, the implementation of the SM contains the amino acid sequence STFPFGMF (SEQ ID NO: 361). In some embodiments, the implementation of the CM contains the amino acid sequence PVGYTSSL (SEQ ID NO: 362). In some embodiments, the implementation of the SM contains the amino acid sequence DWLYWPGI (SEQ ID NO: 363). In some embodiments, the implementation of the CM contains the amino acid sequence ISSGLLSS (SEQ ID NO: 364). In some embodiments, the implementation of the CM contains the amino acid sequence LKAAPRWA (SEQ ID NO: 365). In some embodiments, the implementation of the SM contains the amino acid sequence GPSHLVLT (SEQ ID NO: 366). In some embodiments, the implementation of the SM contains the amino acid sequence LPGGLSPW (SEQ ID NO: 367). In some embodiments, the implementation of the CM contains the amino acid sequence MGLFSEAG (SEQ ID NO: 368). In some embodiments, the implementation of the CM contains the amino acid sequence

- 24 039736 SPLPLRVP (SEQ ID NO: 369). In some embodiments, the implementation of the SM includes the amino acid sequence RMHLRSLG (SEQ ID NO: 370). In some embodiments, the implementation of the SM contains the amino acid sequence LAAPLGLL (SEQ ID NO: 371). In some embodiments, the implementation of the CM contains the amino acid sequence AVGLLAPP (SEQ ID NO: 372). In some embodiments, the implementation of the CM contains the amino acid sequence LLAPSHRA (SEQ ID NO: 373). In some embodiments, the implementation of the SM contains the amino acid sequence PAGLWLDP (SEQ ID NO: 374). In some embodiments, the implementation of the CM contains the amino acid sequence MIAPVAYR (SEQ ID NO: 894). In some embodiments, the implementation of the CM contains the amino acid sequence RPSPMWAY (SEQ ID NO: 895). In some embodiments, the implementation of the SM contains the amino acid sequence WATPRPMR (SEQ ID NO: 896). In some embodiments, the implementation of the CM contains the amino acid sequence FRLLDWQW (SEQ ID NO: 897). In some embodiments, the implementation of the SM contains the amino acid sequence ISSGL (SEQ ID NO: 898). In some embodiments, the implementation of the CM contains the amino acid sequence ISSGLLS (SEQ ID NO: 899). In some embodiments, the implementation of the CM contains the amino acid sequence ISSGLL (SEQ ID NO: 900).

In some embodiments, SM is a substrate for thrombin. In some embodiments, the implementation of the SM is a substrate for thrombin and contains the sequence of GPRSFGL (SEQ ID NO: 375) or GPRSFG (SEQ ID NO: 376). In some embodiments, the implementation of the SM contains the amino acid sequence of GPRSFGL (SEQ ID NO: 375). In some embodiments, the implementation of the SM contains the amino acid sequence GPRSFG (SEQ ID NO: 376).

In some embodiments, the implementation of the CM contains an amino acid sequence selected from the group consisting of NTLSGRSENHSG (SEQ ID NO: 435); NTLSGRSGNHGS (SEQ ID NO: 436); TSTSGRSANPRG (SEQ ID NO: 437); TSGRSANP (SEQ ID NO: 438); VAGRSMRP (SEQ ID NO: 439); WPEGRRS (SEQ ID NO: 440); ILPRSPAF (SEQ ID NO: 441); MVLGRSLL (SEQ ID NO: 442); QGRAITFI (SEQ ID NO: 443); SPRSIMLA (SEQ ID NO: 444); SMLRSMPL (SEQ ID NO: 445).

In some embodiments, the implementation of the CM contains the amino acid sequence NTLSGRSENHSG (SEQ ID NO: 435). In some embodiments, the implementation of the SM contains the amino acid sequence NTLSGRSGNHGS (SEQ ID NO: 436). In some embodiments, the implementation of the SM contains the amino acid sequence TSTSGRSANPRG (SEQ ID NO: 437). In some embodiments, the implementation of the CM contains the amino acid sequence TSGRSANP (SEQ ID NO: 438). In some embodiments, the implementation of the SM contains the amino acid sequence VAGRSMRP (SEQ ID NO: 439). In some embodiments, the implementation of the CM contains the amino acid sequence WPEGRRS (SEQ ID NO: 440). In some embodiments, the implementation of the CM contains the amino acid sequence of ILPRSPAF (SEQ ID NO: 441). In some embodiments, the implementation of the CM contains the amino acid sequence MVLGRSLL (SEQ ID NO: 442). In some embodiments, the implementation of the CM contains the amino acid sequence QGRAITFI (SEQ ID NO: 443). In some embodiments, the implementation of the CM contains the amino acid sequence SPRSIMLA (SEQ ID NO: 444). In some embodiments, the implementation of the CM contains the amino acid sequence SMLRSMPL (SEQ ID NO: 445).

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

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

ISSGLLSGRSDNH (SEQ ID NO: 377); ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 378);

IAVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 379) ; TSTSGRSANPRGGGAVGLAPP (SEQ ID NO: 380); VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 381);

TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 382); AVGLLAPPGGLSGRSDNH (SEQ ID NO: 383); LSGRSDNHGGAVGLLAPP (SEQ ID NO: 384);

VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 385) ; LSGRSDNHGGVHMPLGFLGP (SEQ

ID NO: 386); LSGRSDNHGGGGSISSGLLSS (SEQ ID NO: 387);

- 25 039736

LSGRSGNHGGGGSISSGLLSS (SEQ ID NO: 388); ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 389) ; LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 390) ;

QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 391); LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 392); QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 393);

ISSGLLSGRSGNH (SEQ ID NO: 394); ISSGLLSGRSANPRG (SEQ ID NO

901); AVGLLAPPTSGRSANPRG (SEQ ID NO: 902) ; AVGLLAPPSGRSANPRG (SEQ ID NO: 903); ISSGLLSGRSDDH (SEQ ID NO: 904); ISSGLLSGRSDDIH (SEQ ID NO: 905) ; ISSGLLSGRSDQH (SEQ ID NO: 906); ISSGLLSGRSDTH (SEQ ID NO: 907); ISSGLLSGRSDYH (SEQ ID NO: 908); ISSGLLSGRSDNP (SEQ ID NO: 909) ; ISSGLLSGRSANP (SEQID NO: 910); ISSGLLSGRSANI

(SEQ ID NO: 911); AVGLLAPPGGLSGRSDDDH (SEQ ID NO: 912);

AVGLLAPPGGLSGRSDIH (SEQ ID NO: 913) ; AVGLLAPPGGLSGRSDQH (SEQ ID

NO: 914); AVGLLAPPGGLSGRSDTH (SEQ ID NO: 915);

AVGLLAPPGGLSGRSDYH (SEQ ID NO: 916) ; AVGLLAPPGGLSGRSDNP (SEQ ID

NO: 917); AVGLLAPPGGLSGRSANP (SEQ ID NO: 918);

AVGLLAPPGGLSGRSANI (SEQ ID NO: 919); ISSGLLSGRSDNI (SEQ ID NO:

920); AVGLLAPPGGLSGRSDNI (SEQ ID NO: 921); GLSGRSDNHGGAVGLLAPP (SEQ ID NO: 1009); and/or GLSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 1010) .

In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSDNH (SEQ ID NO: 377), which also refers here to substrate 2001. In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 378), which also refers here to substrate 1001/LP'/000l, where LP' used in this substrate CM1-CM2 represents the amino acid sequence GGSGGS (SEQ ID NO: 922). In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 379), which also refers here to substrate 2015 and/or substrate 1004/LP'/0003, where LP' used in this substrate CM1-CM2 is amino acid sequence GG. In some embodiments, the CM1-CM2 substrate contains the sequence TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 380), which also refers here to substrate 0003/LP'/1004, where LP' used in this CM1-CM2 substrate is the GG amino acid sequence. In some embodiments, the CM1-CM2 substrate contains the sequence VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 381), which also refers here to substrate 1003/LP'/0003, where LP' used in this CM1-CM2 substrate is the GG amino acid sequence. In some embodiments, the CM1-CM2 substrate contains the sequence TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 382), which also refers here to substrate 0003/LP'/1003, where LP' used in this CM1-CM2 substrate is the GG amino acid sequence. In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGLSGRSDNH (SEQ ID NO: 383), which also refers here to substrate 3001 and/or substrate 1004/LP'/0001, where LP' used in this CM1CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1CM2 substrate contains the sequence LSGRSDNHGGAVGLLAPP (SEQ ID NO: 384), which also refers here to substrate 0001/LP'/1004, where LP' used in this CM1-CM2 substrate is the GG amino acid sequence. In some embodiments, the CM1-CM2 substrate contains the sequence VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 385), which also refers here to substrate 1003/LP'/0001, where LP' used in this CM1-CM2 substrate is the GG amino acid sequence. In some embodiments, the CM1-CM2 substrate contains the sequence LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 386), which also refers here to substrate 0001/LP'/1003, where LP' used in this CM1-CM2 substrate is the GG amino acid sequence. In some embodiments, the CM1-CM2 substrate contains the sequence LSGRSDNHGGGGSISSGLSS (SEQ ID NO: 387), which also refers here to substrate 0001/LP'/1001, where LP' used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 922). In some embodiments, the CM1-CM2 substrate contains the sequence LSGRSGNHGGGGSISSGLSS (SEQ ID NO: 388), which

- 26 039736 also refers here to substrate 0002/LP'/1001, where LP' used in this substrate CM1-CM2 represents the amino acid sequence GGSGGS (SEQ ID NO: 922). In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 389), which also refers to substrate 1001/LP'/0002 herein, where LP' used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 922). In some embodiments, the CM1-CM2 substrate contains the sequence LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 390), which also refers here to substrate 0001/LP'/1002, where LP' used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 922). In some embodiments, the CM1-CM2 substrate contains the sequence QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 391), which also refers here to substrate 1002/LP'/0001, where LP' used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 922). In some embodiments, the CM1-CM2 substrate contains the sequence LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 392), which also refers here to substrate 0002/LP'/1002, where LP' used in this CM1CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: : 922). In some embodiments, the CM1-CM2 substrate contains the sequence QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 393), which also refers here to substrate 1002/LP'/0002, where LP' used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 922). In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSGNH (SEQ ID NO: 394), which also refers here to substrate 2002. In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSANPRG (SEQ ID NO: 901), which also refers here to 2003 substrate. In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPTSGRSANPRG (SEQ ID NO: 902), which also refers to substrate 2004 herein. In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPSGRSANPRG (SEQ ID NO: 903), which also refers here to the 2005 substrate. In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSDDH (SEQ ID NO: 904), which also refers to the 2006 substrate here. In some embodiments, the CM1CM2 substrate contains the sequence ISSGLLSGRSDIH (SEQ ID NO: 905), which also refers here to the substrate 2007. In some embodiments, the implementation of the substrate CM1-CM2 contains the sequence ISSGLLSGRSDQH (SEQ ID NO: 906), which also refers here to the substrate 2008. In some embodiments, the implementation of the substrate CM1-CM2 contains the sequence ISSGLLSGRSDTH (SEQ ID NO: 907), which also refers here to the substrate 2009. In some embodiments, the implementation the CM1-CM2 substrate contains the sequence ISSGLLSGRSDYH (SEQ ID NO: 908), which also refers here to the 2010 substrate. In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSDNP (SEQ ID NO: 909), which also refers to the 2011 substrate here. In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSANP (SEQ ID NO: 910), which is also referred to herein as 2012 substrate. In some embodiments, the CM1-CM2 substrate contains the sequence ISSGLLSGRSANI (SEQ ID NO: 911), which is also referred to here to the 2013 substrate. In some embodiments, the CM1-CM2 substrate contains the sequence AVGLL APPGGLSGRSDDH (SEQ ID NO: 912), which also refers to substrate 3006 herein. In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGLSGRSDIH (SEQ ID NO: 913), which also refers to substrate 3007 herein. In some embodiments, the CM1 substrate -CM2 contains the sequence AVGLLAPPGGLSGRSDQH (SEQ ID NO: 914), which also refers here to substrate 3008. In some embodiments, the substrate CM1-CM2 contains the sequence AVGLLAPPGGLSGRSDTH (SEQ ID NO: 915), which also refers here to substrate 3009. In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGLSGRSDYH (SEQ ID NO: 916), which also refers here to the substrate 3010. In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGLSGRSDNP (SEQ ID NO: 917), which also refers to the substrate here 3011. In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPG GLSGRSANP (SEQ ID NO: 918), which also refers to substrate 3012 herein. In some embodiments, the CM1-CM2 substrate contains the sequence AVGLLAPPGGLSGRSANI (SEQ ID NO: 919), which also refers to substrate 3013 herein. In some embodiments, the CM1 substrate -CM2 contains the sequence ISSGLLSGRSDNI (SEQ ID NO: 920), which also refers here to substrate 2014. In some embodiments, the substrate CM1-CM2 contains the sequence AVGLLAPPGGLSGRSDNI (SEQ ID NO: 921), which also refers here to substrate 3014. In some embodiments, embodiments, the CM1-CM2 substrate contains the sequence GLSGRSDNHGGAVGLLAPP (SEQ ID NO: 1009), which also refers here to substrate 0001/LP'/1004, where LP' used in this CM1-CM2 substrate represents the GG amino acid sequence. In some embodiments, the CM1-CM2 substrate contains the sequence GLSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 1010), which also refers here to substrate 0001/LP'/1003, where LP' used in this CM1-CM2 substrate is amino

- 27 039736 acid sequence GG.

In some embodiments, the CM is a substrate for at least two proteases. In some embodiments, each protease is selected from the group consisting of the proteases shown in Table 12. In some embodiments, SM is a substrate for at least two proteases, where one of the proteases is selected from the group consisting of MMP, thrombin, neutrophil elastase, cysteine protease, uPA, legumain, and matriptase, and other serine proteases, and the other protease is selected from the group , consisting of the proteases shown in table.12. In some embodiments, SM is a substrate for at least two proteases selected from the group consisting of MMP, thrombin, neutrophil elastase, cysteine protease, uPA, legumain, and matriptase, and other serine proteases.

In some embodiments, the activated antibody comprises at least a first CM and a second CM. In some embodiments, the first CM and the second CM are each a polypeptide no more than 15 amino acids in length. In some embodiments, the first CM and the second CM in the activated antibody in the uncleaved state have the following structural organization from the N-terminus to the C-terminus: MM-CM1-CM2-AB or AB-CM2-CM1-MM. In some embodiments, at least one of the first CM and the second CM is a polypeptide that functions as a substrate for a protease selected from the group consisting of MMP, thrombin, neutrophil elastase, cysteine protease, uPA, legumain, and matriptase, and other serine proteases . In some embodiments, the first CM is cleaved by a first cleaving agent selected from the group consisting of MMP, thrombin, neutrophil elastase, cysteine protease, uPA, legumain, and matriptase, and other serine proteases in the target tissue, and the second CM is cleaved by a second cleaving agent in the tissue -targets. In some embodiments, the other protease is selected from the group consisting of the proteases shown in Table 12. In some embodiments, the first cleaving agent and the second cleaving agent are the same protease selected from the group consisting of MMP, thrombin, neutrophil elastase, cysteine protease, uPA, legumain, and matriptase, and other serine proteases, and the first CM and the second CM represent different substrates for the enzyme. In some embodiments, the first cleaving agent and the second cleaving agent are the same protease selected from the group consisting of the proteases shown in Table 12. In some embodiments, the first cleaving agent and the second cleaving agent are different proteases. In some embodiments, the first disintegrating agent and the second disintegrating agent are localized together in the target tissue. In some embodiments, the first CM and the second CM are cleaved by at least one cleaving agent in the target tissue.

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

In some embodiments, the activated antibody also includes an AB conjugated agent. In some embodiments, the agent conjugated to an AV or AV-activated antibody is a therapeutic agent. In some embodiments, the agent is an antineoplastic agent. In some embodiments, the agent is a toxin or fragment thereof. As used here, a fragment of a toxin is a fragment that retains toxic activity. In some embodiments, the agent is conjugated to the AB via a cleavable linker. In some embodiments, the agent is conjugated to the AB via a linker that includes at least one CM1-CM2 substrate sequence. In some embodiments, the agent is conjugated to the AB via a non-cleavable linker. In some embodiments, the agent is conjugated to the AB via a linker that is cleavable in the intracellular or lysosomal environment. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylator, a DNA cleaving agent, a DNA crosslinking agent, or a DNA intercalator, or another DNA damaging agent. In some embodiments, the implementation of the agent is an agent selected from the group shown in table. 11. In some embodiments, the agent is dolastatin. In some embodiments, the agent is auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is pyrrolobenzodiazepine. In some embodiments, the agent is a dimer of pyrrolobenzodiazepine.

- 28 039736

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

In some embodiments, the activated antibody contains one or more site-specific amino acid sequence modifications such that the number of lysine residues and/or cysteine residues increases or decreases compared to the original amino acid sequence of the activated antibody, thus, in some embodiments, the number of agents that can be conjugated to an activated antibody, or in some embodiments, the conjugation of agents to an activated antibody in a site-specific manner is restricted. In some embodiments, the modified, activated antibody is modified with one or more non-natural amino acids in a site-specific manner such that, in some embodiments, conjugation of the agents is restricted to only the non-natural amino acid sites.

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

In some embodiments, the implementation of the activated antibody also includes a detectable group. In some embodiments, the detection group is a diagnostic agent.

In some embodiments, the implementation of the activated antibody also includes a signal peptide. In some embodiments, the signal peptide is conjugated to an activated antibody via a spacer. In some embodiments, the spacer is conjugated to an activated antibody in the absence of a signal peptide. In some embodiments, the implementation of the spacer is connected directly to the MM activated antibodies. In some embodiments, the implementation of the spacer is connected directly to the MM activated antibodies in the following structural organization from the N-terminus to the C-terminus: spacer-MM-CM-AB.

An exemplary spacer directly connected to the N-terminus of an MM of an activated antibody is selected from the group consisting of

QGQSGS (SEQ ID NO: 923); GQSGS (SEQ ID NO: 1192); QSGS (SEQ ID NO:

1193); SGS (SEQ ID NO: 1194); GS (SEQ ID NO: 1195); S; QGQSGQG (SEQ ID NO: 924); GQSGQG (SEQ ID NO: 395); QSGQG (SEQ ID NO:

925); SGQG (SEQ ID NO: 926); GQG (SEQ ID NO: 927); QG (SEQID

NO: 928); G; QGQSGQ (SEQ ID NO: 1196); GQSGQ (SEQ ID NO: 1197);

QSGQ (SEQ ID NO: 1198); SGQ (SEQ ID NO: 616); GQ (SEQ ID NO:

1199); and Q.

In some embodiments, the spacer contains at least the amino acid sequence QGQSGS (SEQ ID NO: 923). In some embodiments, the spacer contains at least the amino acid sequence GQSGS (SEQ ID NO: 1192). In some embodiments, the spacer contains at least the amino acid sequence QSGS (SEQ ID NO: 1193). In some embodiments, the spacer contains at least the amino acid sequence SGS (SEQ ID NO: 1194). In some embodiments, the spacer contains at least the amino acid sequence GS (SEQ ID NO: 1195). In some embodiments, the spacer contains at least the amino acid sequence S. In some embodiments, the spacer contains at least the amino acid sequence QGQSGQG (SEQ ID NO: 924). In some embodiments, the spacer contains at least the amino acid sequence GQSGQG (SEQ ID NO: 395). In some embodiments, the spacer contains at least the amino acid sequence QSGQG (SEQ ID NO: 925). In some embodiments, the spacer contains at least the amino acid sequence SGQG (SEQ ID NO: 926). In some embodiments, the spacer

- 29 039736 contains at least the amino acid sequence GQG (SEQ ID NO: 927). In some embodiments, the spacer contains at least the amino acid sequence QG (SEQ ID NO: 928). In some embodiments, the spacer contains at least the G amino acid sequence. In some embodiments, the spacer contains at least the QGQSGQ amino acid sequence (SEQ ID NO: 1196). In some embodiments, the spacer contains at least the amino acid sequence GQSGQ (SEQ ID NO: 1197). In some embodiments, the spacer contains at least the amino acid sequence QSGQ (SEQ ID NO: 1198). In some embodiments, the spacer contains at least the amino acid sequence SGQ (SEQ ID NO: 1198). In some embodiments, the spacer contains at least the GQ amino acid sequence (SEQ ID NO: 616). In some embodiments, the spacer contains at least the Q amino acid sequence. In some embodiments, the activated antibody does not include a spacer sequence.

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

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

In some embodiments, the activated anti-PDL1 antibody and/or the conjugated anti-PDL1 activated antibody is monospecific. In some embodiments, the activated anti-PDL1 antibody and/or the conjugated anti-PDL1 activated antibody is multispecific, eg, by way of non-limiting example, bispecific or trifunctional. In some embodiments, an activated anti-PDL1 antibody and/or

- 30 039736 a conjugated, activated anti-PDL1 antibody is formulated as a component of a probispecific molecule, T-cell activator (BITE), i.e. BITE includes a masking fragment and a split fragment. In some embodiments, the activated anti-PDL1 antibody and/or the conjugated anti-PDL1 activated antibody is formulated as a component of a prochimeric antigen receptor (CAR) that modifies a T cell, modifies a NK cell, or modifies another immune effector cell. In some embodiments, the activated anti-PDL1 antibody is formulated as a component of another engineered receptor on an immune effector cell; those. The npo-CAR or other engineered receptor includes a masking moiety and a cleavable moiety.

In some embodiments, the activated antibody or antigen-binding fragment thereof is included in a multispecific activated antibody or antigen-binding fragment, wherein at least one arm of the multispecific activated antibody specifically binds to PDL1. In some embodiments, the activated antibody or antigen-binding fragment thereof is included in a bispecific antibody or antigen-binding fragment thereof, wherein at least one arm of the bispecific activated antibody specifically binds to PDL1.

In some embodiments, at least one arm of a multispecifically activated antibody, such as a bispecifically activated antibody, comprises a heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, and 56. In some embodiments, at least one arm of a multispecific activated antibody, e.g. a light chain comprising SEQ ID NO: 12 or SEQ ID NO: 58. In some embodiments, at least one arm of a polyspecifically activated antibody, such as a bispecifically activated antibody, comprises an amino acid sequence comprising SEQ ID NO: 12. In some embodiments, at least one arm of a multispecifically activated antibody, such as a bispecifically activated antibody, comprises an amine a light chain acid sequence comprising SEQ ID NO: 58.

In some embodiments, at least one arm of a multispecifically activated antibody, such as a bispecifically activated antibody, comprises a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, and 56, and a light chain amino acid sequence comprising SEQ ID NO: 58.

In some embodiments, at least one arm of a multispecifically activated antibody, such as a bispecifically activated antibody, comprises a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, and 56, and a light chain amino acid sequence comprising SEQ ID NO: 12.

In some embodiments, at least one arm of a multispecifically activated antibody, e.g., a bispecifically activated antibody, comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 , 52, 54, and 56. In some embodiments, at least one arm of a multispecifically activated antibody, e.g., a bispecifically activated antibody, comprises a light chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, at least one arm of a polyspecifically activated antibody, such as a bispecifically activated antibody, contains the amino acid sequence b a light chain that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, at least one arm of a polyspecifically activated antibody , for example, a bespecifically activated antibody, contains a heavy chain 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 : 46, 48, 50, 52, 54, and 56, and a light chain amino acid sequence that is at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO : 58. In some embodiments, at least one arm of the multispecifically activated antibody, e.g., the bispecifically activated antibody, contains a heavy chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 9 9% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54 and 56, and a light chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12.

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

- 31 039736 of a mutable antibody, e.g., a bespecifically activated antibody, comprises the heavy chain amino acid sequence of SEQ ID NO: 46 and the light chain amino acid sequence of SEQ ID NO: 58. In some embodiments, at least one arm of the multispecific activated antibody, e.g. contains the amino acid sequence of the heavy chain of SEQ ID NO: 46 and the amino acid sequence of the light chain of SEQ ID NO: 12.

In some embodiments, at least one arm of a multispecifically activated antibody, such as a bispecifically activated antibody, contains a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid sequence of SEQ ID NO: 46. In some embodiments, at least one arm of a polyspecifically activated antibody, such as a bispecifically activated antibody, comprises a light chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96 , 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, at least one arm of a polyspecifically activated antibody, such as a bispecifically activated antibody, contains a light chain amino acid sequence that is at least 90, 91, 92 , 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence SEQ ID NO: 12. In some embodiments, at least one arm of a multispecifically activated antibody, e.g., a bispecifically activated antibody, comprises a heavy chain 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 SEQ ID NO: 46 and a light chain amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical amino acid sequence of SEQ ID NO: 58. In some embodiments, at least one arm of a multispecifically activated antibody, such as a bispecifically activated antibody, comprises a heavy chain 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 SEQ ID NO: 46 and a light chain amino acid sequence that is at least 90, 91, 92 , 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence of SEQ ID NO: 12.

In some embodiments, at least one arm of a polyspecifically activated antibody, e.g., a bispecifically activated antibody, comprises a combination of a heavy chain variable region 1 (VH CDR1, also referred to herein as CDRH1) complementarity determining region sequence, a heavy chain variable region complementarity determining region sequence chain 2 (VH CDR2, also referred to here as CDRH2), the sequence of the complementarity determining region of the heavy chain variable region 3 (VH CDR3, also referred to here as CDRH3), the sequence of the complementarity determining region of the light chain variable region 1 (for example, VL CDR1, also referred to here as CDRL1), light chain variable region complementarity determining region 2 (VL CDR2, also referred to here as CDRL2), complementarity determining light chain variable region 3 (VL CDR3, also referred to as th here CDRL3), wherein at least one CDR sequence is selected from the group consisting of the VH CDR1 sequence shown in Table 16; the VH CDR2 sequence shown in Table 16; the VH CDR3 sequence shown in Table 16; the VL CDR1 sequence shown in Table 16; the VL CDR2 sequence shown in Table 16; and the VL CDR3 sequence shown in Table 16.

In some embodiments, at least one arm of a polyspecifically activated antibody, such as a bispecifically activated antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one sequence The CDR is selected from the group consisting of a VH CDR1 sequence that includes a sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the VH CDR1 sequence shown in Table .16; a VH CD2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR2 sequence shown in Table 16; a VH CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR3 sequence shown in Table 16; a VL CDR1 sequence that 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 shown in Table 16; a VL CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR2 sequence shown in Table 16; and a VL CDR3 sequence that 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 shown in Table 16.

In some embodiments, at least one arm of a multispecifically activated antibody, such as a bispecifically activated antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2, and a VL CDR3 sequence, wherein the combination is a combination of six CDR sequences (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3) shown in one line in the table. 16.

In some embodiments, the antibody comprises a heavy chain that contains a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, where the combination is a combination of three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown on the same line in Table .16.

In some embodiments, at least one arm of a polyspecifically activated antibody, such as a bispecifically activated antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where each CDR sequence in combination contains a sequence that 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 six CDR sequences (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3) shown on the same line in Table 16.

In some embodiments, the antibody comprises a heavy chain that contains a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VR CDR3 sequence, where each CDR sequence in the combination contains a sequence that 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 three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in one line in Table 16.

In some embodiments, at least one arm of a polyspecifically activated antibody, such as a bispecifically activated antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one sequence The CDR is selected from the group consisting of a VL CDR1 sequence containing RASQSISSYLN (SEQ ID NO: 209); a VL CDR2 sequence containing AASSLQS (SEQ ID NO: 215); a VL CDR3 sequence containing DNGYPST (SEQ ID NO: 228) ; sequence VH CDR1 containing SYAMS (SEQ ID NO: 212); a VH CDR2 sequence containing SSIWRNGIVTVYADS (SEQ ID NO: 246) ; and a VH CDR3 sequence containing WSAAFDY (SEQ ID NO: 235).

In some embodiments, at least one arm of a multispecific activated antibody, such as a bispecific activated antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR2 sequence comprises SSIWRNGIVTVYADS (SEQ ID NO: 246). In some embodiments, at least one arm of a multispecifically activated antibody, such as a bispecifically activated antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR3 sequence contains WSAAFDY (SEQ ID NO: 235). In some embodiments, at least one arm of a multispecific activated antibody, such as a bispecific activated antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR2 sequence comprises SSIWRNGIVTVYADS (SEQ ID NO: 246) and the VH CDR3 sequence contains WSAAFDY (SEQ ID NO: 235).

In some embodiments, at least one arm of a polyspecifically activated antibody, such as a bispecifically activated antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises a VL CDR1 sequence. containing RASQSISSYLN (SEQ ID NO: 209); a VL CDR2 sequence containing AASSLQS (SEQ ID NO: 215); a VL CDR3 sequence containing DNGYPST (SEQ ID NO: 228); a VH CDR1 sequence containing SYAMS (SEQ ID NO: 212); a VH CDR2 sequence containing SSIWRNGIVTVYADS (SEQ ID NO: 246); and a VH CDR3 sequence containing WSAAFDY (SEQ ID NO: 235).

In some embodiments, anti-PDL1 antibodies, anti-PDL1-conjugated antibodies, anti-PDL1-activated antibodies, and/or anti-PDL1-activated conjugated antibodies described herein are used as the sole active agents. In some embodiments, the anti-PDL1 antibodies, anti-PDL1-conjugated antibodies, anti-PDL1-activated antibodies, and/or anti-PDL1-activated conjugated antibodies described herein are used in combination with one or more additional agents, or a combination of additional agents. agents. Suitable additional agents include currently available pharmaceutical and/or surgical treatments for the intended use, such as, for example, cancer. For example, anti-PDL1 antibodies, conjugated anti-PDL1 antibodies, activated anti-PDL1 antibodies and/or conjugated activated anti-PDL1 antibodies described herein are used in combination with an additional chemotherapeutic or antineoplastic agent.

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

In some embodiments, the additional agent(s) is an immune checkpoint inhibitor, a kinase inhibitor, an agent that targets inhibitors to the tumor microenvironment, and/or a T cell or NK cell agonist. In some embodiments, the additional agent(s) is radiation therapy, alone or in combination with other additional agent(s), such as a chemotherapy or antineoplastic drug. In some embodiments, the additional agent(s) is a vaccine, an oncovirus, and/or a DC activating agent such as, but not limited to, a Toll-like receptor (TLR) agonist and/or a-CD40. In some embodiments, the additional agent(s) is a tumor-targeting antibody designed to eliminate the tumor by ADCC or by direct conjugation to a toxin (eg, an antibody drug conjugate (ADC).

In some embodiments, the immune checkpoint inhibitor is a target inhibitor selected from the group consisting of CTLA-4, LAG-3, PD1 (also includes PD-1), PDL1, TIGIT, TIM-3, B7H4, and Vista. In some embodiments, the kinase inhibitor is selected from the group consisting of B-RAFi, MEKi, and Btk inhibitors, such as ibrutinib. In some embodiments, the kinase inhibitor is crizotinib. In some embodiments, the tumor microenvironment inhibitor is selected from the group consisting of an IDO inhibitor, an α-CSFIR inhibitor, an α-CCR4 inhibitor, TGF-beta, a myeloid-derived suppressor cell, or a regulatory T cell. In some embodiments, the agonist is selected from the group consisting of 0x40, GITR, CD137, ICOS, CD27, and HVEM.

In some embodiments, the inhibitor is a CTLA-4 inhibitor. In some embodiments, the inhibitor is a LAG-3 inhibitor. In some embodiments, the implementation of the inhibitor is a PD1 inhibitor. In some embodiments, the inhibitor is a PDL1 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 B-RAFi inhibitor is vemurafenib. 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-CSFIR inhibitor. In some embodiments, the inhibitor is an a-CCR4 inhibitor. In some embodiments, the inhibitor is a TGF-beta inhibitor. In some embodiments, the inhibitor is a suppressor cell of myeloid origin. In some embodiments, the implementation of the inhibitor is a T-regulatory cell.

In some embodiments, the implementation of the agonist is 0x40. 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.

In some embodiments, the anti-PDL1 antibody, anti-PDL1 conjugated antibody, anti-PDL1 activated antibody, and/or anti-PDL1 activated conjugated antibody are administered before and/or during and/or after treatment in combination with one or several additional agents, such as, for example, a chemotherapeutic agent, an anti-inflammatory agent and/or an immunosuppressive agent. In some embodiments, the anti-PDL1 antibody, the anti-PDL1 conjugated antibody, the anti-PDL1 activated antibody, and/or the conjugated anti-PDL1 activated antibody and the additional agent are formulated as a single therapeutic composition, and the anti-PDL1 antibody, the anti-PDL1-conjugated antibody, the anti-PDL1-activated antibody, and/or the anti-PDL1-activated conjugated antibody and the additional agent are administered simultaneously. Alternatively, an anti-PDL1 antibody, an anti-PDL1-conjugated antibody, an anti-PDL1-activated antibody, and/or an anti-PDL1-activated conjugated antibody, and an additional

- 34 039736 the agent is separated from each other, for example, each of them is formulated in a separate therapeutic composition, and an anti-PDL1 antibody, an anti-PDL1 conjugated antibody, an anti-PDL1 activated antibody and/or an anti-PDL1 conjugated activated antibody and an additional agent administered simultaneously, or the anti-PDL1 antibody, the anti-PDL1 conjugated antibody, the anti-PDL1 activated antibody and/or the conjugated anti-PDL1 activated antibody and the additional agent are administered at different times during the treatment regimen. For example, an anti-PDL1 antibody, an anti-PDL1 conjugated antibody, an anti-PDL1 activated antibody, and/or an anti-PDL1 activated conjugated antibody is administered prior to administration of an additional agent, an anti-PDL1 antibody, an anti-PDL1 conjugated antibody, an anti-PDL1 activated antibody and/or the anti-PDL1-activated conjugated antibody is administered after administration of the additional agent, or the anti-PDL1 antibody, the anti-PDL1-conjugated antibody, the anti-PDL1-activated antibody and/or the anti-PDL1-activated conjugated antibody and the additional agent are administered in turn. As described herein, the anti-PDL1 antibody, anti-PDL1 conjugated antibody, anti-PDL1 activated antibody and/or anti-PDL1 activated conjugated antibody, and the additional agent are administered in single doses or in multiple doses.

In some embodiments, the anti-PDL1 antibody, anti-PDL1 conjugated antibody, anti-PDL1 activated antibody, and/or anti-PDL1 activated conjugated antibody, and additional agent(s) are administered simultaneously. For example, the anti-PDL1 antibody, anti-PDL1 conjugated antibody, anti-PDL1 activated antibody, and/or anti-PDL1 activated conjugated antibody and additional agent(s) may be formulated as a single composition or administered as two or more separate compositions. . In some embodiments, the anti-PDL1 antibody, the anti-PDL1 conjugated antibody, the anti-PDL1 activated antibody, and/or the anti-PDL1 conjugated activated antibody and additional agent(s) are administered sequentially, or the anti-PDL1 antibody conjugated with anti- The PDL1 antibody, the anti-PDL1 activated antibody and/or the conjugated anti-PDL1 activated antibody and the additional agent are administered at different times during the treatment regimen.

In some embodiments, the anti-PDL1 antibody, anti-PDL1 conjugated antibody, anti-PDL1 activated antibody, and/or anti-PDL1 activated conjugated antibody1 are administered before and/or during and/or after treatment in combination with one or more additional agents, for example, but not limited to, a chemotherapeutic drug, an anti-inflammatory drug, and/or an immunosuppressive agent such as an alkylating agent, an antimetabolite, an antimicrotubule agent, a topoisomerase inhibitor, a cytotoxic antibiotic, and/or any other nucleic acid damaging agent. In some embodiments, the additional agent is a taxane, such as paclitaxel (eg, Abraxane®). In some embodiments, the implementation of the additional agent is an antimetabolite, such as gemcitabine. In some embodiments, the additional agent is an alkylating agent, such as a platinum-based chemotherapy drug such as carboplatin or cisplatin. In some embodiments, the additional agent is a target agent such as a kinase inhibitor such as sorafenib or erlotinib. In some embodiments, the additional agent is a target agent, such as another antibody, such as a monoclonal antibody (eg, bevacizumab), a bispecific antibody, or a polyspecific antibody. In some embodiments, the additional agent is a proteasome inhibitor such as bortezomib or carfilzomib. In some embodiments, the additional agent is an immunomodulatory agent such as lenalidomide or IL-2. In some embodiments, the implementation of the additional agent is radiation therapy. In some embodiments, the implementation of the additional agent is an agent considered as a standard of care by those skilled in the art. In some embodiments, the additional agent is a chemotherapy drug well known to those skilled in the art.

In some embodiments, the additional agent is another antibody or antigen-binding fragment, another conjugated antibody or antigen-binding fragment, another activated antibody or antigen-binding fragment, and/or another conjugated activated antibody or antigen-binding fragment. In some embodiments, the additional agent is another antibody or antigen-binding fragment thereof, another conjugated antibody or antigen-binding fragment thereof, another activated antibody or antigen-binding fragment thereof, and/or another conjugated activated antibody or antigen-binding fragment thereof against the same target as the first antibody, or an antigen-binding fragment thereof, a first conjugated antibody or antigen-binding fragment thereof, an activated antibody or antigen-binding fragment thereof, and/or a conjugated activated antibody or antigen-binding fragment thereof, for example against PDL1. In some embodiments, the additional agent is another antibody or antigen-binding fragment, another conjugated antibody or antigen-binding fragment, another activated antibody or antigen-binding fragment, and/or another conjugated activated antibody, or

- 35 039736 antigen-binding fragment thereof against a target other than the target of the first antibody or its antigen-binding fragment, the first conjugated antibody or its antigen-binding fragment, an activated antibody or its antigen-binding fragment and/or a conjugated activated antibody or its antigen-binding fragment (i.e. target, different from PDL1). In some embodiments, the additional agent is a chimeric antigen receptor modified T cell, modified NK cell, or other modified immune effector cell, or an immune effector cell modified with another receptor. In some embodiments, the additional agent is a prochimeric antigen receptor, a modified T cell, a modified NK cell, or another modified immune effector cell, or an immune effector cell modified with another receptor.

As a non-limiting example, an antibody or antigen-binding fragment and/or an AB activated antibody is a binding partner for any of the targets listed in Table 22. In some embodiments, the additional agent is ipilimumab, an ipilimumab CTLA4 binding fragment, and/or an ipilimumab activated antibody.

Table 22. Exemplary Targets

1-92-LFA-3 CD52 DL44 HVEM LIF-R STEAP1 Alpha-4integrin CD56 DLK1 Hyaluronidase Lewis X STEAP2 Alpha-Vintegrin CD64 DLL4 ICOS LIGHT TAG-72 Alpha-4-beta-1integrin CD70 DPP-4 IFN-alpha LRP4 TAPA1 Alpha-4-beta-7integrin CD71 DSG1 IFN-beta LRRC26 TGF-beta

- 36 039736

AGR2 CD74 EGFR IFN-gamma MCSP TIGIT Anti-Lewis-Y EGFRviii IgE Mesothels H TIM-3 Apelin J receptor CD80 Endothelin B receptor IgE receptor (FceRI) MRP 4 TLR2 APRIL CD81 ENPP3 IGF MUC1 TLR4 B7-H4 CD86 yer cam IGF1R Mucin-16 (MUC16, CA-125) TLR6 BAFF CD95 ERHA2 IL1B Na/K ATPase TLR7 BT LA CD117 ERNV2 ILIR Neutrophilic TLR8 Complement C5 CD125 ERBB3 IL2 NGF TLR9 S-242 CD132 (IL-2RG) F-protein RSV IL11 Nikastri Η TMEM31 SA9 CD133 FAP IL12 Notch receptor TNF-alpha SA19-9 (Lewis a) CD137 FGF-2 IL12p40 Notch 1 TNFR Carbonic anhydrase 9 CD138 FGF8 IL-12R, IL-12R6eTa1 Notch 2 TNFRS12 A CD2 CD166 FGFR1 IL13 Notch 3 TRAIL-RI CD3 CD172A FGFR2 IL13R Notch 4 TRAIL-R2 CD6 CD248 FGFR3 IL15 NOV Transferrin CD9 CDH6 FGFR4 IL17 OSM-R Transferrin receptor CDlla SEACAM5 (CEA) folate receptor IL18 OX-4 0 TRK-A CD19 SEACAM6 (NCA-90) GAL3ST1 IL21 PAR2 TRK-B CD2 0 CLAUDIN-3 G-CSF IL23 PDGF-AA uPAR CD22 CLAUDIN-4 G-CSFR IL23R PDGF-BB VAP1 CD24 cmet GD2 IL27/IL27R (wsxl) PDGFRalpha VCAM-1 CD25 Collagen GITR IL29 PDGFRbeta VEGF CD27 Crypto GLUT1 IL-31R PD-1 VEGF-A CD2 8 CSFR GLUT4 IL31/IL31R PD-L1 VEGF-B CD30 CSFR-1 GM-CSF IL2R PD-L2 VEGF-C CD33 CT LA-4 GM-CSFR IL4 Phosphatide yl-serine VEGF-D

- 37 039736

CD38 CTGF GP Ilb/lIIa receptors IL4R P1GF VEGFR1 CD40 CXCL10 Gpl3 0 IL6, IL6R PSCA VEGFR2 CD40L CXCL13 GPIIB/IIIA insulin receptor PSMA VEGFR3 CD41 CXCR1 GPNMB Jagged ligands RAAG12 VISTA CD44 CXCR2 GRP78 Jagged 1 RAGE WISP-1 CD44v6 HER2/neu Jagged 2 SLC44A4 WISP-2 CD47 CXCR4 HGF LAG-3 Sphingosi n-l- WISP-3 CD51 CYR61 hGH

As a non-limiting example, the antibody or antigen-binding fragment and/or AB activated antibody is or is derived from the antibodies shown in Table 23.

Table 23. Exemplary sources of AB

Trade name of the antibody Target Avastin™ (bevacizumab) VEGF Lucentis™ (ranibizumab) VEGF Erbitux™ (cetuximab) EGFR Vectibix™ (panitumumab) EGFR Remicade™ (infliximab) TNFa Humira™ (adalimumab) TNFa Tysabri™ (natalizumab) Integrin-α-4 Simulect™ (basiliximab) IL2R Sobris™ (eculizumab) Complement C5 Raptiva™ (efalizumab) CDlla Bexxar™ (tositumomab) CD20 Zevalin™ (ibritumomab tiuxetan) CD20 Rituxan™ (rituximab) CD20 (Ocrelizumab) CD20 Arzerra™ (ofatumumab) CD20 Gazyva™ (obinutuzumab) CD20 Zenapax™ (daclizumab) CD25 Adcetris™ (brentuximab vedotin) CD30 Myelotarq™ (gemtuzumab) CD33 Mylotarq™ (gemtuzumab ozogamicin) CD33 Campath™ (alemtuzumab) CD52 ReoPro™ (abciximab) Glycoprotein receptor Xolair™ (omalizumab) IRE Herceptin™ (trastuzumab) Neg2 Kadcyla™ (trastuzumab emtansine) Neg2 Synaqis™ (palivizumab) F-protein RSV (ipilimumab) CTLA-4 (tremelimumab) CTLA-4 Ni5s8 CD40L (Pertuzumab) Her2-neu

- 38 039736

(Ertumaxomab) CD3/Her2-neu Orencia™ (abatacept) CTLA-4 (tanezumab) NGF (Bavituximab) Phosphatidylserine (Zalutumumab) EGFR (Mapatumumab) EGFR (Matuzumab) EGFR (Nitotuzumab) EGFR ICR62 EGFR mAb 528 EGFR CH806 EGFR MDX-447 EGFR/CD64 (Edrecolomab) ErSAM RAV12 RAAG12 huJ591 PSMA Enbrel™ (etanercept) TNF-R Amevive™ (alefacept) 1-92-LFA-3 Antril™, Kineret™ (anakinra) IL-IRa GC1008 TGFbeta Notch, e.g. Notch 1 Jaqqed 1 or Jaqqed 2 (Adecatumumab) Epcam (Figitumumab) IGF1R (Tocilizumab) IL-6 receptor Stelara™ (ustekinumab) IL-12/IL-23 Proha™ (denosumab) RANKL

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

The invention also provides methods for producing an anti-PDL1 antibody polypeptide and/or an activated anti-PDL1 antibody by culturing a cell under conditions that result in the expression of the polypeptide, wherein the cell contains an isolated nucleic acid molecule encoding an antibody and/or an activated antibody described herein. , and/or vectors that include such isolated nucleic acid sequences. The invention provides methods for producing an antibody and/or an activated antibody by culturing a cell under conditions that result in the expression of an antibody and/or an activated antibody, wherein the cell contains an isolated nucleic acid molecule encoding an antibody and/or an activated antibody described herein and/or vectors, which include such isolated nucleic acid sequences.

The invention also provides a method for producing activated antibodies that, when activated, bind to PDL1 by (a) culturing a cell containing a nucleic acid construct that encodes an activated antibody under conditions that result in the expression of an activated antibody, where the activated antibody contains a masking fragment (MM) , a cleavable fragment (CM), and an antibody or antigen-binding fragment (AB) thereof that specifically binds to PDL1, (i) where CM is a polypeptide that functions as a substrate for a protease; and (ii) where the CM is located in the activated antibody such that when the activated antibody is in an uncleaved state, the MM interferes with specific binding of AB to PDL1, and in the cleaved state, MM does not interfere with or compete for specific binding of AB to PDL1; and (b) isolating the activated antibody. Suitable AB, MM and/or CM include any of the AB, MM and/or CM described herein.

In some embodiments, the activated antibody in the uncleaved state has the following structural organization from the N-terminus to the C-terminus: MM-CM-AB or AB-CM-MM. In some embodiments, the activated antibody comprises a linker peptide between MM and CM. AT

- 39 039736 in some embodiments, the implementation of the activated antibody contains a linker peptide between SM and AB. In some embodiments, the activated antibody comprises a first linker peptide (LP1) and a second linker peptide (LP2), and wherein the activated antibody in an uncleaved state has the following structural organization from the N-terminus to the C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CMLP1-MM. In some embodiments, the activated antibody in the uncleaved state has the following structural organization from the N-terminus to the C-terminus: spacer-MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM-spacer. In some embodiments, the two linker peptides need not be identical to each other.

In some embodiments, at least one of LP1 or LP2 contains an amino acid sequence selected from the group consisting of (GS) _n , (GGS) _n , (GSGGS) _n (SEQ ID NO: 191) and (GGGS) _n (SEQ ID NO: 192), where n is an integer, at least one.

In some embodiments, at least one of LP1 or LP2 contains an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 193), GGSGG (SEQ ID NO: 194), GSGSG (SEQ ID NO: 195), GSGGG (SEQ ID NO: 196), GGGSG (SEQ ID NO: 197) and GSSSG (SEQ ID NO: 198).

In some embodiments, LP1 contains the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 199), GSSGGSGGSGG (SEQ ID NO: 200), GSSGGSGGSGGS (SEQ ID NO 201), GSSGGGGGGGGGGGS (SEQ ID NO: 202), GSSGGSGGSG (SEQ ID NO: 203 ) or GSSGGSGGSGS (SEQ ID NO: 204).

In some embodiments, LP2 contains the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 205), GSSGT (SEQ ID NO: 206), or GSSG (SEQ ID NO: 207).

The invention relates to methods for preventing, slowing the progression, treating, ameliorating a symptom, or otherwise alleviating a PDL1 mediated disease in a subject by administering a therapeutically effective amount of an anti-PDL1 antibody, a conjugated anti-PDL1 antibody, an activated anti-PDL1 antibody, and/or a conjugated activated anti -PDL1-antibody described here, the subject in need of it.

The invention also relates to methods for preventing, slowing the progression, treating, ameliorating a symptom, or otherwise alleviating cancer in a subject by administering a therapeutically effective amount of an anti-PDL1 antibody, an anti-PDL1 conjugated antibody, an anti-PDL1-activated antibody, and/or a conjugated anti-PDL1-activated antibody. an antibody described here to a subject in need thereof. PDL1 is known to be expressed in many cancers, such as melanoma, non-small cell lung cancer, nasopharyngeal cancer, glioblastoma/mixed glioma, colon adenocarcinoma, hepatocellular carcinoma, urothelial cancer, multiple myeloma, ovarian cancer, gastric cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma (RCC), breast cancer, lymphomas and leukemias (see, for example, Chen et al., Molecular Pathways: NextGeneration Immunotherapy - Inhibiting Programmed Death-Ligand 1 and Programmed Death-1, Clin. Can. Res., vol.18: 6580-6587 (2012), the contents of this publication are incorporated herein by reference in their entirety).

In some embodiments, the cancer is bladder cancer, bone cancer, breast cancer, carcinoid, cervical cancer, colon cancer, endometrial cancer, glioma, head and neck cancers, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer. , pancreatic cancer, prostate cancer, renal cell carcinoma, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, urogenital cancer and/or urothelial cancer.

In some embodiments, the cancer is selected from the group consisting of melanoma (MEL), renal cell carcinoma (RCC), squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer (CRC), castration-resistant prostate cancer (CRPC), hepatocellular carcinoma (HCC), head and neck squamous cell carcinoma, carcinoma of the esophagus, ovary, gastrointestinal tract, and breast, or hematologic malignancy such as multiple myeloma, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma/primary mediastinal B-cell lymphoma and chronic myeloid leukemia. In some embodiments, the cancer is a PDL1-expressing cancer.

The invention also relates to methods for treating cancer patients with an autoimmune or inflammatory disease by administering a therapeutically effective amount of an anti-PDL1 antibody, an anti-PDL1 conjugated antibody, an anti-PDL1-activated antibody, and/or an anti-PDL1-activated conjugated antibody described herein. , the subject in need of it. In some embodiments, the autoimmune disease is colitis, RA, pancreatitis, diabetes, or pneumonitis.

The anti-PDL1 antibody, conjugated anti-PDL1 antibody, activated anti-PDL1 antibody, and/or conjugated anti-PDL1 activated antibody used in any of the embodiments of these methods and uses can be administered at any stage of the disease. For example, such an anti-PDL1 antibody, an anti-PDL1 conjugated antibody, an activated anti-PDL1 antibody, and/or

- 40 039736 conjugated activated anti-PDL1 antibody can be administered to a patient suffering from cancer at any stage, from early to metastatic stage. The terms subject and patient are used interchangeably herein.

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

An anti-PDL1 antibody, an anti-PBL1 conjugated antibody, an anti-PDL1-activated antibody, and/or an anti-PDL1-activated conjugated antibody, and therapeutic compositions thereof, are administered to a subject suffering from or at risk of developing a disease or disorder associated with abnormal expression and/or PDL1 activity. A subject suffering from or at risk of developing a disease or disorder associated with abnormal expression and/or activity of PDL1 is identified using any of the various methods known in the art. For example, subjects suffering from cancer or other neoplastic conditions are identified using any of a variety of clinical and/or laboratory tests, such as a physical examination and blood, urine, and/or stool testing to assess health status. For example, subjects suffering from inflammation and/or an inflammatory disorder are identified using any of a variety of clinical and/or laboratory tests, such as a physical examination and/or body fluid analysis, such as a blood, urine, and/or stool test, to evaluate health status.

Administration of an anti-PDL1 antibody, an anti-PDL1 conjugated antibody, an activated anti-PDL1 antibody, and/or an activated anti-PDL1 conjugated antibody to a patient suffering from a disease or disorder associated with abnormal expression and/or activity of PDL1 is considered successful if any of a number of laboratory or clinical uses. For example, administering an anti-PDL1 antibody, an anti-PDL1 conjugated antibody, an activated anti-PDL1 antibody, and/or an activated anti-PDL1 conjugated antibody to a patient suffering from a disease or disorder associated with abnormal PDL1 expression and/or activity is considered successful if one or more symptoms associated with the disease or disorder are alleviated, reduced, suppressed, or do not progress further, i.e. to the worst condition. Administration of an anti-PDL1 antibody, an anti-PDL1 conjugated antibody, an activated anti-PDL1 antibody, and/or an activated anti-PDL1 conjugated antibody to a patient suffering from a disease or disorder associated with abnormal expression and/or activity of PDL1 1 is considered successful if the disease or disorder goes into remission or does not progress further, i.e. to the worst condition.

In some embodiments, the anti-PDL1 antibody, anti-PDL1 conjugated antibody, anti-PDL1 activated antibody, and/or anti-PDL1 activated conjugated antibody are administered before and/or during and/or after treatment in combination with one or more additional agents such as, for example, a chemotherapeutic drug, an anti-inflammatory agent, and/or an immunosuppressive agent. In some embodiments, the anti-PDL1 antibody, anti-PDL1 conjugated antibody, anti-PDL1 activated antibody, and/or anti-PDL1 activated conjugated antibody, and additional agent(s) are administered simultaneously. For example, the anti-PDL1 antibody, anti-PDL1 conjugated antibody, anti-PDL1 activated antibody, and/or anti-PDL1 activated conjugated antibody and additional agent(s) may be formulated as a single composition or administered as two or more separate compositions. . In some embodiments, the anti-PDL1 antibody, anti-PDL1 conjugated antibody, anti-PDL1 activated antibody, and/or anti-PDL1 activated conjugated antibody, and additional agent(s) are administered sequentially.

The invention also provides methods and kits for using activated anti-PDL1 antibodies and/or conjugated activated anti-PDL1 antibodies in various diagnostic and/or prophylactic indications. For example, the invention provides methods and kits for detecting the presence or absence of a cleavage agent and a target of interest in a subject or sample by (i) contacting the subject or sample with an anti-PDL1-activated antibody, wherein the anti-PDL1 antibody includes a masking moiety ( MM), a cleavable fragment (CM), which is cleaved by a cleaving agent, and its antigen-binding site or fragment (AB), which specifically binds to the target of interest, where the activated anti-PDL1 antibody in the non-cleaved, non-activated state has the following structural organization from the N-terminus to C - end: MM-CM-AB or AB-CM-MM; (a) where MM is a peptide that inhibits AB binding to PDL1, and where MM does not have the amino acid sequence of a naturally occurring AB binding partner and is not a modified form of a natural AB binding partner; and (b) where, when AV is in a non-cleaved, non-activated state, then MM prevents specific binding of AV to PDL1, and when AV is in a cleaved, activated state, then MM does not prevent or compete for specific binding of AV to PDL1 ; and (ii) measuring the level of activated activated anti-PDL1 antibody in the subject or sample, wherein the detectable level of activated activated anti-PDL1 antibody in the subject or sample indicates that the degrading agent and PDL1 are present in the subject or sample, and where an undetectable level of activated anti-PDL1-activated antibody in the subject or sample indicates that the cleaving agent, PDL1, or both the cleaving agent and PDL1 are absent from the subject or sample.

In some embodiments, the activated anti-PDL1 antibody is an activated anti-PDL1 antibody to which the therapeutic agent is conjugated. In some embodiments, the activated anti-PDL1 antibody is not conjugated to an agent. In some embodiments, the activated anti-PDL1 antibody contains a detectable label. In some embodiments, the implementation of the detectable label is located in the AB. In some embodiments, the level of an activated anti-PDL1 antibody in a subject or sample is measured using a secondary reagent that specifically binds to the activated antibody, where the reagent contains a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

In some embodiments of these methods and kits, the activated anti-PDL1 antibody comprises a detectable label. In some embodiments of these methods and kits, the detectable label includes an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments of these methods and kits, the imaging agent comprises 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 contrast agent includes iodine, gadolinium, or iron oxide. In some embodiments of these methods and kits, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments of these methods and kits, the fluorescent label includes yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red dimeric fluorescent protein (RFP tdimer2), HCRED or a europium derivative. In some embodiments of these methods and kits, the luminescent label includes an N-methylacridium derivative. In some embodiments of these methods, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments of these methods and kits, the ligand-based label includes biotin, avidin, streptavidin, or one or more haptens.

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

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

In some embodiments of the methods and kits, the method is used to identify or otherwise refine a patient population suitable for treatment with an activated anti-PDL1 antibody of the invention, followed by treatment with the administration of that activated anti-PDL1 antibody and/or a conjugated, activated anti-PDL1 antibody to a subject in need of in that. For example, patients who test positive for the target (eg, PDL1) and for the protease that cleaves the substrate in the cleavable fragment (CM) of the activated anti-PDL1 antibody that is tested in these methods are identified as suitable candidates for treatment with such activated anti-PDL1. an antibody containing such a SM, and then a therapeutically effective amount of an activated anti-PDL1 antibody and/or a conjugated activated anti-PDL1 antibody that has been tested is administered to the patient. Similarly, patients who test negative for one or both of a target (eg, PDL1) and a protease that cleaves a substrate in the CM in an activated antibody that is tested using these methods may be identified as suitable candidates for another type of treatment. In some embodiments, such patients may be tested with other anti-PDL1-activated antibodies until a suitable anti-PDL1-activated antibody for treatment is identified (e.g., an activated anti-PDL1 antibody containing CM that is cleaved in the patient at the lesion). diseases). In some embodiments, the patient is then administered

- 42 039736 a therapeutically effective amount of an activated and/or conjugated anti-PDL1 antibody for which the patient has a positive result. Suitable AB, MM and/or CM include any of the AB, MM and/or CM described herein.

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

Brief description of the figures

In FIG. 1 is a graph showing the results of an ELISA-based binding assay indicating that PDL1 c60 ScFv phage specifically binds to human and mouse PDL1.

In FIG. 2 is a graph showing that hPDl inhibits the binding of clones 12, 18 and 60 to hPDL1 in an ELISA format and furthermore that mPD1 inhibits the binding of clone 60 to mPDL1.

In FIG. 3 is a graph showing the results of the saturation binding assay and it demonstrates that the 60:IgG anti-PDL1 clone binds to human PDL1 with a binding affinity of ~1 nM and to mouse PDL1 of 30 nM.

In FIG. 4A and B are a series of graphs showing H2 specificity and cross-reactivity (FIG. 4A) and H3W specificity and cross-reactivity (FIG. 4B).

In FIG. 5A and B are a series of graphs showing that combining mature heavy chain variable region CDR3 with heavy chain variable region CDR2 results in antibodies with increased affinity for human PDL1.

In FIG. 6A, B, C and D are a series of graphs showing that the anti-PDL1 antibody C5H9 binds to human and mouse PDL1 with almost equal affinity and that the anti-PDL1 antibodies C5B10 and C5E10 only bind to human PDL1.

In FIG. 7 is a graph showing the ability of the C5H9 anti-PDL1 antibody to bind with high and equal affinity to human, mouse PDL1 and Cynomolgus monkey PDL1.

In FIG. 8A and B are a series of graphs showing that the C5H9v2 anti-PDL1 antibody has a lower potential for immunogenicity and improved processability. These graphs show a decrease in predicted immunogenicity, an increase in expression levels (3x), and an increase in the percentage of monomers in the human IgG1 format.

In FIG. 9A, B and C are a series of graphs showing the ability of the anti-PDL1 antibody C5H9v2 to bind with high affinity to (A) mouse, (B) cynomolgus monkey and (C) human PDL1. In FIG. 9A-C show that both anti-PDL1 antibodies C5H9 and C5H9v2 bind to human, mouse PDL1 and cynomolgus PDL1 with almost the same affinity.

In FIG. 10 is a graph showing the specificity of C5H9v2 for a panel of human and mouse proteins. In FIG. 10 shows that the anti-PDL1 antibody C5H9v2 only binds to human and mouse PDL1, showing PDL1 specificity.

In FIG. 11 is a series of graphs showing B7-1 and PD1 blocking. In FIG. 11 shows that both C5H9 anti-PDL1 antibody and C5H9v2 anti-PDL1 antibody are effective blockers of B7-1 or PD1 binding to PDL1, and this blocking includes all three species of PDL1, human, cynomolgus monkey and mouse with one EC ₅₀ value. at the nM level.

In FIG. 12 is a graph showing that anti-PDL1 antibody C5H9 accelerates the onset of diabetes in NOD mice, similar to anti-PDL1 antibody 10F9G2 used as a positive control.

In FIG. 13 is a graph showing that anti-PDL1 antibody C5H9v2 accelerates the onset of developmental diabetes in NOD mice in a dose-dependent manner.

In FIG. 14 is a graph showing that anti-PDL1 antibody C5H9v2 inhibits the growth of syngeneic MC38 tumors, similarly to anti-PDL1 antibody 10F9G2 used as a positive control.

In FIG. 15 is a graph showing binding isotherms for activated anti-PDL1 antibodies, which include anti-PDL1 antibody C5H9v2.

In FIG. 16 is a graph showing binding isotherms for activated anti-PDL1 antibodies, which include anti-PDL1 antibody C5H9v2.

In FIG. 17 is a graph showing binding isotherms for activated anti-PDL1 antibodies, which include the anti-PDL1 antibody C5H9v2.

In FIG. 18A and B are a series of graphs showing that PL15-0003-C5H9v2 activated anti-PDL1 antibody at 1 mg/kg did not induce diabetes, and PL18-0003-C5H9v2 activated anti-PDL1 antibody at 1 mg/kg. kg delayed the induction of diabetes compared to anti-PBL1 antibody C5H9v2 administered at 1 mg/kg in NOB mice (FIG. 18A); in addition, the activated anti-PDL1 antibody PL15-0003-C5H9v2, when administered at a dose of 3 mg/kg, induced the development of diabetes in 2 of 8 NOB mice (75% without diabetes), compared with the administration of anti-PDL1 antibody C5H9v2 at a dose of 1 mg/kg, which provided 38% of NOB mice without diabetes (Fig. 18B).

In FIG. 19 is a graph showing that the activated anti-PDL1 antibodies PL15-0003C5H9v2 and PL18-0003-C5H9v2 inhibit the growth of syngeneic MC38 tumors, similar to the anti-PDL1-43 039736 antibody C5H9v2.

In FIG. 20A, B, C and D are a series of graphs showing the percentage of aHTu-PDL1 antibody C5H9v2 and upregulated anti-PDL1 antibody PL15-0003-C5H9v2 associated with CD4+ and CD8+ T cells from peripheral blood (FIG. 20A, B) or spleen (Fig. 20C, D). In all graphs, circles represent isotype antibody, squares represent C5H9v2 anti-PDL1 antibody, and triangles represent PL15-0003-C5H9v2 activated anti-PDL1 antibody.

In FIG. 21A and B are a series of graphs showing that the presence of tumor-derived proteases capable of cleaving the activated anti-PDL1 antibody PL15-0003-C5H9v2 (shown as squares) did not result in high blood levels of activated activated antibody compared to anti-PDL1. PDL1 antibody C5H9v2 (shown by circles) or isotype antibody (shown by triangles) (FIG. 21A), despite the fact that plasma concentrations of activated antibody are higher than antibodies (FIG. 21B).

In FIG. 22 is a graph showing the ability of activated anti-PDL1 antibody PL07-2001-C5H9v2 to increase CMV-stimulated IFN-gamma secretion compared to control hIgG4, but with reduced activity compared to parental anti-PDL1 antibody C5H9v2.

In FIG. 23A, B, C and D are a series of graphs showing the ability of the activated anti-PDL1 antibody PL15-0003-C5H9v2 to activate and bind to frozen mouse MC38 cancer tissue using an in situ imaging technique.

In FIG. 24 shows that the activated anti-PDL1 antibody PL15-0003-C5H9v2 had a higher EC ₅₀ for PDL1 binding and blocking PD1 than the anti-PDL1 antibody as determined by ELISA. The figure also shows that activation of the activated anti-PDL1 antibody PL15-0003-C5H9v2 by matriptase completely restored PDL1 binding and PD1 blocking to levels comparable to those determined for the anti-PDL1 antibody C5H9v2.

In FIG. 25A and B are a series of graphs showing the ability of activated anti-PDL1 antibodies, herein designated PL07-2001-C5H9v2 and PF07-3001-C5H9v2, and an anti-PDL1 antibody, herein designated C5H9, to bind to human or mouse PDL1.

In FIG. 26A, B, C are a series of graphs showing the potency of PL07-2001-C5H9v2 anti-PDL1-activated antibody, PL072001-C5H9v2 uPA-activated anti-PDL1-activated antibody, PL07-2001-C5H9v2-activated anti-PDL1 antibody, and PL07-2001-C5H9v2 anti-PDL1 activated antibody. C5H9v2 antibodies block the binding of human PD1 (PD-1) to human PDL1 (Fig. 26A), cynomolgus monkey PD1 (PD-1) to cynomolgus monkey PDL1 (Fig. 26B), or rat PD1 (PD-1) to rat PDL1 (Fig. 26B). Fig. 26C).

In FIG. 27A and B are a series of graphs showing the potency of PL07-2001-C5H9v2 anti-PDL1-activated antibody, PL072001-C5H9v2 uPA-activated anti-PDL1-activated antibody, PL07-2001-C5H9v2-activated anti-PDL1 antibody, and PL07-2001-C5H9v2 anti-PDL1 antibody. C5H9v2 block the binding of human PD1 (PD-1) to human PDL1 (FIG. 27A), cynomolgus monkey PD1 (PD-1) to cynomolgus monkey PDL1 (FIG. 27B), or rat PD1 (PD-1) to rat PDL.

In FIG. 28 is a graph showing the ability of activated anti-PDL1 antibodies PL150003-C5H9v2, PL15-2001-C5H9v2, and PL15-3001-C5H9v2 to inhibit the growth of syngeneic MC38 tumors, similar to the anti-PDL1 antibody C5H9v2 used as a positive control.

In FIG. 29A, B, C are a series of graphs showing that the presence of tumor-derived proteases capable of cleaving C5H9v2 anti-PDL1 activated antibody or PL15-0003-C5H9v2, PL15-2001-C5H9v2 or PL15-3001 activated anti-PDL1 antibody -C5H9v2 did not result in high blood levels of activated antibodies.

In FIG. 30 is an image showing the ability of proteases in an implanted mouse tumor to activate the anti-PDL1-activated antibody PL07-2001-C5H9v2.

Detailed description of the invention

The present invention relates to monoclonal antibodies (mAbs) and activated monoclonal antibodies that specifically bind to ligand 1 of a programmed death polypeptide. PDL1 is a 40 kDa type I transmembrane protein that forms a complex with its programmed cell death (PD1) polypeptide receptor, also known as CD279. The interaction of PDL1 with its PD1 receptor on T cells provides a signal that inhibits TCR-mediated activation of IL-2 production and T cell proliferation. Aberrant expression and/or activity of PDL1 and the PDL1-associated signaling pathway are involved in the pathogenesis of many diseases and disorders such as cancer, inflammation, and autoimmunity.

Activated anti-PDL1 antibodies are used in methods of treating, preventing, slowing the progression, attenuating and/or alleviating a symptom of a disease or disorder associated with abnormal expression and/or activity of PDL1. For example, activated anti-PDL1 antibodies are used in methods of treating, preventing, slowing the progression, attenuating and/or alleviating a symptom of a cancer or other neoplastic condition.

Activated antibodies include an antibody or antigen-binding fragment thereof that specifically binds to PDL1 associated with a masking fragment (MM) such that the MM compound reduces the ability of the antibody or antigen-binding fragment to bind to

PDL1. In some embodiments, the MM is linked through a sequence that includes a substrate for a protease, such as a protease that is co-localized with PDL1 at the site being treated in the subject.

Exemplary activated anti-PDL1 antibodies of the invention include, for example, activated antibodies that comprise a heavy chain and a light chain that are or are derived from the antibodies described in the examples, for example, Example 3 and Example 4.

In some embodiments, the activated anti-PDL1 antibody comprises a heavy chain that contains or is derived from an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 and 56, and a light chain that contains or is derived from the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 58.

In some embodiments, the antibody comprises a combination of a heavy chain variable region 1 complementarity determining region (VH CDR1, also referred to herein as CDRH1), a heavy chain variable region complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, a heavy chain variable region complementarity determining region 3 (VH CDR3, also referred to here as CDRH3), light chain variable region complementarity determining region 1 (VL CDR1, also referred to here as CDRL1), complementarity determining region sequence, light chain variable region chain 2 (VL CDR2, also referred to herein as CDRL2), and a light chain variable complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein at least one CDR sequence is selected from the group consisting of a VH sequence. CDR1 shown in Table 15; the VH CDR2 sequence shown in Table 15; the VH CDR3 sequence shown in Table 15; the VL CDR1 sequence shown in Table 15; the VL CDR2 sequence shown in Table 15; and the VL CDR3 sequence shown in Table 15.

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

In some embodiments, the antibody comprises a heavy chain that contains a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, where the combination is a combination of three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown on the same row in Table .fifteen.

In some embodiments, the antibody comprises a light chain that contains a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, where the combination is a combination of three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) shown on the same line in Table .fifteen.

In some embodiments, the activated anti-PDL1 antibody comprises a heavy chain that contains or is derived from the heavy chain amino acid sequence shown in Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a light chain that contains or is derived from the light chain amino acid sequence shown in Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a heavy chain that contains or is derived from the heavy chain amino acid sequence shown in Table 15 and a light chain that contains or is derived from the amino acid sequence shown in Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain variable region and light chain variable region sequences from the combinations shown in group A in Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a combination of the heavy chain variable region and light chain variable region sequences shown in group B of Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a combination of the heavy chain variable region and light chain variable region sequences shown in group C of Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a combination of the heavy chain variable region and light chain variable region sequences shown in group B of Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a combination of the heavy chain variable region and light chain variable region sequences shown in group E in Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a combination of the heavy chain variable region and light chain variable region sequences shown in group F in Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy variable region sequences

- 45 039736 loy chain and variable region of the light chain, shown in group G in table.15. In some embodiments, the activated anti-PDL1 antibody comprises a combination of the heavy chain variable region and light chain variable region sequences shown in group H in Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a combination of the heavy chain variable region and light chain variable region sequences shown in group I of Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a combination of the heavy chain variable region and light chain variable region sequences shown in group J of Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a combination of the heavy chain variable region and light chain variable region sequences shown in group K of Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a combination of the heavy chain and light chain sequences shown in group K in Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a combination of the heavy chain variable region and light chain variable region sequences shown in group L in Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a combination of the heavy chain variable region and light chain variable region sequences shown in group M in Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a combination of the heavy chain variable region and light chain variable region sequences shown in group N in Table 15. In some embodiments, the activated anti-PDL1 antibody comprises a combination of the heavy chain variable region and light chain variable region sequences shown in group O in Table 15.

Table 15: Heavy chain variable region (VH) and light chain variable region (VL) sequences for activated antibodies that bind to PDL1 (CDR sequences are underlined; CDR sequences disclosed here are identified according to definitions by the Kabat numbering system, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991))).

Table 15

Group A vh QVQLVQ S GAEVKKP GASVKVS CKAS GYT FTDYGFSWVRQAPGQGLEWMGWITAYNGNTNYAQKLQGRVTM TTDTSTSTVYMELRSLRSDDTAVYYCARDYFYGMDVWGQGTTVTVSS (SEQ ID NO: 248) vh QVQLVQSGAEVKKPGS SVKVS CKTSGDTFSTYAISWVRQAPGQGLEWMGGII PIFGKAHYAQKFQGRVTI TADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS (SEQ ID NO: 249) vh QVQLVQ S GAEVKKP GASVKVS CKAS GYT FT5YDVHWVRQAPGQRLEWMGWLHADTGITKF5QKFQGRVTI TRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGT (SEQ ID NO: 250) vh QVQLVQSGAEVKKPGS SVKVSCKVSGGIFSTYAINWVRQAPGQGLEWMGGII PIFGTANHAQKFQGRVTI TADESTSTAYMELSSLRSEDTAVYYCARDQGIAAALFDYWGQGTLVTVSS (SEQ ID NO: 251) vh EVQLVESGGGLVQPGRSLRLSCAVSGFTFDDYWHWVRQAPGKGLEWVSGNSGNGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAVPFDYWGQGTLVTVSS (SEQ ID NO: 252)

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vh QVQLVQSGAEVKKP GS SVKVSCKTSGDTFSSYAISWVRQAPGQGLEWMGGII PIFGRAHYAQKFQGRVTI TADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS (SEQ ID NO: 253) vh QVQLVQSGAEVKKPGS SVKVSCKTSGGTFSSYAISWVRQAPGQGLEWMGGII PIFGKAHYAQKFQGRVTI TADESTTTAYMELSSLRSEDTAVYYCARKYDYVSGSPFGMDVWGQGTTVTVSS (SEQ ID NO: 254) vh QVQLVQSGAEVKKPGS SVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGII PIFGSANYAQKFQDRVTI TADESTSAAYMELSSLRSEDTAVYYCARDSSGWSRYYMDVWGQGTTVTVSS (SEQ ID NO: 255) vh QVQLVQ S GAEVKEP GS SVKVS CKAS GGT FNSYAISWVRQAP GQGLEWMGGIIPLFGIAHYAQKFQGRVTI TADESTNTAYMDLSSLRSEDTAVYYCARKYSYVSGSPFGMDVWGQGTTVTVSS (SEQ ID NO: 256) vh EVQLVE S GGGLVQP GRS LRL S CAAS GITFDDYGMHWVRQAPGKGLEWVSGISWNRGRIEYADSVKGRFTI SRDNAKNSLYLQMNSLRAEDTALYYCAKGRFRYFDWFLDYWGQGTLVTVSS (SEQ ID NO: 257) vh QMQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARDYFWSGFSAFDIWGKGTLVTVS (SEQ ID NO: 449) VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLVWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIK (SEQ ID NO: 258) VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK (SEQ ID NO: 259) VL DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIK (SEQ ID NO: 260) VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 261) VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYGSSPFGGGTKVEIK (SEQ ID NO: 262) VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPTFGQGTRLEIK (SEQ ID NO: 263) VL AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQFNSYPFTFGPGTKVDIK (SEQ ID NO: 264) DIVMTQSPSTLSASVGDRVTITCRASQGISSWLAWYQQKPGRAPKVLIYKASTLESGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKLEIK (SEQ ID NO: 450) Group B vh EVQL VESGGGL VQPGGSL RL SCAASGFTFSRYWMSWVRQAPGKGL EWVANIKQDGSEKYYVDSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS (SEQ ID NO: 265) VL EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIK (SEQ ID NO: 266) Group C vh EVQLVE S GGGLVQP GGS LRL S CAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO: 267) vh EVQLVESGGGLVQPGGSLRLSCAASGFTFSGSWIHWVRQAPGKGLEWVAWILPYGGSSYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO: 451)

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VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 268) VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYYNVPWTFGQGTKVEIKR (SEQ ID NO: 452) VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYYAPPWTFGQGTKVEIKR (SEQ ID NO: 453) VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYYTVPWTFGQGTKVEIKR (SEQ ID NO: 454) VL DIQMTQSPSSLSASVGDRVTITCRASQVINTFLAWYQQKPGKAPKLLIYSASTLASGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYYTVPRTFGQGTKVEIKR (SEQ ID NO: 455) VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQGYGVPRTFGQGTKVEIKR (SEQ ID NO: 456) VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYLFTPPTFGQGTKVEIKR (SEQ ID NO: 457) VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYFITPTTTFGQGTKVEIKR (SEQ ID NO: 458) VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYYYTPPTFGQGTKVEIKR (SEQ ID NO: 459) VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQFFYTPPTFGQGTKVEIKR (SEQ ID NO: 460) VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQSLFTPPTFGQGTKVEIKR (SEQ ID NO: 461) VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQSLYTPPTFGQGTKVEIKR (SEQ ID NO: 462) VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQSWYHPPTFGQGTKVEIKR (SEQ ID NO: 463) VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYFYIPPTFGQGTKVEIKR (SEQ ID NO: 464) VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYWYTPTTFGQGTKVEIKR (SEQ ID NO: 465) VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQSYFIPPTFGQGTKVEIKR (SEQ ID NO: 466) Group D vh METGLRWLLLVAVLKGVQCLSVEESGGRLVTPGTPLTLTCTASGFTITNYHMFWVRQAPGKGLEWIGVIT SSGIGSSSTTYYATWAKGRFTISKTSTTVNLRITSPTTEDTATYFCARDYFTNTYYALDIWGPGTLVTVS S (SEQ ID NO: 467) vh QVQLVQSGAEVKKPGS SVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGII PIFGKAHYAQKFQGRVTI TADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS (SEQ ID NO: 249)

- 48 039736

vh QVQLVQ S GAEVKKP GASVKVS CKAS GYT FTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTI TRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSS (SEQ ID NO: 468) vh QVQLVQSGAEVKKPGS SVKVSCKVSGGIFSTYAINWVRQAPGQGLEWMGGIIPIFGTANHAQKFQGRVTI TADESTSTAYMELSSLRSEDTAVYYCARDQGIAAALFDYWGQGTLVTVSS (SEQ ID NO: 251) vh EVQLVESGGGLVQPGRSLRLSCAVSGFTFDDYWHWVRQAPGKGLEWVSGISGNSGNIGYADSVKGRFTI SRDNAKNSLYLQMNSLRAEDTALYYCAVPFDYWGQGTLVTVSS (SEQ ID NO: 469) vh QVQLVQSGAEVKKPGS SVKVS CKTSGDTFSSYAISWVRQAPGQGLEWMGGII PIFGRAHYAQKFQGRVTI TADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS (SEQ ID NO: 253) vh QVQLVQSGAEVKKPGS SVKVSCKTSGGTFSSYAISWVRQAPGQGLEWMGGII PIFGKAHYAQKFQGRVTI TADESTTTAYMELSSLRSEDTAVYYCARKYDYVSGSPFGMDVWGQGTTVTVSS (SEQ ID NO: 254) vh QVQLVQSGAEVKKPGS SVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGII PIFGSANYAQKFQDRVTI TADESTSAAYMELSSLRSEDTAVYYCARDSSGWSRYYMDVWGQGTTVTVSS (SEQ ID NO: 255) vh QVQLVQ S GAEVKEP GS SVKVS CKAS GGT FNSYAISWVRQAPGQGLEWMGGIIPLFGIAHYAQKFQGRVTI TADESTNTAYMDLSSLRSEDTAVYYCARKYSYVSGSPFGMDVWGQGTTVTVSS (SEQ ID NO: 256) vh EVQLVESGGGLVQPGRSLRLSCAASGITFDDYGMHWVRQAPGKGLEWVSGISWNRGRIEYADSVKGRFTI SRDNAKNSLYLQMNSLRAEDTALYYCAKGRFRYFDWFLDYWGQGTLVTVSS (SEQ ID NO: 257) VL MDTRAPTQLLGLLLLWLPGARCALVMTQTPSSTSTAVGGTVTIKCQASQSISVYLAWYQQKPGQPPKLLI YSASTLASGVPSRFKGSRSGTEYTLTISGVQREDAATYYCLGSAGS (SEQ ID NO: 470) VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLVWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIK (SEQ ID NO: 258) VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK (SEQ ID NO: 259) VL DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIK (SEQ ID NO: 260) VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 261) VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQYGSSPFGGGTKVEIK (SEQ ID NO: 262) VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPTFGQGTRLEIK (SEQ ID NO: 263) VL AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQFNSYPFTFGPGTKVDIK (SEQ ID NO: 264) Group E vh EVQL L ESGGGL VQPGGSL RL SCAASGFTFSSYIMMWVRQAPGKGL EWVSSIYPSGGITFYADTVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS (SEQ ID NO: 471) VL QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSSGSKSG NTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL (SEQ ID NO: 472)

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Group F vh EVKLQESGPSLVKPSQTLSLTCSVTGYSITSDYWNWIRKFPGNKLEYVGYISYTGSTYYNPSLKSRISIT RDTSKNQYYLQLNSVTSEDTATYYCARYGGWLSPFDYWGQGTTLTVSS (SEQ ID NO: 473) vh EVQLQESGPGLVAPSQSLSITCTVSGFSLTTYSINWIRQPPGKGLEWLGyMWAGGGTIISNSyLKSRLIIS KDNSKSQVFLKMNSLQTDDTARYYCARYYGNSPYYAIDYWGQGTSVTVSS (SEQ ID NO: 474) vh EVKLQESGPSLVKPSQTLSLTCSVTGYSIISDYWNWIRKFPGNKLEYLGYISYTGSTYYNPSLKSRISIT RDTSKNQYYLQLNSVTTEDTATYYCARRGGWLLPFDYWGQGTTLTVSS (SEQ ID NO: 475) vh EVKLQESGPSLVKPGASVKLSCKASGYTFTSYDINWVKQRPGQGLEWIGWIFPRDNNTKYNENFKGKATL TVDTSSTTAYMELHSLTSEDSAVYFCTKENWVGDFDYWGQGTTLTLSS (SEQ ID NO: 476) vh EVQLQQSGPDLVTPGASVRISCQASGYTFPDYYMNWVKQSHGKSLEWIGDIDPNYGGTTYNQKFKGKAIL TVDRSSSTAYMELRSLTSEDSAVYYCARGALTDWGQGTSLTVSS (SEQ ID NO: 477) vh EIVLTQSPATLSLSPGERATLSCRASSSVSYIYWFQQKPGQSPRPLIYAAFNRATGIPARFSGSGSGTDY TLTISSLEPEDFAVYYCQQWSNNPLTFGQGTKVEIK (SEQ ID NO: 478) vh QVQLVQSGAEVKKPGASVKVSCKASGYTFPDYYMNWVRQAPGQGLEWMGDIDPNYGGTNYAQKFQGRVTM TRDTSISTAYMELSRLRSDDTAVYYCARGALTDWGQGTMVTVSS (SEQ ID NO: 479) vh QVQLVQSGAEVKKPGASVKVSOKASGYTFPDYYMNWVRQAPGQSLEWMGDIDPNYGGTNYNQKFQGRVTM TRDTSISTAYMELSRLRSDDTAVYYCARGALTDWGQGTMVTVSS (SEQ ID NO: 480) vh EVQLVQSGAEVKKPGASVKVSOKASGYTFPDYYMNWVRQAPGQSLEWMGDIDPNYGGTNYNQKFQGRVTM TVDRSSSTAYMELSRLRSDDTAVYYCARGALTDWGQGTMVTVSS (SEQ ID NO: 481) vh EVQLVESGGGLVQPGRSLRLSCTASGYTFPDYYMNWVRQAPGKGLEWVGDIDPNYGGTTYAASVKGRFTI SVDRSKSIAYLQMSSLKTEDTAVYYCTRGALTDWGQGTMVTVSS (SEQ ID NO: 482) vh EVQLVESGGGLVQPGRSLRLSCTASGYTFPDYYMNWVRQAPGKGLEWVGDIDPNYGGTTYNASVKGRFTI SVDRSKSIAYLQMSSLKTEDTAVYYCARGALTDWGQGTMVTVSS (SEQ ID NO: 483) VL DIVMTQSHKL MSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKL L IYWASTRHTGVPDRFTGSGSGTD FTLTISNVQSEDLADYFCQQDSSYPLTFGAGTKVELK (SEQ ID NO: 484) VL DIVTTQSHKLMTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTD FTLTISNVQSEDLADYFCQQDSSYPLTFGAGTKVELK (SEQ ID NO: 485) VL DIVMTQSPSSLAVSVGEKVSMGCKSSQSLLYSSNQKNSLAWYQQKPGQSPKLLIDWASTRESGVPDRFTG SGSGTDFTLTISSVKAEDLAVYYCQQYYGYPLTFGAGTKLELK (SEQ ID NO: 486) VL DIVMTQSPAIMSASPGEKVTMTCSASSSIRYMHWYQQKPGTSPKRWISDTSKLTSGVPARFSGSGSGTSY ALTISSMEAEDAATYYCHQRSSYPWTFGGGTKLEIK (SEQ ID NO: 487) VL QIVLSQSPAILSASPGEKVTMTCRASSSVSYIYWFQQKPGSSPKPWIYATFNLASGVPARFSGSGSGTSY SLTISRVETEDAATYYCQQWSNNPLTFGAGTKLELK (SEQ ID NO: 488) VL EIVLTQSPATLSLSPGERATLSCRASSSVSYIYWFQQKPGQAPRLLIYAAFNRATGIPARFSGSGSGTDY TLTISSLEPEDFAVYYCQQWSNNPLTFGQGTKVEIK (SEQ ID NO: 489) VL QIVLTQSPATLSLSPGERATLSCRASSSVSYIYWFQQKPGQSPRPLIYATFNLASGIPARFSGSGSGTSY TLTISRLEPEDFAVYYCQQWSNNPLTFGQGTKVEIK (SEQ ID NO: 490) VL DIQLTQSPSSLSASVGDRVTITCRASSGVSYIYWFQQKPGKAPKLLIYAAFNLASGVPSRFSGSGSGTEY TLTISSLQPEDFATYYCQQWSNNPLTFGQGTKVEIK (SEQ ID NO: 491)

- 50 039736

VL DIQLTQSPSSLSASVGDRVTITCRASSGVSYIYWFQQKPGKAPKPLIYAAFNLASGVPSRFSGSGSGTEY TLTISSLQPEDFATYYCQQWSNNPLTFGQGTKVEIK (SEQ ID NO: 492) VL DIQLTQSPSILSASVGDRVTITCRASSSVSYIYWFQQKPGKAPKPLIYATFNLASGVPSRFSGSGSGTSY TLTISSLQPEDFATYYCQQWSNNPLTFGQGTKVEIK (SEQ ID NO: 493)

Group G vh QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTM TTDTSTSTAYMELRSLRSDDTAVYYCARALPSGTILVGGWFDPWGQGTLVTVSS (SEQ ID NO: 494) vh EVQLVQSGGGWQPGRSLRLSCAASGFTFSSYALSWVRQAPGKGLEWVSAISGGGGSTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKDVFPETFSMNYGMDVWGQGTLVTVSS (SEQ ID NO: 495) vh QVQLVQSGGGWQPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSLISGDGGSTYYADSVKGRFTI SRDNSKNSLYLQMNSLRTEDTALYYCAKVLLPCSTSTSCYGSVGAFDIWGQGTTVTVSS (SEQ ID NO : 4 9 6) vh QVQLVQSGGSWRPGESLRLSCVASGFIFDNYDMSWVRQVPGKGLEWVSRVNWNGGSTTYADAVKGRFTI SRDNTKNSLYLQMNNLRAEDTAVYYCVREFVGAYDLWGQGTTVTVSS (SEQ ID NO: 497) vh QVQLVQSGAEVKKPGATVKVSCKVFGDTFRGLYIHWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTI TTDESTSTAYMELSSLRSEDTAVYYCASGLRWGIWGWFDPWGQGTLVTVSS (SEQ ID NO: 498) vh EVQLVQSGAELKKPGSSVKVSCKAFGGTFSDNAISWVRQAPGQGPEWMGGIIPIFGKPNYAQKFQGRVTI TADESTSTAYMVLSSLRSEDTAVYYCARTMVRGFLGVMDVWGQGTTVTVSS (SEQ ID NO: 499) vh QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKDQFVTIFGVPRYGMDVWGQGTTVTVSS (SEQ ID NO: 500) vh QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTI TADKSTSTAYMELSSLRSEDTAVYYCARGRQMFGAGIDFWGPGTLVTVSS (SEQ ID NO: 501) vh EVQLVESGAEVKKPGSSVKVSCKVSGGTFGTYALNWVRQAPGQGLEWMGRIVPLIGLVNYAHNFEGRISI TADKSTGTAYMELSNLRSDDTAVYYCAREVYGGNSDYWGQGTLVTVSS (SEQ ID NO: 502) vh QVQLVQSGGEVKKPGASVKVSCKASGYTLSSHGITWVRQAPGQGLEWMGWISAHNGHASNAQKVEDRVTM TTDTSTNTAYMELRSLTADDTAVYYCARVHAALYYGMDVWGQGTLVTVSS (SEQ ID NO: 503) vh QVQLQESGGGWQPGRSLRLSCSASGFTFSRHGMHWVRQAPGKGLEWVAVISHDGSVKYYADSMKGRFSI SRDNSNNTLYLQMDSLRADDTAVYYCARGLSYQVSGWFDPWGQGTLVTVSS (SEQ ID NO: 504) vh NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDTS SNSASLTISGLKTKDEADYYCQSYDGITVIFGGGTKLTVL (SEQ ID NO: 505) vh NFMLTQPHSVSGSPGKTVTLPCTRSSGSIASHYVQWYQQRPGSAPTTVIYEDNKRPSGVPDRFSGSIDSS SNSASLSISGLKTEDEADYYCQSYDSSNRWVFGGGTKLTVL (SEQ ID NO: 506) vh LPVLTQPASLSASPGASASLTCTLRSGLNVGSYRIYWYQQKPGSRPQYLLNYKSDSNKQQASGVPSRFSG SKDASANAGILLISGLQSEDEADYYCMIWYSSAWFGGGTKLTVL (SEQ ID NO: 507) VL NFMLTQPHSVSESPGKTVTISCTRSSGNIASNYVQWYQQRPGSAPTTVIYEDNQRPSGVPDRFSGSIDSS SNSASLTISGLKTEDEADYYCQSYDSSNLWVFGGGTKLTVL (SEQ ID NO: 508)

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VL SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTA SLTITGAQAEDEADYYCNSRDSSGNHYVFGTGTKVTVL (SEQ ID NO: 509) VL LPVLTQAPSVSVAPGKTARITCGGSDIGRKSVHWYQQKPGQAPALVIYSDRDRPSGISERFSGSNSGNTA TLTISRVEAGDEADYYCQVWDNNSDHYVFGAGTELIVL (SEQ ID NO: 510) VL QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSG NTASLTISGLQAEDEADYYCSSYTSSTLPFGGGTKLTVL (SEQ ID NO: 511) VL EIVLTQSPATLSLSPGERATLSCRASQSIGNSLAWYQQKPGQAPRLLMYGASSRATGIPDRFSGSGAGTD FTLTISSLEPEDFATYYCQQHTIPTFSFGPGTKVEVK (SEQ ID NO: 512) VL DIVMTQTPSFLSASIGDRVTITCRASQGIGSYLAWYQQRPGEAPKLLIYAASTLQSGVPSRFSGSGSGTD FTLTISNLQPEDFATYYCQQLNNYPITFGQGTRLEIK (SEQ ID NO: 513) VL QSALTQPPSVSVSPGQTANIPCSGDKLGNKYAYWYQQKPGQSPVLLIYQDIKRPSRIPERFSGSNSADTA TLTISGTQAMDEADYYCQTWDNSWFGGGTKLTVL (SEQ ID NO: 514) VL NFMLTQPHSVSESPGKTVTISCTRSSGSIDSNYVQWYQQRPGSAPTTVIYEDNQRPSGVPDRFSGSIDSS SNSASLTISGLKTEDEADYYCQSYDSNNRHVIFGGGTKLTVL (SEQ ID NO: 515) VL NFMLTQPHSVSESPGKTVTISCTRSSGNIGTNYVQWYQQRPGSAPVALIYEDYRRPSGVPDRFSGSIDSS SNSASLIISGLKPEDEADYYCQSYHSSGWEFGGGTKLTVL (SEQ ID NO: 516) VL QSVLTQPPSVSVAPGQTARITCGGNNIGSKGVHWYQQKPGQAPVLWYDDSDRPSGIPERFSGSNSGNTA TLTISRVEAGDEADYYCQVWDSSSSDHWVFGGGTKLTVL (SEQ ID NO: 517) VL NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVIYEDNQRPSGVPDRFSGSIDSS SNSASLTISGLKTEDEADYYCQSYDSTTPSVFGGGTKLTVL (SEQ ID NO: 518) VL QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWTSPHNGLTAFAQILEGRVTM TTDTSTNTAYMELRNLTFDDTAVYFCAKVHPVFSYALDVWGQGTLVTVSS (SEQ ID NO: 519) VL EVQLVESGAEVMNPGSSVRVSCRGSGGDFSTYAFSWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTI TADKSTSTAYMELSSLRSDDTAVYYCARDGYGSDPVLWGQGTLVTVSS (SEQ ID NO: 520) VL EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKVQGRVTM TTDTSTSTGYMELRSLRSDDTAVYYCARGDFRKPFDYWGQGTLVTVSS (SEQ ID NO: 521) Group H vh EVQLVQSGPELKKPGASVKMSCKASGYTFTSYVMHWVKQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATL TSDKSTSTAYMELSSLRSEDSAVYYCARgAWGYPWGQGTLVTVSS (SEQ ID NO: 522) vh EVQLVQSGAEVKKPGASVKMSCKASGYTFTSYVMHWVKQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATL TSDKSTSTAYMELSSLRSEDTAVYYCARgAWGYPWGQGTLVTVSS (SEQ ID NO: 523) vh EVQLVQSGAEVKKPGASVKMSCKASGYTFTSYymWRQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATL TSDKSTSTAYMELSSLRSEDTAVYYCARQAWGYPWGQGTLVTVSS (SEQ ID NO: 524) vh EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATL TSDKSTSTAYMELSSLRSEDTAVYYCARQAWGYPWGQGTLVTVSS (SEQ ID NO: 525) vh EVQLVQ S GAEVKKP GASVKVS OKAS GYT FTSYVMHWVRQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATI TSDKSTSTAYMELSSLRSEDTAVYYCARgAWGYPWGQGTLVTVSS (SEQ ID NO: 526) VL DIVLTQSPASLALSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSG SGTDFTLTINSLEEEDAAMYFCQQSRRVPYTFGQGTKLEIK (SEQ ID NO: 527)

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VL DIVLTQSPATLSLSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSG SGTDFTLTINSLEAEDAAMYFCQQSRRVPYTFGQGTKLEIK (SEQ ID NO: 528) VL EIVLTQSPATLSLSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSG SGTDFTLTINSLEAEDAAMYFCQQSRRVPYTFGQGTKLEIK (SEQ ID NO: 529) VL DIVLTQSPATLSLSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSG SGTDFTLTINSLEAEDAATYFCQQSRRVPYTFGQGTKLEIK (SEQ ID NO: 530) Group 1 vh EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTI TADKSTSTAYMELSSLRSEDTAVYYCAREGTIYDSSGYSFDYWGQGTLVTVSS (SEQ ID NO: 531) vh EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVSM TRDTSTSTVYMELSSLTSEDTAVYYCARDLFPHIYGNYYGMDIWGQGTTVTVSS (SEQ ID NO: 532) vh QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTI TADKSTSTAYMELSSLRSEDTAVYYCARLAVPGAFDIWGQGTMVTVSS (SEQ ID NO: 533) vh EVQLVESGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLAVISYDGSNKYYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAVYYCARGQWLVTELDYWGQGTLVTVSS (SEQ ID NO: 534) vh EVQLVESGSEVEKPGSSVKVSCKASGGTFSDSGISWVRQAPGQGLEWMGGIIPMFATPYYAQKFQDRVTI TADESTSTVYMELSGLRSDDTAVFYCARDRGRGHLPWYFDLWGRGTLVTVSS (SEQ ID NO: 535) vh EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTI TADESTSTAYMELSSLRSEDTAVYYCARAPYYYYYMDVWGQGTTVTVSS (SEQ ID NO: 536) vh EVQLLESGAEVKKPGSSVKVSCKASGGTLSRYALSWVRQAPGQGPEWVGAIIPIFGTPHYSKKFQDRVII TVDTSTNTAFMELSSLRFEDTALYFCARGHDEYDISGYHRLDYWGQGTLVTVSS (SEQ ID NO: 537) vh QVQLVQSGSELKKPGSSVKVSCKASGYSFSGYYIHWVRQAPGQGLEWMGWIDPNSGVTNYVRRFQGRVTM TRDTSLSTAYMELSGLTADDTAVYYCARDENLWQFGYLDYWGQGTLVTVSS (SEQ ID NO: 538) vh QVQLVQSGAEVKKPGSSVKVSCKASGGTFSRYGVHWVRQAPGQGLEWMGRLIPIVSMTNYAQKFQDRVSI TTDKSTGTAYMELRSLTSEDTALYYCASVGQQLPWVFFAWGQGTLVTVSS (SEQ ID NO: 539) vh QVQLVESGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISFDGSNKYYADSVRGRFTI SRDNSKNTLYLQMNSLRTEDTAVYYCARGWLDRDIDYWGQGTLVTVSS (SEQ ID NO: 540) vh EVQLVESGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISFDGSNKYYADSVRGRFTI SRDNSKNTLYLQMNSLRTEDTAVYYCARGWLDRDIDYWGQGTLVTVSS (SEQ ID NO: 541) vh EVQLVQSGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQPPGKGLEWLAVISYDGSYKIHADSVQGRFTI SRDNAKNSVFLQMNSLKTEDTAVYYCTTDRKWLAWHGMDVWGQGTTVTVSS (SEQ ID NO: 542) vh EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTI TADESTSTAYMELSSLRSEDTAVYYCARDGIVADFQHWGQGTLVTVSS (SEQ ID NO: 543) vh EVQLVESGAEVKKPGASVKVSCKASGDTFSRYGITWVRQAPGRGLEWMGNIVPFFGATNYAQKFQGRLTI TADKSSYTSYMDLSSLRSDDTAVYYCARDHFYGSGGYFDYWGQGTLVTVSS (SEQ ID NO: 544) vh EVQLLESGAEVKKPGASVKVSCKASGYTFNSYDINWVRQAPGQGLEWMGGIIPVFGTANYAESFQGRVTM TADHSTSTAYMELNNLRSEDTAVYYCARDRWHYESRPMDVWGQGTTVTVSS (SEQ ID NO: 545)

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vh EVQLVESGGGLVRPGGSLRLACAASGFSFSDYYMTWIRQAPGRGLEWIAYISDSGQTVHYADSVKGRFTI SRDNTKNSLFLQVNTLRAEDTAVYYCAREDLLGYYLQSWGQGTLVTVSS (SEQ ID NO: 546) vh QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSR ITINPDTSKNQFSLQLNSVTPEDTAVYYCARDEPRAVAGSQAYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 547) vh EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSDGSTSYAQKFQGRVTM TRDTSTSTVHMELSSLRSEDTAVYYCARDLFPHIYGNYYGMDIWGQGTTVTVSS (SEQ ID NO: 548) vh QMQLVQSGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISFDGSNKYYADSVRGRFTI SRDNSKNTLYLQMNSLRTEDTAVYYCARGWLDRDIDYWGQGTLVTVSS (SEQ ID NO: 549) vh QVQLVQSGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISFDGSNKYYADSVRGRFTI SRDNSKNTLYLQMNSLRTEDTAVYYCARGWLDRDIDYWGQGTLVTVSS (SEQ ID NO: 550) VL QSVLTQPPSVSAAPGQKVTISCSGNNSNIANNYVSWYQQLPGTAPKLLIYDNNYRPSGIPDRFSGSKSSGT SATLDITGLQTGDEADYYCGVWDGSLTTGVFGGGTKLTVL (SEQ ID NO: 551) VL AIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLESGVPSRFSGSGSGTD FTLTISSLQPEDLATYYCQQLHTFPLTFGGGTKVEIK (SEQ ID NO: 552) VL QPVLTQPPSASGSPGQSVTISCTGTSSDVGAYNFVSWYRQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSG NTASLTVSGLQAEDEADYYCSSYAGTNSLGIFGTGTKLTVL (SEQ ID NO: 553) VL QSWTQPPSVSAAPGQKVTISCSGSSSDIGNHYVSWYQQLPGTAPKLLIYDNNQRPSGIPDRFSGSKSGT SATLAITGLQTGDEADYYCGTWDNSLSPHLLFGGGTKLTVL (SEQ ID NO: 554) VL QSVLTQPPSVSAAPGQKVTISCSGSSNMGNNYVSWYKQVPGTAPKLLIYENDKRPSGIPDRFSGSKSGT SATLGITGLQTGDEADYYCGTWDNSLSGFVFASGTKVTVL (SEQ ID NO: 555) VL QSALTQPASVSGSLGQSVTISCTGSSSDVGSYNLVSWYQQHPGKAPNLMIYDVSKRSGVSNRFSGSKSGN TASLTISGLQAEDEADYYCSSYTGISTWFGGGTKLTVL (SEQ ID NO: 556) VL QSVLTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVSKRPSGVSNRFSSGSKSG NTASLTISGLQAEDEADYYCSSYGGFNNLLFGGGTKLTVL (SEQ ID NO: 557) VL DIVMTQSPSSLSASIGDRVTITCRASQRISAYVNWYQQKPGKAPKVLIYAASSLRSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQTYSSPWTFGQGTKVEIK (SEQ ID NO: 558) VL QSVLTQPPSASGSPGQSVTISCTGTSSDIGGYDSVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSGSKSG NTASLTISGLQAEDEADYYCSSYTSSSIFFYVFGTGTKVTVL (SEQ ID NO: 559) VL LPVLTQPASVSGSPGQSITISCTGTTSDIGGYDYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSSGSKSG NTASLTISGLQAEDEADYYCSSYTSSSTHVFGTGTKLTVL (SEQ ID NO: 560) VL QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSG NTASLTISGLQAEDEADYYCSSYRSSTLGPVFGGGTKLTVL (SEQ ID NO: 561) VL QAGLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSSGSKSGT SASLAISGLQSEDEADYYCAAWDDSLNGYVFGTGTKLTVL (SEQ ID NO: 562) VL QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSG NTASLTISGLQAEDEADYYCSSYTSSTTHVFGTGTKVTVL (SEQ ID NO: 563) VL QSWTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSSGT SATLGITGLQTGDEADYYCGTWDSSLSVWVFGGGTQLTVL (SEQ ID NO: 564)

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VL QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGRAPRLMIYDVSNRPSGVSNRFSSGSKSG NTASLTISGLQAEDEGDYYCSSYTSGGTLGPVFGGGTKLTVL (SEQ ID NO: 565) VL QAGLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSSGSKSGT SASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVL (SEQ ID NO: 566) VL AIRMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQTYSTPYTFGQGTKLEIK (SEQ ID NO: 567) VL QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYRQHPGKAPKLMIYDVSYRPSGVSNRFSGSKSG NTASLTISGLQAEDEADYYCSSYTDSSTRYVFGTGTKLTVL (SEQ ID NO: 568) VL QPVLTQPPSASGTPGQRVAISCSGSRSNIEINSVNWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSSGT SATLGITGLQTGDEADYYCGSWDSSLSADVFGTGTKLTVL (SEQ ID NO: 569) VL QSVLTQPPSVSAAPGKKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYRNNQRPSGVPDRFSSGSKSGT SASLAISGLQSEDEADYYCATWDDSLNGWVFGGGTKLTVL (SEQ ID NO: 570) VL QSWTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNNNRHSGVPDRFSGSKSG TSASLAITGLQAEDEAEFFCGTWDSRLTTYVFGSGTKLTVL (SEQ ID NO: 571) VL QSWTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSSGT SATLGITGLQTGDEADYYCGTWDSSLSAWFGGGTKLTVL (SEQ ID NO: 572) VL VIWMTQSPSSLSASVGDRVTITCAASSLQSWYQQKPGKAPKLLIYEASTLESGVPSRFSGSGSGTEFTLT ISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK (SEQ ID NO: 573) VL QSWTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQVPGTAPKLLIYDNNKRPSGIPDRFSGSNSDT SATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL (SEQ ID NO: 574) VL QSWTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSSGT SATLGITGLQTGDEADYYCGTWDSSLSAGSWFGGGTKLTVL (SEQ ID NO: 575) VL SYELMQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTA TLTISRVEAGDEADYYCLVWDSSSDHRIFGGGTKLTVL (SEQ ID NO: 576) VL SYELMQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTA TLTISRVEAGDEADYYCQVWDSSSDHRIFGGGTKLTVL (SEQ ID NO: 577) VL SYELMQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTA TLTISRVEAGDEADYYCQVWDSSSDHRIFGGGTKLTVL (SEQ ID NO: 578) VL SYELMQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTA TLTISRVEAGDEADYYCQVWDSSSDHRIFGGGTKLTVL (SEQ ID NO: 579) HC Group J* QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTL TTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSE STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 580)

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NS QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTI SRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKR VESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK (SEQ ID NO: 581) LC EIVLTQSPATLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSG SGTDFTLTISSLEPEDFAVYYCQHSRDDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKGLVYACEVQN: NOVECTHQN: LC EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLNOTLSKADYEKHKVYACESFTHQGLSSPV3 (ID) Group K vh EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO: 735) vh EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 736) NS EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 737) VL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 268) LC DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACESFQ3 Group L vh EVQLVE S GGGLVQP GGS LRL S CAASGFTFSRFWMSWVRQAPGKGLEWVANINQDGTEKYYVDSVKGRFTI SRDNAKNSLYLQMNSLRAGDTAVYYCANTYYDFWSGHFDYWGQGTLVTVSS (SEQ ID NO: 739) vh QEHLVESGGGWQPGRSLRLSCEASGFTFSNFGMHWVRQAPGKGLEWVAALWSDGSNKYYADSVKGRVTI SRDNSKNTLYLQMNSLRAEDTAVYYCARGRGAPGIPIFGYWGQGTLVTVSS (SEQ ID NO: 74 0)

- 56 039736

vh EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKRKTDGGTTDYAAPVKGRF TISRDDSKNTLHLQMNSLKTEDTAVYYCTTDDIVWPAVMREYYFGMDVWGQGTTVTVSS (SEQ ID NO: 741) vh QVQLVQ S GAEVKKP GASVQVS CKASGYSFTGYYIHWVRQAPGQGLEWMGWINPNSGTKKYAHKFQGRVTM TRDTSIDTAYMILSSLISDDTAVYYCARDEDWNFGSWFDSWGQGTLVTVSS (SEQ ID NO: 742) vh QVHLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGHGLEWMGWLNPNTGTTKYIQNFQGRVTM TRDTSSSTAYMELTRLRSDDTAVYYCARDEDWNYGSWFDTWGQGTLVTVSS (SEQ ID NO: 743) vh EVQLVESGGGWRPGGSLRLSCAASGFTFDDYGMTWVRQAPGRGLEWVSGIHWHGKRTGYADSVKGRFTI SRDNAKKSLYLQMNSLKGEDTALYHCVRGGMSTGDWFDPWGQGTLVIVSS (SEQ ID NO: 744) vh EVQLVESGGGWRPGGSLRLSCAASGFTFDDYGMTWVRQVPGKGLEWVSGIHWSGRSTGYADSVKGRFTI SRDNAKNSLYLQMNSLRAEDTALYYCARGGMSTGDWFDPWGQGTLVTVSS (SEQ ID NO: 745) vh EVQLVESGGGLVQPGGSLRLSCAASGFTVGSNYMNWVRQAPGKGLEWVSVIYSGGSTYYADSVKGRFTIS RLTSKNTLYLQMSSLRPEDTAVYYCARGIRGLDVWGQGTTVTVSS (SEQ ID NO: 746) vh EERLVESGGDLVQPGGSLRLSCAASGITVGTNYMNWVRQAPGKGLEWVSVISSGGNTHYADSVKGRFIMS RQTSKNTLYLQMNSLETEDTAVYYCARGIRGLDVWGQGTMVTVSS (SEQ ID NO: 747) vh QVQLVQSGAEVKMPGS SVRVSCKASGGIFSSSTISWVRQAPGQGLEWMGEIIPVFGTVNYAQKFQDRVIF TADESTTTAYMELSSLKSGDTAVYFCARNWGLFSFYIWGQGTMVTVSS (SEQ ID NO: 748) vh EVQLVESGGDLVHPGRSLRLSCAASGFPFDEYAMHWVRQVPGKGLEWVSGISWSNNNIGYADSVKGRFTI S RDNAKNS LYLQMNSLRPEDTAFYYCAKSGIFDSWGQGTLVTVSS (SEQ ID NO: 74 9) vh EVQLVE S GGGWQP GRS LRL S CAASGFTFSSYGMHWVRQAPGKGLEWVT LISYEGRNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKDRTLYGMDVWGQGTTVTVSS (SEQ ID NO: 750) vh QVTLRESGPALVKTTQTLTLTCTFSGFSLSTNRMCVTWIRQPPGKALEWLARIDWDGVKYYNTSLKTRLT ISKPTSKNQWLTMTNMDPVDTATFYCARSTSLTFYYFDYWGQGTLVTVSS (SEQ ID NO: 751) vh EVQLVESGGGLVQPGGSLRLSCAASEFTVGTNHMNWVRQAPGKGLEWVSVIYSGGNTFYADSVKGRFTIS RHTSKNTLYLQMNSLTAEDTAVYYCARGLGGMDVWGQGTTVTVSS (SEQ ID NO: 752) vh EVQLVESGGGLVQRGESLRLYCAASGFTFSKYWMNWVRQAPGKGLEWVANIKGDGSEKYYVDSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARDYWGSGYYFDFWGQGTLVTVSS (SEQ ID NO: 753) vh EVQLVESGGGLVQSGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTI SRDNAKNSLYLQMNSLRADDTAVYYCARDDIVWPAPMGYYYYYFGMDVWGQGTTVTVSS (SEQ ID NO: 754) vh EVQLVESGGGLVQPGRSLRLSCAASGFTFDDFAMHWVRQAPGKGLEWVSGISWTGGNMDYANSVKGRFTI SREDAKNSLYLQMNSLRAADTALYYCVKDIRGIVATGGAFDIWGRGTMVTVSS (SEQ ID NO: 755) vh EVQLVESGGGLVQPGGSLRLSCAASGFTVGTNYMNWVRQAPGKGLEWISVIYSGGSTFYADSVKGRFTIS RQTSQNTLYLQMNSLRPEDTAVYYCARGIRGFDIWGQGTMVTVSS (SEQ ID NO: 756) vh EVQLVESGGGLVQPGGSLRLSCAASGFTISTNYMNWVRQAPGKGLEWVAVIYSSGSTYYIDSVKGRFTIS RLTSKNTVYLQMSSLNSEDTAVYYCARGIRGFDIWGQGTMVTVSS (SEQ ID NO: 757) vh EVQLVESGGGLVQPGRSLRLSCAASGFTIDDSAMHWVRQTPGKGLEWVSGISWKSGSIGYADSVRGRFTI SRDNAKNSLYLQMNSLRVEDTALYYCVKDIRGNWNYGGNWFDPWGQGTLVTVSS (SEQ ID NO: 758)

- 57 039736

vh EVQLVESGGGLVQPGGSLRLSCEASGFTVGVNHMNWVRQAPGKGLEWVSVIFSSGRTFYGDYVKGRLTIF RQTSQNTVYLQMNSLRSEDTAIYYCARGIGGLDIWGRGTMVTVSS (SEQ ID NO: 759) vh EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYALHWVRQAPGKGLEWVSGISWTGGTIDYADSVKGRFTI SRDNAKNSLYLQMSSLRTEDTAIYYCTRDIRGNWKYGGWFDPWGQGTLVTVSS (SEQ ID NO: 760) vh QVQLVQSGTEVKKPGASVKVSCKASGYTFTAYYMHWVRQAPGQGLDWMGWISPNSGFTNYAQKFQGRVTM TRDTSINTFYMELSGLRSDDTAVYYCAREGSTHHNSFDPWGQGTLVTVSS (SEQ ID NO: 761) vh EVQLVESGGGLVQPGGSLRLSCAASGFTVGTNFMNWVRQAPGKGLEWVSAIYSGGTANYADSVKGRFTIS RDTSRNTLYLQMNSLRTEDTAVYYCARGGGMDVWGQGTTVTVSS (SEQ ID NO: 762) vh QVQLVQSGAEVKKPGSSVKVSCKASGGTFNTYVLSWVRQAPGQGLEWMGEIIPILGAANYAQNFQGRVTF TTDESTNTAYMDLSSLRSEDTAVYYCARDRTSGGFDPWGQGTLVTVSS (SEQ ID NO: 763) vh QVQLVQSGAEVEKPGASVKVSCKASGYIFTHYGISWVRQAPGQGLEWVGWISPYNGYTDYAQKLQGRVTL TTDTSTTTAYMELRNLRSDDTAMYYCSRGRGPYWSFDLWGRGTLVTVSS (SEQ ID NO: 764) VL DIQMTQSPSTLSASVGDRVTITCRASQSISNWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTE FTLTISSLQPDDFATYYCQQYHSYSYTFGQGTKEIK (SEQ ID NO: 765) VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYTASSLQSGVPSRFSGSGSGTE FTLTISSLQPEDFATYYCLQHNSYPLTFGGGTKVAIK (SEQ ID NO: 766) VL DIQMTQSPSSLSASVGDRVTITCRTSQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTE FTLTISSLQPEDFATYYCLQHNNYPYTFGQGTKLEIK (SEQ ID NO: 767) VL DIVMTQTPLSSPVTLGQPASISCRSSQTLVHGDGNTYLSWIQQRPGQPPRLLIYKVSNQFSGVPDRFSGS GAGTDFTLKISRVEAEDVGLYFCMQATHFPITFGQGTRLEIK (SEQ ID NO: 768) VL DIVMTQTPLSSPVTLGQPASISCRSSPSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGS GAGTDFTLKISRVEAEDVGVYYCMQATHFPITFGQGTRLEIR (SEQ ID NO: 769) VL DIQMTQSPSSLSASLGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTE FTLTISNLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 770) VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 771) VL DIQMTQSPSSLSASVGDRVTITCRASQTINIYLNWYQQKPGRAPRLLIYAASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCHQSYSTPPITFGQGTRLEIK (SEQ ID NO: 772) VL DIQMTQSPSSLSASVGDRVTITCRASQSMSSYLNWYQQKPGRAPKLLIFAASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 773) VL EIVLTQSPGTLSLSPGERATLSCRASQSFNFNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT DFTLTINRLEPEDFGVFYCQQYESAPWTFGQGTKVEIK (SEQ ID NO: 774) VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKLLIYAASSLQSGVPSRFSGGGSGTDFTL TISSLRPEDFATYYCQQSYCTPPITFGQGTRLEIK (SEQ ID NO: 775) VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 776) VL DRVTITCRASQVISNYLAWYQQKPGKVPRLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATY YCQKYNSAPRTFGQGTKVEIK (SEQ ID NO: 777) VL DIQMTQSPSSLSASVGDRVTITCRASQNINNYLNWYQQKPGKAPKLLIYAASSFQNAVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQSYNTPLTFGGGTKVEIK (SEQ ID NO: 778)

- 58 039736

VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTE FTLTISSLQPEDFATYYCLQHNSYPYTFGQGTKLEIK (SEQ ID NO: 779) VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 780) Group Μ vh QSLEESGGRLVKPDETLTITCTVSGIDLSSNGLTWVRQAPGEGLEWIGTINKDASAYYASWAKGRLTISK PSSTKVDLKITSPTTEDTATYFCGRIAFKTGTSIWGPGTLVTVSS (SEQ ID NO: 1108) VL AIVMTQTPSPVSAAVGGTVTINCQASESVYSNNYLSWFQQKPGQPPKLLIYLASTLASGVPSRFKGSGSG TQFTLTISGVQCDDAATYYCIGGKSSSTDGNAFGGGTEVWR (SEQ ID NO: 1109) Group N vh QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGII PIFGTANYAQKFQGRVTI TADESTSTAYMELSSLRSEDTAVYYCARGNIVATITPLDYWGQGTLVTVSS (SEQ ID NO: 1110) vh QPVLTQPPSVSAAPGQKVTISCSGSSSNIANNYVSWYQQLPGTAPKLLIFANNKRPSGIPDRFSGSKSSGT SAALDITGLQTGDEADYYCGTWDSDLRAGVFGGGTKLTVL (SEQ ID NO: 1111) vh EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGII PIFGTANYAQKFQGRVTI TADKSTSTAYMELSSLRSEDTAVYYCAREGTIYDSSGYSFDYWGQGTLVTVSS (SEQ ID NO: 1112) vh QVQLVESGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISFDGSNKYYADSVRGRFTI SRDNSKNTLYLQMNSLRTEDTAVYYCARGWLDRDIDYWGQGTLVTVSS (SEQ ID NO: 1113) vh EVQLVESGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISFDGSNKYYADSVRGRFTI SRDNSKNTLYLQMNSLRTEDTAVYYCARGWLDRDIDYWGQGTLVTVSS (SEQ ID NO: 1114) vh QVQLVESGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISFDGSNKYYADSVRGRFTI SRDNSKNTLYLQMNSLRTEDTAVYYCARGWLDRDIDYWGQGTLVTVSS (SEQ ID NO: 1115) vh EVQLVESGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISFDGSNKYYADSVRGRFTI SRDNSKNTLYLQMNSLRTEDTAVYYCARGWLDRDIDYWGQGTLVTVSS (SEQ ID NO: 1116) vh EVQLVESGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISFDGSNKYYADSVRGRFTI SRDNSKNTLYLQMNSLRTEDTAVYYCARGWLDRDIDYWGQGTLVTVSS (SEQ ID NO: 1117) vh QVQLVESGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISFDGSNKYYADSVRGRFTI SRDNSKNTLYLQMNSLRTEDTAVYYCARGWLDRDIDYWGQGTLVTVSS (SEQ ID NO: 1118) vh QVQLVQSGAEVKKPGSSVKVSCKASGGTFSRYGVHWVRQAPGQGLEWMGRLIPIVSMTNYAQKFQDRVSI TTDKSTGTAYMELRSLTSEDTALYYCASVGQQLPWVFFAWGQGTLVTVSS (SEQ ID NO: 1119) vh QMQLVQSGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISFDGSNKYYADSVRGRFTI SRDNSKNTLYLQMNSLRTEDTAVYYCARGWLDRDIDYWGQGTLVTVSS (SEQ ID NO: 1120) vh QVQLVQSGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISFDGSNKYYADSVRGRFTI SRDNSKNTLYLQMNSLRTEDTAVYYCARGWLDRDIDYWGQGTLVTVSS (SEQ ID NO: 1121) vh QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAYSWVRQAPGQGLEWMGGII PSFGTANYAQKFQGRVTI TADESTSTAYMELSSLRSEDTAVYYCARGPIVATITPLDYWGQGTLVTVSS (SEQ ID NO: 1122) vh QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAYSWVRQAPGQGLEWMGGII PIFGTANYAQKFQGRVTI TADESTSTAYMELSSLRSEDTAVYYCARGPIVATITPLDYWGQGTLVTVSS (SEQ ID NO: 1123)

- 59 039736

vh QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAYSWVRQAPGQGLEWMGGIIPSFGTANYAQKFQGRVTI TADESTSTAYMELSSLRSEDTAVYYCARGPIVATITPLDYWGQGTLVTVSS (SEQ ID NO: 1124) vh QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPAFGTANYAQKFQGRVTI TADESTSTAYMELSSLRSEDTAVYYCARGPIVATITPLDYWGQGTLVTVSS (SEQ ID NO: 1125) VL SYELMQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTA TLTISRVEAGDEADYYCQVWDSSSDHRIFGGGTKLTVL (SEQ ID NO: 1126) VL AIRMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYTTSSLKSGVPSRFSGSGSGTD FTLTISRLQPEDFATYYCQQSYSSTWTFGRGTKVEIK (SEQ ID NO: 1127) VL QSVLTQPPSVSAAPGQKVTISCSGNNSNIANNYVSWYQQLPGTAPKLLIYDNNYRPSGIPDRFSGSKSSGT SATLDITGLQTGDEADYYCGVWDGSLTTGVFGGGTKLTVL (SEQ ID NO: 1128) VL LPVLTQPASVSGSPGQSITISCTGTTSDIGGYDYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSSGSKSG NTASLTISGLQAEDEADYYCSSYTSSSTHVFGTGTKLTVL (SEQ ID NO: 1129) VL QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSG NTASLTISGLQAEDEADYYCSSYRSSTLGPVFGGGTKLTVL (SEQ ID NO: 1130) VL QAGLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSSGSKSGT SASLAISGLQSEDEADYYCAAWDDSLNGYVFGTGTKLTVL (SEQ ID NO: 1131) VL QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSG NTASLTISGLQAEDEADYYCSSYTSSTTHVFGTGTKVTVL (SEQ ID NO: 1132) VL QSWTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSSGT SATLGITGLQTGDEADYYCGTWDSSLSVWVFGGGTQLTVL (SEQ ID NO: 1133) VL QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGRAPRLMIYDVSNRPSGVSNRFSGSKSG NTASLTISGLQAEDEGDYYCSSYTSGGTLGPVFGGGTKLTVL (SEQ ID NO: 1134) VL QSWTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGT SATLGITGLQTGDEADYYCGTWDSSLSAWFGGGTKLTVL (SEQ ID NO: 1135) VL QSWTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQVPGTAPKLLIYDNNKRPSGIPDRFSGSNSDT SATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL (SEQ ID NO: 1136) VL QSWTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGT SATLGITGLQTGDEADYYCGTWDSSLSAGSWFGGGTKLTVL (SEQ ID NO: 1137) VL SYELMQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTA TLTISRVEAGDEADYYCLVWDSSSDHRIFGGGTKLTVL (SEQ ID NO: 1138) VL SYELMQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTA TLTISRVEAGDEADYYCQVWDSSSDHRIFGGGTKLTVL (SEQ ID NO: 1139) VL SYELMQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTA TLTISRVEAGDEADYYCQVWDSSSDHRIFGGGTKLTVL (SEQ ID NO: 1140) VL SYELMQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTA TLTISRVEAGDEADYYCQVWDSSSDHRIFGGGTKLTVL (SEQ ID NO: 1141) Group O vh QVQLVQSGSEVKKSGSSVKVSCKTSGGTFSITNYAINWVRQAPGQGLEWMGGILPIFGAAKYAQKFQDRV TITADESTNTAYLELSSLTSEDTAMYYCARGKRWLQSDLQYWGQGTLVTVSS (SEQ ID NO: 1142)

VL QPVLTQPASVSGSPGQSITISCTGSSSDVGSYDLVSWYQQSPGKVPKLLIYEGVKRPSGVSNRFSSGSKSG NTASLTISGLQAEDEADYYCSSYAGTRNFVFGGGTQLTVL (SEQ ID NO: 1143)

It should be noted that the sequences given for group J represent the amino acid sequences of the heavy chain and light chain; the sequences given for group K include the amino acid sequences of the heavy chain variable region, variable

- 60 039736 light chain, heavy chain and light chain regions; all other sequences shown in Table 15 represent the heavy chain variable region and light chain variable region sequences.

In some embodiments, the activated anti-PDL1 antibody comprises a heavy chain that contains or is derived from the heavy chain amino acid sequence shown in US Pat. Nos. 7,943,743; 8779108; 8217149; 8460927; 7794710; 8741295 and 8981063; in U.S. Patent Application Publications No. 2014356353; 20130323249; 2014341917; 20130309250; 20110280877 and 20090317368 (currently issued as US Patent No. 8981063 above); and in PCT International Application Publications WO 2014/100483; WO2012/145493; WO2014/100439; WO 2014/195852; WO2014/100079; WO 2013/173223 and WO 2014/55897, the content of each source is incorporated herein by reference in its entirety. In some embodiments, the activated anti-PDL1 antibody comprises a light chain that contains or is derived from the light chain amino acid sequence shown in US Pat. Nos. 7,943,743; 8779108; 8217149; 8460927; 7794710; 8741295 and 8981063; in U.S. Patent Application Publications No. 2014356353; 20130323249; 2014341917; 20130309250; 20110280877 and 20090317368 (currently issued as US Patent No. 8981063 above); and in PCT International Application Publications WO 2014/100483; WO2012/145493; WO2014/100439; WO 2014/195852; WO2014/100079; WO 2013/173223 and WO 2014/55897, the contents of each source are incorporated herein by reference in their entirety. In some embodiments, the activated anti-PDL1 antibody comprises a heavy chain and a light chain that comprise or are derived from the heavy chain amino acid sequence and the light chain amino acid sequence shown in US Pat. Nos. 7,943,743; 8779108; 8217149; 8460927; 7794710; 8741295 and 8981063; in U.S. Patent Application Publications No. 2014356353; 20130323249; 2014341917; 20130309250; 20110280877 and 20090317368 (now issued as US Patent No. 8 981063 above); in PCT International Application Publications WO 2014/100483; WO2012/145493; WO2014/100439; WO2014/195852; WO2014/100079; WO 2013/173223 and WO 2014/55897, the content of each source is incorporated herein by reference in its entirety.

In some embodiments, the activated antibody comprises an anti-PDL1 antibody that contains at least one CDR sequence selected from the CDR sequences (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, VL CDR3) from the CDR sequences shown in US patent No. 7943743. In some embodiments, the implementation of the activated antibody includes an anti-PDL1 antibody that contains at least one CDR sequence, at least two, at least three, at least four, at least five and/or six CDR sequences selected from the following sequences given in US patent No. 7943743: SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 51, SEQ ID NO: 61 and SEQ ID NO: 71. In some embodiments, the activated antibody comprises an anti-PDL1 antibody that contains at least one CDR sequence, at least two, at least three, at least four, at least five, and/or six sequences of CDRs selected from SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, and SEQ ID NO: 72. In some embodiments, the activated antibody comprises an anti- PDL1 antibody that contains at least one CDR sequence, at least two, at least three, at least four, at least five and/or six CDR sequences selected from SEQ ID NO: 23, SEQ ID NO: 33 , SEQ ID NO: 43, SEQ ID NO: 53, SEQ ID NO: 63 and SEQ ID NO: 73.

In some embodiments, the activated antibody comprises an anti-PDL1 antibody that contains at least one CDR sequence, at least two, at least three, at least four, at least five, and/or six CDR sequences selected from the following sequences are given in US patent No. 7943743: VH CDR1 sequence containing SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23; a VH CDR2 sequence containing SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33; a VH CDR3 sequence containing SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43; a VL CDR1 sequence containing SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53; a VL CDR2 sequence containing SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63; and a VL CDR3 sequence containing SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

In some embodiments, the activated antibody comprises an anti-PDL1 antibody that binds to a polypeptide that contains or is derived from SEQ ID NO: 1 or SEQ ID NO: 3 of US Pat. No. 8,460,927.

In some embodiments, the activated antibody comprises an anti-PDL1 antibody that binds to a human PDL polypeptide that contains or is derived from SEQ ID NO: 1 of US Pat. No. 84,60927.

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MRIFAVFIFMTYWHLLNAFTVTVPKDLYWEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDL

KVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILWDPVT

SEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTA

ELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ

ID NO: 5 8 5)

In some embodiments, the activated antibody comprises an anti-PDL1 antibody that binds to a mouse PDL1 polypeptide that contains or is derived from SEQ ID NO: 3 of US Pat. No. 8,460,927.

MRIFAGIIFTACCHLLRAFTITAPKDLYWEYGSNVTMECRFPVERELDLLALWYWEKEDEQVIQFVAGEEDL

KPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATS

EHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAE

LIIPELPATHPPQNRTHWVLLGSILLFLIWSTVLLFLRKQVRMLDVEKCGVEDTSSKNRNDTQFEET (SEQ

ID NO: 586)

In some embodiments, the activated antibody comprises an anti-PDL1 antibody that binds to a polypeptide that contains or is derived from SEQ ID NO: 36 or SEQ ID NO: 38 of PCT International Application Publication WO 2014/10079.

In some embodiments, the activated antibody comprises an anti-PDL1 antibody that binds to a human PDL1 polypeptide that contains or is derived from SEQ ID NO: 36 of PCT International Application Publication WO 2014/10079.

MRIFAVFIFMTYWHLLNAFTVTVPKDLYWEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDL

KVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILWDPVT

SEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTA

ELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKCGIQDTNSKQSDTHLEET (SEQ ID

NO: 587)

In some embodiments, the activated antibody comprises an anti-PDL1 antibody that binds to a human PDL1 extracellular domain polypeptide that contains or is derived from SEQ ID NO: 38 of PCT International Publication WO 2014/10079.

FTVTVPKDLYWEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKN11QFVHGEEDLKVQHS SYRQRARLLKDQL

SLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILWDPVTSEHELTCQAEGYPKAEVI

WTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERT (SEQ ID NO: 588)

In some embodiments, the activated antibody comprises an anti-PDL1 antibody that represents or is derived from BMS-936559 (MDX-1105). In some embodiments, the activated antibody comprises an anti-PDL1 antibody that is or is derived from MPDL3280A (RG7446). In some embodiments, the activated antibody comprises an anti-PDL1 antibody that is or is derived from MEDI4736. In some embodiments, the activated antibody comprises an anti-PDL1 antibody that is or is derived from AMP224. In some embodiments, the activated antibody comprises an anti-PDL1 antibody that is or is derived from MSB0010718C.

In some embodiments, the activated anti-PDL1 antibody comprises or is derived from an antibody that is synthesized, secreted, or otherwise produced by a hybridoma, such as, for example, the hybridoma(s) disclosed in U.S. Patent No. 8,779,108 and deposited with the National Collections of Industrial and Marine Bacteria (NCIMB) under accession number 41598, as well as hybridoma(s) disclosed in US patent No. 8741295 and deposited with the National Collection of Cultures of Microorganisms (CNCM) under accession number I-4080, I-4081 and/or I-4122.

In some embodiments, the activated anti-PDL1 antibody comprises the CDR sequence shown in Table 16, a combination of VL CDR sequences (VL CDR1, VL CDR2, VL CDR3) selected from the group consisting of the combinations shown on the same line in Table 16. 16, a combination of VH CDR sequences (VH CDR1, VH CDR2, VH CDR3) selected from the group consisting of the combinations shown in Table 16, 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 the combinations shown in the table. 16.

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Table 16. CDR sequences for antibodies and activated antibodies that bind to PDL1

VL AB Name CDR1 (SEQID NO) CDR2 (SEQ ID NO) CDR3 (SEQID NO) C8 RASQSISSYLN (209) KASRLQS (210) RALKPVT (211) C12 RASQSISSYLN (209) AASSLQS (215) SYSTPNT (216) C16 RASQSISSYLN (209) SASQLQS (219) ANSRPST (220) C2 0 RASQSISSYLN (209) NASSLQS (223) YPYGPG (224)

vh CDR1 (SEQ ID NO) CDR2 (SEQID NO) CDR3 (SEQ ID NO) SYAMS DITASGQRTTYADS SKAIFDY (212) (213) (214) SYAMS SINKDGHYTSYADS NLDEFDY (212) (217) (218) SYAMS SIMATGAGTLYADS DGAGFDY (212) (221) (222) SYAMS TITSSGAATYYADS NYTGFDY (212) (225) (226)

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C60 RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) Cl RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) DI RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) G7 RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) H9 RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) bio RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) E10 RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) A05 RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) C05 RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) CIO RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) D08 RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) G09 RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) GIO RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) G12 RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) El RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) DOI RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) H06 RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) C5H9 RASQSISSYLN (209) YASTLQS (227) DNGYPST (228)

SYAMS (212) SIYSTGGATAYADS (229) SSAGFDY (230) SSAGQSRP SYAMS SSIYSTGGATAYADS GFDY (212) (231) (232) SSAGQSWP SYAMS SSIYSTGGATAYADS GFDY (212) (231) (233) SSAGQSFP SYAMS SSIYSTGGATAYADS GFDY (212) (231) (234) SYAMS SSIYSTGGATAYADS WSAAFDY (212) (231) (235) SYAMS SSIYSTGGATAYADS WSAGYDY (212) (231) (236) SYAMS SSIYSTGGATAYADS WSKGFDY (212) (231) (237) SYAMS SSIWKQGIVTVYDS SSAGFDY (212) (238) (230) SYAMS SSIWRNGIVTVYDS SSAGFDY (212) (239) (230) SYAMS SDIWKQGMVTVYDS SSAGFDY (212) (240) (230) SYAMS SSIWRQGLATAYDS SSAGFDY (212) (241) (230) SYAMS SEIVATGILTSYDS SSAGFDY (212) (242) (230) SYAMS SSIGRQGLITVYDS SSAGFDY (212) (243) (230) SYAMS SSIWYQGLVTVYDS SSAGFDY (212) (244) (230) SYAMS SDIWKQGFATADS SSAGFDY (212) (245) (230) SYAMS SSIWKQGIVTVYDS SSAGFDY (212) (238) (230) SYAMS SSIWRQGLATAYDS SSAGFDY (212) (241) (230) SYAMS SSIWRNGIVTVYADS WSAAFDY (212) (246) (235)

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C5B10 RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) C5E10 RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) G12H9 RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) G12B10 RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) G12E10 RASQSISSYLN (209) YASTLQS (227) DNGYPST (228) C5H9 v2 RASQSISSYLN (209) AASSLQS (215) DNGYPST (228)

SIAMS (212) SSIWRNGIVTVYADS (246) WSAGYDY (236) SYAMS SSIWRNGIVTVYADS WSKGFDY (212) (246) (237) SYAMS SSIWYQGLVTVYADS WSAAFDY (212) (247) (235) SYAMS SSIWYQGLVTVYADS WSAGYDY (212) (247) (236) SYAMS SSIWYQGLVTVYADS WSKGFDY (212) (247) (237) SYAMS SSIWRNGIVTVYADS WSAAFDY (212) (246) (235)

In some embodiments, the activated aHTu-PDL1 antibody comprises the CDR sequence shown in Table 17, a combination of VL CDR sequences selected from the group consisting of the combinations shown in Table 17, and/or a combination of VH CDR sequences selected from the group , consisting of the combinations shown in Table 17.

In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group A in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group A in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group A in Table 1. 17 and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group A in Table 17.

In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group B in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group B in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group B of Table 1. 17 and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group B in Table 17.

In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group C in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group C in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group C in Table 1. 17 and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group C in Table 17.

In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group D in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group D in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group D in Table 17 and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group D in Table 17.

In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group E in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group E in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group E in Table 17 and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group E in table 17.

In some embodiments, an activated anti-PDL1 antibody comprises a combination

- 65 039736 heavy chain CDR sequences selected from the group consisting of the combinations shown in group F in Table 17. In some embodiments, the anti-PDL1-activated antibody comprises a combination of light chain CDR sequences selected from the group consisting of combinations shown in Group F of Table 17. In some embodiments, the anti-PDL1-activated antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group F in Table 17 and the combination of light chain CDR sequences selected from the group consisting of the combinations shown in group F in Table 17.

In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group G in Table 17. In some embodiments, the anti-PDL1-activated antibody comprises a combination of light chain CDR sequences selected from the group consisting of combinations shown in group G of Table 17. In some embodiments, the anti-PDL1-activated antibody comprises a combination of heavy chain CDR sequences selected from a group consisting of the combinations shown in group G in Table 17 and a combination of light chain CDR sequences selected from the group consisting of combinations shown in group G in Table 17.

In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group H in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group H in Table 17. In some embodiments, the activated anti-PDLl antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of combinations shown in group H in Table 17 and a combination of light chain CDR sequences selected from the group consisting of combinations shown in group H in Table 17.

In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group I in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group I in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group I in Table 17 and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group I in Table 17.

In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group J in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group J in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group J in Table 17 and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group J in Table 17.

In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group K in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group K in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group K in Table 17 and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group K in Table 17.

In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group L in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group L in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group L in Table 17 and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group L in Table 17.

In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group M in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group M in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group M in Table 17 and a combination of light chain CDR sequences,

- 66 039736 selected from the group consisting of the combinations shown in group M in table.17.

In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group N in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group N in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group N in Table 17 and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group N in Table 17.

In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group O in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group O in Table 17. In some embodiments, the activated anti-PDL1 antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of combinations shown in group O in Table 17 and a combination of light chain CDR sequences selected from the group consisting of combinations shown in group O in Table 17.

In some embodiments, the activated anti-PDL1 antibody comprises a heavy chain CDR1 sequence selected from the group consisting of the sequences shown in group P in Table 17.

Table 17 Additional CDR Sequences for Antibodies and Activated Antibodies that Bind to PDL1

Group A VL vh CDR1 (SEQ ID NO:) CDR2 (SEQID NO:) CDR3 (SEQ ID NO:) CDR1 (SEQID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQID NO:) RASQSVSSYLV (304) DASNRAT (293) QQRSNWPRT (278) DYGFS (296) WITAYNGNTNYAQKLQG (284) DYFYGMDV (269) RASQSVSSYLA (305) AASSLQS (215) QQRSNWPT (279) TYAIS (297) GIIPIFGKAHYAQKFQG (285) KFHFVSGSPFG MDV (270)

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RASQGISSWLA (306) GASSRAT (294) QQYNSYPYT (280) SYDVH (298) WLHADTGITKFSQKFQG (286) ERIQLWFDY (271) RASQSVSSSYLA (307) DASSLES (295) QQYGSSPWT (281) TYAIN (299) GIIPIFGTANHAQKFQG (287) DQGIAAALFDY (272) RASQGISSALA (308) KASTLES (447) QQYGSSP (282) DYWH (300) GISGNGSGNIGYADSVKG (288) PFDY (273) QQFNSYPFT (283) SYAIS (301) GIIPIFGRAHYAQKFQG (289) KYDYVSGSPFG MDV (274) QQSYSTPWT (448) SYAIN (302) GIIPIFGSANYAQKFQD (290) DSSGWSRYYMD V (275) DYGMH (303) GIIPLFGIAHYAQKFQG (291) KYSYVSGSPFG MDV (276) GISWNRGRIEYADSVKG (292) GRFRYFDWFLD Y (277) DYFWSGFSAFD I (446)

Group B VL vh CDR1 (SEQ ID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQID NO:) CDR1 (SEQ ID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQ ID NO:) SGDALPQKYVF EDSKRPS YSTDRSGNHRV TYSMN SISSSGDYIYYADSVK DLVTSMVAFDY (312) (313) (314) (309) (310) (311) RASQRVSSSYLA (318) DASSRAT (319) QQYGSLPWT (320) RYWMS (315) NIKQDGSEKYYVDSVKG (316) EGGWFGELAFD Y (317) RASQSVSSNYLA GTSSRAT QQYGSSIFT SYWMS NIKQDGGEQYYVDSVK DWNYGYYDMDV (324) (325) (326) (321) (322) (323)

Group C VL vh CDR1 (SEQ ID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQ ID NO:) CDR1 (SEQ ID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQ ID NO:)

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RASQ(D/V) (V/I)(S/N)T (A/F)(V/L)A (327 ) SAS (F/T) L(Y/A) S (328) QQ(Y/G/F/S) (L/Y/F/W)(Y /N/A/T/G/F/ I)(H/V/P/T/ I)P(A/W /R/P /Т ) T (329) GFTFS(D/G) SWIH (330) AWI(S/L)PYGGS(T/S) YYADSVKG (331) RHWPGGF DY(332) RASQDVSTAVA (333) SASFLYS (334) QQYLYHPAT (335) GFTFSDSWIH (336) AWISPYGGSTYYADSVKG (337)

Group D VL vh CDR1 (SEQID NO:) CDR2 (SEQID NO:) CDR3 (SEQID NO:) CDR1 (SEQID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQID NO:) KSSQSLL (H/N)(S/T) (R/S)TRKNYL A (584) WASTRES (589) (Q/K) QSYDW T (590) SYW(I/M) H (591) YINPSS(D/G)Y(H/N) E Y(S/N)(E/Q)KF(I/M) D (592) SGWL(I/V)HGDYY FD(F/Y) (593) KSSQSLLNSRT RKNYLA (594) QQSYDWT (595) SYWMH (596) YINPSSDYNEYSEKFMD (597) SGWLVHG DYYFDY (598) KSSQSLLHTST RKNYLA (599) KQSYDWT (600) GYIFTSYWMH (601) YINPSSGYHEYNQKFID (602) SGWLIHG DYYFDF(603) SWIH (604) GTTFTSWIH (605)

Group E VL vh CDR1 (SEQID NO:) CDR2 (SEQID NO:) CDR3 (SEQ ID NO:) CDR1 (SEQID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQ ID NO:)

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TGT(N/S)(T/ R/ S)DVG(A/G )YNYVS (606) (E/D)V(I /N/D)(D/H/N)RPS (607) SS(F/Y)T(N/S) (R/T/S) (G/S)(I/T)RV (608) (K/R/T/Q/G /A/W/M/I/S )Y(V/R/K/L /M/I)M(H/T /N/Q/A/V/Y /W/F/M) (609) SIYPSGG(F/I)TFYAD( S/T)VKG (610) IKLGTVT TV(E/D) Y (611) TGTSSDVGGYN YVS (612) DVSNRPS (613) SSYTSSSTRV (614) SYIMM (615) SIY P S GGITFYADTVKG (617) IKLGTVT TVDY (618)

Group F VL vh CDR1 (SEQ ID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQ ID NO:) CDR1 (SEQ ID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQ ID NO:) MYMMM (619) SIYPSGGITFYADSVKG (620) TGTSSDV GAYNYVS (621)

Group G VL vh CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 (SEQID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:) (SEQ ID NO:) NO:) NO:) NO:) NO:) (R/S/K)(A/S X(A/C/G/ G(Y/F)(T/S X(I/L/M/F/V)X(P or (A/R/N/ )S(R/N/D/Q/ S/T/V)(A (R/N/Q/E/H) ) (I/L/M/F) Absent)(A/R/N/Q/E/ Q/E/H/K E/H/K/M/S)( /C/H/I/L QX(A/R/N/C/ (A/R/N/D/C H/K/M/S/T/Y/V)(A/R /M/S/T/ SLLYSS OR /M/F/S/T Q/E/H/I/L/K /Q/E/H/I/L /N/D/C/Q/E/G/H/K/M Y)(A/R/ missing) /M/F/S/T/Y/ /K/M/P/S/T /F/P/S/T/Y/V)(N/G/ N/D/C/Q (N/D or /Y/V)(R/ missing) N/D/Q/E/ V)(N/G/S)(A /W/Y/V)(N/ S)(N/G/S)T(A/R/N/Q /E/G/H/ (A/q K/S/T) (A /R/N/D/C/Q/ D/Q/E/K/P/ /E/H/K/M/S/T/Y)(A/ K/M/F/P /E/L/K/M/R/ /R/N/D/Q E/G/H/I/L/K S/T)(A/R/N R/N/C/Q/E/H/I/L/K/ /S/T/Y/ S/T/Y/V)X(R /M/F/R/S/T/ /D/C/Q/E/G M/F/S/T/W/Y/V)N(A/ V)(Q/G/ /E/G/H/K /N/S/T)(A/R /M/F/P/S W/Y/V)P(A/R /H/1/L/K/M D/Q/E/K/P/S/T)(A/R H/T/W)X /N/C/Q/E/N/ /T/Y)(A/ /N/D/Q/E/G/ /F/P/S/T/Y /N/D/C/Q/E/G/H/I/L XXX(F/Y I/L/K/M/F/S N/S/T) H/K/M/P/S/T /V)(A/R/N/ /K/M/F/P/S/T/Y/V)( ) (AI or /T/Y/V)(I/L /Y) T (624) D/C/Q/E/G/ I/L/M/F)K(A/G/S missing) (623) /M/V)(A/R/N H/1/L/K/M/ (626) (627)

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/Q/E/H/I/K/ M/F/S/T/Y) (622) F/P/S/T/Y/V)(A/R/C/Q /E/H/1/L/K /M/F/S/T/W /Y/V)N (625) RASSSVSYIY ATFNLAS HQRSSYPWT GYTFPDYYMN DIDPNYGGTTYNQKFKG gal (628) (629) (630) (631) (632) (633) SASSSIRYMH DTSKLTS QQDSSYPLT GYTFTSYDIN WIFPRDNNTKYNENFKG ENWVGDF (634) (635) (636) (637) (638) (639) KASQDVGTAVA WASTRHT QQYYGYPLT GYSITSDYWN YISYTGSTYYNPSLKS YGGWLSP (640) (641) (642) (643) (644) F(645) KSSQSLLYSSN WASTRES GYSIISDYWN RGGWLLP QKNSL (646) (589) (647) F(648) GFSLTTYSIN (649) VMWAGGGTNNSVLKS (650) YYGNSPY YAI (651)

Group Η

VL vh CDR1 (SEQ ID NO:) CDR2 (SEQID NO:) CDR3 (SEQID NO:) CDR1 (SEQID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQID NO:) TRSSSGSIGSNY VQ (652) EDNQRPS (653) QSYDSSTWV (654) SYAIS (301) WISPIGGSTNYAQK VQG (655) GLXXXXXXXXX XXXXXXDV (656) TRSSGNIASNY GKNNRPS QSYDSSNLWV SYGIS WISAYNGNTNYAQK ALPSGTILVGG VQ (657) (658) (659) (660) LED (661) WFDP(662) QGDSLRSYYAS SDRDRPS NSRDSSGNHYV SYALS AISGGGGSTYYADS DVFPETFSMNY (663) (664) (665) (666) VKD (667) GMDV(668) GGSDIGRKSVH (669) DVSNRPS (613) QVWDNNSDHYV (670) DYAMH (671) LISGDGGSTYYADS VKD (672) VLLPCSTSCY GSVGAFDI (673) TGTSSDVGGYN GASSRAT SSYTSSTLP NYDMS RVNWNGGSTTYADA EFVGAYDL YVS (612) (294) (674) (675) VKD (676) (677) RASQSIGNSLA AASTLQS QQHTIPTFS GLYIH WIIPIFGTANYAQK GLRWGGIWGWFD (678) (679) (680) (681) FED (682) P(683) RASQGIGSYLA QDIKRPS QQLNNYPIT DNAIS WIIPIFGKPNYAQK TMVRGFLGVMD (684) (685) (686) (687) FED (688) V (689)

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SGDKLGNKYAY (690) EDYRRPS (691) QTWDNSW(692) SYAMS (212) AISGSGGSTYYADS VKD (693) DQFVTIFGVPR YGMDV (694) TRSSGSIDSNY VQ (695) DDSDRPS (696) QSYDSNNNRHVI (697) TYALN (698) RIVPLIGLVNYAHN FED (699) GRQMFGAGIDF (700) TRSSGNIGTNY VQ (701) EDNKRPS (702) QSYHSSGWE (703) SHGIT (704) WISAHNGHASNAQK VED (705) EVYGGNSDY (706) GGNNIGSKGVH (707) YKSDSNKQ QAS (708) QVWDSSSSDHWV (709) RHGMH (710) VISHDGSVKYYADS MKD (711) VHAALYYGMDV (712) TRSSGSIASNY VQ (713) QSYDSTTPSV (714) WTSPHNGLTAFAQI LED (715) GLSYQVSGWFD P(716) TRSSGSIASHY VQ (717) QSYDGITVI (718) RIIPILGIANYAQK FED (719) VHPVFSYALDV (720) TLRSGLNVGSY R.I.Y. (721) QSYDSSNNRWV (722) TYAFS (723) WISAYNGNTNYAQK VED (724) DGYGSDPVL (725) MIWYSSAW (726) NYGIS (727) GDFRKPFDY (728)

Group 1 VL vh CDR1 (SEQID NO:) CDR2 (SEQID NO:) CDR3 (SEQID NO:) CDR1 (SEQ ID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQID NO:) RATESVEYYGT AASSVDS QQSRRVPYT SYVMH YVN P FNDGT KYNEMFKG QAWGYP SLVQ (729) (730) (731) (732) (733) (734)

Group J VL vh CDR1 (SEQID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQID NO:) CDR1 (SEQID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQ ID NO:) RASQDVSTAVA (333) SASFLYS (334) QQYLYHPAT (335) GFTFSDSWIH (336) AWISPYGGSTYYADSVKG (337) RHWPGGF DY(332)

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Group K VL vh CDR1 (SEQID NO:) CDR2 (SEQID NO:) CDR3 (SEQ ID NO:) CDR1 (SEQ ID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQID NO:) QSISNW (781) KAS (782) QQYHSYSYT (783) GFTFSRFW (784) INQDGTEK (785) ANTYYDFWSGH FDY (786) QGIRND (787) TAS (788) LQHNSYPLT (789) GFTFSNFG (790) LWSDGSNK (791) ARGRGAPGIPI FGY (792) QGIRND (787) AAS (793) LQHNNYPYT (794) GFTFSNAW (795) IKRKTDGGTT (796) TTDDIVWPAV MREYYFGMDV (797) QTLVHGDGNTY (798) KVS (799) MQATHFPIT (800) GYSFTGYY (801) INPNSGTK (802) ARDEDWNFGSW FDS(803) PSLVHSDGNTY (804) KIS (805) MQATHFPIT (800) GYTFTGYY (806) LNPNTGTT (807) ARDEDWNYGSW FDT (808) QSINSY(809) VAS (810) QQSYSTPPIT(811) GFTFDDYG (812) IHWHGKRT (813) VRGGMSTGDWF D.P. (814) QSISSY (815) VAS (810) QQSYSTPPIT(811) GFTFDDYG (812) IHWSGRST (816) ARGGMSTGDWF D.P. (817) QTINY (818) AAS (793) HQSYSTPPIT (819) GFTVGSNY (820) IYSGGST (821) ARGIRGLDV (822) QSMSSY(1011) AAS (793) QQSYSTPPIT (811) GITVGTNY (1012) ISSGGNT (1013) ARGIRGLDV (822) QSFNFNY (823) GAS (824) QQYESAPWT (825) GGIFSSST (826) IIPVFGTV (827) ARNWGGLSFYI (828) QSISSY (815) AAS (793) QQSYCTPPIT (829) GFPFDEYA (830) ISWSNNNI (831) AKSGIFDS (832) QSISSY (815) AAS (793) QQSYSTPPIT(811) GFTFSSYG (833) ISYEGRNK (834) AKDRTLYGMDV (835) QVISNY (836) AAS (793) QKYNSAPRT (837) GFSLSTNRMC (838) IDWDGVK (839) ARSTSLTFYYF DY(840) QNINNY (841) AAS (793) QQSYNTPLT (842) EFTVGTNH (843) IYSGGNT (844) ARGLGGMDV (845) QTISTY (846) AAS (793) LQHNSYPYT (847) GFTFSKYW (848) IKGDGSEK (849) ARDYWGSGYYF D.F. (850)

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QSISSY (815) WS (851) QQSYSTPFT (852) GFTFSSYW (853) IKQDGSEK (854) ARDDIVWPAP MGYYYYYFGMD V(855) AAS (793) QQSYSTPPIT (811) GFTFDDFA (856) ISWTGGNM (857) VKDIRGIVATG GAFDI (858) GFTVGTNY (859) IYSGGST (821) ARGIRGFDI (860) GFTISTNY (861) IYSSGST (862) ARGIRGFDI (860) GFTIDDSA (863) ISWKSGSI (864) VKDIRGNWNYG GNWFDP (865) GFTVGVNH (866) IFSSGRT (867) ARGIGGLDI (868) GFTFDDYA (869) ISWTGGTI (870) TRDIRGNWKYG GWFDP(871) GYTFTAYY (872) ISPNSGFT (873) AREGSTHHNSF D.P. (874) GFTVGTNF (875) IIPILGAA (876) ARGGGMDV (877) GGTFNTYV (878) ISPYNGYT (879) ARDRTSGGFDP (880) GYIFTHYG (881) SRGRGPYWSFD L(882)

Group L

VL vh CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 (SEQ ID (SEQ ID (SEQID (SEQ ID (SEQ ID NO:) (SEQID NO:) NO:) NO:) NO:) NO:) QASESVYSNNY LASTLAS IGGKSSSTDG SNGLT TINKDASAYYASWAK IAFKTGTSI LS (1014) (1015) N.A. (1016) (1017) G(1018) (1019) Group M VL vh CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 (SEQID (SEQ ID (SEQID (SEQ ID (SEQ ID NO:) (SEQID NO:) NO:) NO:) NO:) NO:)

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RSSKSLLHSNG ITYLY (1020) QMSNLAS (1021) AQNLEPPLT (1022) DYYTH (1023) WIDPENGKTAYAPKF QG (1024) GGYDVYFLDY (1025)

Group N VL vh CDR1 (SEQID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQ ID NO:) CDR1 (SEQID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQ ID NO:) KASQDVGIWA (1026) WASIRHT (1027) QQYSNYPLYT (1028) GFSLTSYGVH (1029) VIWAGGSTNYNSALM S (1030) AKPYGNSAMDY (1031) VIWAGGSTNYVDSVK G(1032) AKPYGTSAMDY (1033) VIWAGGSTNYADSVK G(1034)

Group 0 VL vh CDR1 (SEQID NO:) CDR2 (SEQID NO:) CDR3 (SEQ ID NO:) CDR1 (SEQ ID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQID NO:) ASQSVSTSSSS YASNLES QHSWEIPYT SYGMS SISSGGSTYYPDSVK GYDSGFAY FMH (1035) (1036) (1037) (1038) G(1039) (1040) RASQSVSTSSS YASNLES QHSWEIPYT SYGMS SISSGGTTYYPDSVK GYDSGFAY SYMH (1041) (1036) (1037) (1038) G(1042) (1040) KASQSVSNDVA YAANRYT QQDYTSPYT TYGVH VIWRGVTTDYNAAFM LGFYAMDY (1043) (1044) (1045) (1046) AS (1047) (1048) KASQSVSNDVG YASNRYS QQDYTSPYT SYGVH VIWSGGVTDYNAAFI LGFYAMDY (1049) (1050) (1045) (1051) S (1052) (1048) RSSQIIVHSNA KVSNRFS FQGSHVPYT TYWMH QINPDSTINTAPSL PGDYGYDFDC NTYLE (1054) (1055) (1056) KD (1057) (1058) (1053) SASSSVSSSYL NTSNLAS HQWRSYPPT SGYWN YISYSGSTYYNPSLK SLLWFSTGFAY Y (1059) (1060) (1061) (1062) S (1063) (1064) SANSSVSYMH DTSKLAS QQWSSNPWT SYGVH YIWSGGITDYNAAFK LGFYAMDY (1065) (1066) (1067) (1051) S (1068) (1048) RASQSVSTSSY YASNLES QNSWEIPYT STGMS SISSGGTTYYLGSVQ GYDAGFAY SYMH (1069) (1036) (1070) (1071) G(1072) (1073)

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KSSQSLLYSSN QKNSLA (1074) WASNRES (1075) QQYYSYPLT (1076) SGYWT (1077) YIYTGSTYYNPSLKS (1078) QRDWLGFAY (1079) RASQSVSTSSY SYVH (1080) YASNLES (1036) QHSWEIPYT (1037) SYGMS (1038) SISSGGSIYYPDSVK G(1081) GYDAGFAF (1082)

Group P VL vh CDR1 (SEQID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQID NO:) CDR1 (SEQID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQ ID NO:) GFTFSMYMMM (1083) GETFSAYAMA (1084) GFTFSAYRMF (1085) GFTFSAYLMV (1086) GFTFSAYVMF (1087) GFTFSAYVMS (1088) GFTFSGYLMV (1089) GFTFSGYQML (1090) GFTFSGYSMF (1091) GFTFSGYWMA (1092) GFTFSQYLMY (1093) GFTFSQYVMF (1094) GFTFSQYYMY (1095) GFTFSSYLMS (1096)

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GFTFSSYLMT (1097) GFTFSSYQMV (1098) GFTFSSYSMA (1099) GFTFSSYVMF (1100) GFTFSSYVMS (1101) GFTFSSYVMY (1102) GFTFSSYYMF (1103) GFTFSSYYMV (1104) GFTFSYYSMV (1105) GFTFSWYLMA (1106) GFTFSWYQMS (1107)

The AV in the activated antibodies of the present invention specifically binds to a PDL1 target, such as, for example, mammalian PDL1 and/or human PDL1. Also included in the invention are ABs that bind to the same PDL1 epitope as the antibody of the invention and/or the activated activable antibody described herein. Also included in the invention are ABs that compete with an anti-PDL1 antibody and/or an activated anti-PDL1 antibody described herein for binding to a PDL1 target, eg, human PDL1. Also included in the invention are ABs that cross-compete with an anti-PDL1 antibody and/or an activated anti-PDL1 activated antibody described herein for binding to a PDL1 target, eg, human PDL1.

The activated anti-PDL1 antibodies provided herein include a masking moiety. In some embodiments, the masking fragment is an amino acid sequence that is linked to or otherwise attached to an anti-PDL1 antibody and is located within an anti-PDL1 antibody to be activated such that the masking fragment reduces the ability of the anti-PDL1 antibody to specifically bind to PDL1. Suitable masking fragments are identified using any of a number of known methods. For example, peptide masking fragments are identified using the methods described in PCT International Application WO 2009/025846, Daugherty et al., the contents of which are incorporated herein by reference in its entirety.

The activated anti-PDL1 antibodies provided herein include a cleavable fragment. In some embodiments, the cleavable fragment includes an amino acid sequence that is a substrate for a protease, typically an extracellular protease. Suitable substrates are identified using any of a number of known methods. For example, peptide substrates are identified using the methods described in US Pat. No. 7,6666,817 to Daugherty et al.; in US patent No. 8563269, Stagliano et al.; and in PCT International Application Publication WO 2014/026136, La Porte et al., the content of each source is incorporated herein by reference in its entirety (see also Boulware et al. Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics , Biotechnol Bioeng 106.3 (2010): 339-46).

Typical substrates include, but are not limited to, substrates cleaved by one or more of the following enzymes or proteases listed in Table 12.

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Table 12 Exemplary Proteases and/or Enzymes

ADAMS, AD AMTS, cysteine proteases, Serine ADAM 8 Cruzipine activated protein C ADAM 9 Legumain Cathepsin A ADAM 10 Otubain-2 Cathepsin G ADAM 12 Himaz ADAM 15 KLKs, for example, protease factors ADAM 17/TACE KLK4 e.g. FVIIa, FIXa, FXa, FXIa, FXIIa) ADAMDEC1 KLK5 AD AMTS 1 KLK6 Elastase ADAMTS4 KLK7 Granzym B ADAMTS 5 KLK8 Guanidinobenzoatase KLK10 HtrAl aspartate protease, KLK11 Neutrophil elastase VACE KLK13 lactoferrin Renin KLK14 Marapsin NS 3/4 A Aspartic cathepsins, like: Metalloproteinases, napshmeg), RACE 4 Cathepsin D Meprin Plasmin Cathepsin E Neprilysin PSA PSMA tPA Caspases, for example: VMP-1 Thrombin Caspase 1 Tryptase Caspase 2 MMPs, for example, and RA Caspase 3 MMP1 Caspase 4 MMP2 Transmembrane serine proteases (TTSPs), e.g. type II Caspase 5 MMRP Caspase 6 MMP7 DESC1 Caspase 7 MMP8 DPP-4 Caspase 8 MMP9 FAP Caspase 9 MMP10 Hepsin Caspase 10 MMP11 Ma tryptase-2 Caspaza 14 MMP12 MT-SP1 /Matriptase MMP13 TMPRSS2 cysteine cathepsins, MMP14 TMPRSS3 Cathepsin B MMP15 TMPRSS4 Cathepsin C MMP16 Cathepsin K MMP17 Cathepsin L MMP19 Cathepsin S MMP2 0 Cathepsin V/L2 MMP23 Cathepsin X/Z/P MMP24 MMP2 6 MMP27

The activated anti-PDL1 antibodies described herein overcome the limitations of antibody therapeutics, in particular antibody therapeutics known to be at least somewhat toxic in vivo. Target-mediated toxicity represents a major limitation for the development of such therapeutic antibodies. The activated anti-PDL1 antibodies provided herein are designed to eliminate the toxicity associated with target inhibition in normal tissues by traditional therapeutic antibodies. These activated anti-PDL1 antibodies remain masked until they are proteolytically activated at the disease site. Initially starting with an anti-PDL1 antibody as the parent therapeutic antibody, activated anti-PDL1 antibodies were constructed according to

- 78 039736 of the invention by linking the antibody to the inhibitory mask through a linker that includes a substrate for the protease.

When AB is modified with MM and is in the presence of a target, specific binding

AV with its target is reduced or inhibited compared to specific binding of AV, unmodified MM, or specific binding of the parent AV to the target.

K _d value of MM-modified AB targeting at least 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000, 000000 times or more higher or 5-10, 10-100, 10-1000, 10-10000, 10-100000, 10-1000000, 10-10000000, 100-1000, 100-10000, 100-100000, 100-1000000, 100 -10000000, 1000-10000, 1000-100000, 1000-1000000, 1000-10000000, 10000100000, 1000-1000000, 1000-10000000, 100000-1000000 or 100000-10000,000 times higher than KD AB, non-modified mm, or parent Av s. target. In contrast, the binding affinity of the MM-modified AB to the target is at least 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, 50000, 100000, 500000, 1000000 . 1001000000, 100-10000000, 1000-10000, 1000-100000, 1000-1000000, 1000-10000000, 10000-100000, 100001000000, 1000-10000000, 100000-1000000 or 100000-10000,000 times lower than the affiliation of binding with the target of AB, unmodified MM, or parent AB.

In some embodiments, the dissociation constant (K _d ) of MM with AB is typically higher than the K _d of AB with the target. The K _d MM with AB can be at least 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, 100000, 1000000 or even 10000000 times higher than the K _d AB with the target. On the other hand, the binding affinity of MM to AB is usually lower than the binding affinity of AB to the target. The binding affinity of MM to AB can be at least 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, 100000, 1000000 or even 10000000 times lower than the binding affinity of AB to the target.

In some embodiments, the dissociation constant (Kd) of MM with AB is approximately equal to the Kd of AB with the target. In some embodiments, the dissociation constant (Kd) of MM with AB is not higher than the dissociation constant of AB with the target.

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

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

In some embodiments, the implementation of the MM has a K _d value for binding to AB, which is not higher than the value of K d for binding AB to the target.

In some embodiments, the implementation of the MM has a Kd value for binding to AB, which is not lower than the Kd value for binding AB to the target.

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

In some embodiments, the implementation of the MM has a Kd value for binding to AB, which is lower than the K _d value for binding AB to the target.

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

In some embodiments, MM has a K _d for binding to AB that is no more than 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1000 times higher than the K _d for binding AB to a target . In some embodiments, MM has a K _d for binding to AB that is 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5100, 10-100, 10-1000, 20 -100, 20-1000 or 100-1000 times higher than the K _d for AB binding to the target.

In some embodiments, the MM has a binding affinity for AB that is lower than the binding affinity of AB for the target.

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

In some embodiments, the MM has a binding affinity for AB that is approximately equal to the binding affinity of AB for the target.

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

In some embodiments, the MM has a binding affinity for AB that is higher than the binding affinity of AB for the target.

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

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

MM inhibits AV binding to the target. MM is associated with the antigen-binding site of AV and inhibits AV binding to the target. MM can sterically inhibit AV binding to the target. MM can allosterically inhibit the binding of AV to its target. In these embodiments, when the AV is modified or bound to the MM and in the presence of the target, there is no binding, or essentially no binding of the AB to the target, or no more than 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 50% target binding of AB compared to target binding of AB, unmodified MM, parental AB or AV not associated with MM 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, or 12 months or more, as determined in an in vivo assay or in vitro.

When an AV is bound or modified by an MM, the MM masks or reduces or otherwise inhibits the specific binding of the AV to the target. When an AV is bound or modified by MM, such linkage or modification can result in a structural change that reduces or inhibits the ability of the AV to specifically bind to its target.

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

(MM) - (AB) (AB) - (MM) (MM)-L-(AB) (AB)-L-(MM) where MM is a masking fragment, AB is an antibody or a fragment of this 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 flexibility.

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

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

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

Kd value of AB modified with MM and SM, targeting at least 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000, 0.00000 50000000 times or more or 5-10, 10-100, 10-1000, 10-100000, 10-1000000, 10-10000000, 100-1000, 100-10000, 100100000, 100-1000000, 100-0.0 1000-10000, 1000-100000, 1000-1000000, 1000-10000000, 10000100000, 10000-1000000, 10000-10000000, 100000-1000000 or 100000 AB d

- 80 039736 unmodified MM and SM, or parent AB, with target. In contrast, the binding affinity of AB modified with MM and SM to the target is at least 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000, 1000000. , 50000000 times or more or 5-10, 10-100, 10-1000, 10-10000, 10-10-1000000, 10-10000000, 100-1000, 100-10000, 100-100000, 100 1000, 100 -100000, 1000-1000000, 1000-000, 10000-100000 times lower than the target binding affinity of AB, unmodified MM and SM, or the parent AB.

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

As used here, the term split state refers to the state of activated antibodies after modification of the SM at least one protease. As used here, the term uncleaved state refers to the state of activated antibodies in the absence of cleavage by the CM protease. As discussed above, the term activated antibodies is used herein to refer to an activated antibody in both its uncleaved (native) state and its cleaved state. One of skill in the art will appreciate that in some embodiments, the cleaved, activated antibody may lack MM as a result of CM protease cleavage, resulting in the release of at least MM (e.g., when the MM is not covalently bonded to the activated antibody (e.g., , disulfide bond between cysteine residues).

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

The CM and AB of the activated antibodies are chosen such that AB is the binding site for the target and CM is the substrate for the protease. In some embodiments, the protease is co-targeted at a therapeutic or diagnostic lesion in a subject. As used here, co-localization refers to a situation where they are in the same place or relatively close together. In some embodiments, the protease cleaves the CM to form an activated antibody that binds to a target adjacent to the cleavage site. The activated antibodies described herein find their particular use when, for example, a protease capable of cleaving a site in the CM, ie. the protease is present at relatively higher levels or in close proximity to the target tissue of a lesion to be treated or diagnosed than in tissues of untreated sites (eg, healthy tissue). In some embodiments, the CM of the invention is also cleaved by one or more different proteases. In some embodiments, it is one or more different proteases that co-localize with the target and that are responsible for the cleavage of CM in vivo.

In some embodiments, activated antibodies provide reduced toxicity and/or reduced adverse side effects that might otherwise result from binding of the AB to untreated sites if the AB were not masked or otherwise inhibited from binding to the target.

In general, an activated antibody can be designed by selecting an AB of interest and designing the remainder of the activated antibody such that when they are conformationally restricted, MM masks the AB or reduces binding of the AB to its target. Structural design criteria may be taken into account to provide such functional performance.

Activated antibodies are provided having a switchable phenotype in the desired dynamic range to bind the target in an inhibited versus uninhibited conformation. Dynamic range generally refers to the ratio of (a) the maximum detected level of a parameter under the first set of conditions to (b) the minimum detected value.

- 81 039736 this parameter under the second set of conditions. For example, in the context of an activated antibody, dynamic range refers to the ratio of (a) the maximum detected level of binding of the target protein to the activated antibody in the presence of at least one protease capable of cleaving the CM of the activated antibody to (b) the minimum detected level of binding of the target protein to activated antibody in the absence of a protease. The dynamic range of an activated antibody can be calculated as the ratio of the dissociation constant when an activated antibody is treated with a cleaving agent (eg, an enzyme) to the dissociation constant when activated antibodies are treated with a cleaving agent. The greater the dynamic range of the activated antibody, the better the switchable phenotype of the activated antibody. Activated antibodies having relatively higher dynamic range values (e.g., greater than 1) exhibit more desirable switching phenotypes such that the activated antibodies bind to the target protein to a greater extent (e.g., predominantly) in the presence of a cleaving agent (e.g., an enzyme ) capable of cleaving the CM of activated antibodies than in the absence of a cleaving agent.

Activated antibodies can be provided in various structural configurations. Exemplary formulas for activated antibodies are shown below. In particular, it is contemplated that the N-terminal to C-terminal order of AB, MM, and CM within an activated antibody can be reversed. Also, in particular, it is envisaged that the SM and MM may overlap in amino acid sequence, for example, so that the SM is within the MM.

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

(MM) - (CM) - (AB) (AB) - (CM) - (MM) where MM is a masking fragment, CM is a cleavable fragment, and AB is an antibody or fragment thereof. It should be noted that while MM and CM are designated as separate components in the formulas above, in all exemplary embodiments (including formulas) disclosed herein, it is contemplated that the amino acid sequences of MM and CM may overlap, for example, by having CM wholly or partly included in the MM. In addition, the above formulas provide additional amino acid sequences that can be located N-terminal or C-terminal to the elements of the activated antibodies.

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

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

For example, in some embodiments, the activated antibody has one of the following formulas (wherein the formulas below represent the amino acid sequence in N- to C-terminal or C- to N-terminal direction):

(MM) -L1- (CM) - (AB) (MM) - (CM) -L2- (AB) (MM) -LI - (CM) -L2 - (AB) where MM, CM and AB have the meanings, defined above; where L1 and L2 are each independently and optionally

- 82 039736 necessarily present or absent, being the same or different flexible linkers, which include at least 1 flexible amino acid (for example, Gly). In addition, the formulas above provide additional amino acid sequences that can be positioned N-terminal or C-terminal to the activated antibody elements. Examples include, but are not limited to, targeting moieties (e.g., a ligand for a receptor on a cell located in a target tissue) and moieties that increase serum half-lives (e.g., polypeptides that bind serum proteins such as immunoglobulin (e.g., IgG) or serum albumin (eg human serum albumin (HAS)).

CM is specifically cleaved by at least one protease at a rate of about 0.0011500x104 M ^-1 s ^-1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250 or 1500x104 M ^-1 s ^-1 . In some embodiments, CM is specifically cleaved at a rate of about 100,000 M ^-1 s ^-1 . In some embodiments, CM is specifically cleaved at a rate of about 1x10E2 to about 1x10E6 (i.e., about ^1x102 to about 1x106 M ^-1 s ^-1 .

For specific cleavage by the enzyme, contact is made between the enzyme and the CM. When an activated antibody containing an AB associated with MM and SM is in the presence of a target and in the presence of sufficient enzyme activity, then SM can be cleaved. Sufficient enzyme activity may refer to the ability of the enzyme to contact the CM and allow degradation. It can be easily assumed that the enzyme can be located near the SM, but will not be able to cleave it due to other cellular factors or modification of the enzyme protein.

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

Representative flexible linkers include (G)n glycine polymers, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 191) and (GGGS)n (SEQ ID NO: 192), where n is an integer, at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine based polymers are relatively unstructured and therefore can serve as a neutral bond between the components. Glycine gets significantly more phi-psi space than even alanine, and is significantly less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem., 11173-142 (1992)). Examples of flexible linkers include, but are not limited to, Gly-Gly-Ser-Gly (SEQ ID NO: 193), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 194), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 195), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 196), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 197), Gly-Ser-Ser-Ser- Gly (SEQ ID NO: 198) and the like. One of ordinary skill in the art understands that the design of activated antibodies may include linkers that are all or part of the flexible, such that the linker may include a flexible linker, as well as one or more regions that give a less flexible structure, to provide the desired structure of the activated antibodies.

The present invention also provides compositions and methods that include an activated anti-PDL1 antibody that includes an antibody or antibody fragment (AB) that specifically binds to PDL1, where the AB is associated with a masking fragment (MM) that reduces the ability of the AB to bind to its target. In some embodiments, the activated anti-PDL1 antibody also includes a cleavable fragment (CM) that is a substrate for the protease. The compositions and methods provided herein allow one or more agents to be attached to one or more cysteine residues in an AB without adversely affecting the activity (eg, masking, activating, or binding activity) of an activated anti-PDL1 antibody. In some embodiments, the compositions and methods provided herein allow one or more agents to be attached to one or more cysteine residues in the AB, without reducing or otherwise breaking one or more disulfide bonds in the MM. The compositions and methods provided herein make it possible to obtain an activated anti-PDL1 antibody that is conjugated to one or more agents, e.g., any of various therapeutic, diagnostic, and/or prophylactic agents, e.g., in some embodiments, without any or an agent(s) that is conjugated to an MM of an activated anti-PDL1 antibody. The compositions and methods provided herein make it possible to prepare conjugated anti-PDL1 activated antibodies in which MM retains the ability to efficiently and optimally mask the AV activated antibody in the uncleaved state. The compositions and methods provided herein make it possible to prepare conjugated anti-PDL1 activated antibodies, wherein the activated antibody is still activated, i.e. cleaved in the presence of a protease that can cleave SM.

Activated anti-PDL1 antibodies have at least one conjugation point for an agent, but in the methods and compositions provided herein, almost all possible conjugation points are available for conjugation with an agent. In some embodiments, one or more conjugation points are sulfur atoms involved in the formation of disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms involved in the formation of interchain disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms involved in the formation of interchain sulfide bonds, but not sulfur atoms involved in the formation of intrachain disulfide bonds. In some embodiments, one or more conjugation points are cysteine sulfur atoms or other amino acid residues containing a sulfur atom. Such residues may occur naturally in the structure of the antibody, or may be incorporated into the antibody by site-directed mutagenesis, chemical transformation, or misplacement of unnatural amino acids.

Also provided are methods for preparing an activated anti-PDL1 antibody conjugate comprising one or more interchain disulfide bonds in AB and one or more intrachain disulfide bonds in MM, and a free thiol-reactive drug. The method typically includes partial reduction of interchain disulfide bonds in the activated antibody using a reducing agent such as TCEP; and conjugating the free thiol-reactive drug to the partially reduced, activated antibody. As used herein, the term partial recovery refers to situations where an activated anti-PDL1 antibody contacts a reducing agent and is reduced to less than all disulfide bonds, eg, less than all possible conjugation sites. In some embodiments, less than 99, 98, 97, 96, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 40, 35, 30, 25, 20, 15, 10 or less 5% of all possible conjugation sites.

In still other embodiments, a method is provided for reconstituting and conjugating an agent, such as a drug, with an activated anti-PDL1 antibody resulting in selective placement of the agent. The method typically involves partially reducing the activated anti-PDL1 antibody with a reducing agent such that any conjugation sites in the masking or other non-AB fragment of the activated antibody are not reduced, and conjugating the agent to the interchain thiols in the AB. The conjugation site(s) are chosen to provide the desired placement of the agent in order for conjugation to take place at the desired site. The reducing agent is, for example, TCEP. Conditions of the reduction reaction, for example, such as the ratio of reducing agent to activated antibody, incubation time, temperature during incubation, pH of the reducing reaction solution, etc., are determined by identifying the conditions under which a conjugated activated antibody is prepared in which MM retains the ability to effectively and optimally mask the AV-activated antibody in the uncleaved state. The ratio of reducing agent to activated anti-PDL1 antibody will vary depending on the activated antibody. In some embodiments, the ratio of reducing agent to activated anti-PDL1 antibody will range from about 20:1 to 1:1, about 10:1 to 1:1, about 9:1 to 1:1, about 8:1 to 1:1, about 7:1 to 1:1, about 6:1 to 1:1, about 5:1 to 1:1, about 4:1 to 1:1, about 3:1 to 1:1, about 2:1 to 1:1, about 20:1 to 1:1.5, about 10:1 to 1:1.5, about 9:1 to 1: 1.5, about 8:1 to 1:1.5, about 7:1 to 1:1.5, about 6:1 to 1:1.5, about 5:1 to 1:1, 5, about 4:1 to 1:1.5, about 3:1 to 1:1.5, about 2:1 to 1:1.5, about 1.5:1 to 1:1, 5 or about 1:1 to 1:1.5. In some embodiments, the implementation of the ratio is in the range from about 5:1 to 1:1. In some embodiments, the implementation of the ratio is in the range from about 5:1 to 1.5:1. In some embodiments, the implementation of the ratio is in the range from about 4:1 to 1:1. In some embodiments, the implementation of the ratio is in the range from about 4:1 to 1.5:1. In some embodiments, the ratio is in the range of about 8:1 to about 1:1. In some embodiments, the ratio is in the range of about 2.5:1 to about 1:1.

In some embodiments, a method is provided for reducing interchain disulfide bonds in an AV of an activated anti-PDL1 antibody and conjugating an agent, e.g., a thiol-containing agent such as a drug, with the resulting interchain thiols to selectively locate the agent(s) in the AV. The method typically includes partial reduction of AB with a reducing agent to form at least two interchain thiols without generating all possible interchain thiols in the activated antibody; and conjugation of the agent with interchain thiols of the partially reduced AB. For example, the AV of an activated antibody body is partially reduced over about 1 hour at a temperature of about 37°C at the desired ratio of reducing agent:activated antibody. In some embodiments, the ratio of reducing agent to activated antibody will be in the range of about 20:1 to 1:1, about 10:1 to 1:1, about 9:1 to 1:1, about 8:1 to 1:1, about 7:1 to 1:1, about 6:1 to 1:1, about 5:1 to 1:1, about 4:1 to 1:1, about 3:1 to 1:1, about 2:1 to 1:1, about 20:1 to 1:1.5, about 10:1 to 1:1.5, about 9:1 to 1:1.5, about 8:1 to 1:1.5, about 7:1 to 1:1.5, about 6:1 to 1:1.5, about 5:1 to 1:1.5, about 4:1 to 1:1.5, about 3:1 to 1:1.5, about 2:1 to 1:1.5, about 1.5:1 to 1:1.5, or about 1:1 to 1:1.5. In some embodiments, the implementation of the ratio is in the range from about 5:1 to 1:1. In some embodiments, the implementation of the ratio is in the range from about 5:1 to 1.5:1. In some embodiments, the implementation of the ratio is in the range from about 4:1 to 1:1. In some embodiments, the implementation of the ratio is in the range from about 4:1 to 1.5:1. In some embodiments, the ratio is in the range of about 8:1 to about 1:1. In some embodiments, the implementation of the ratio is in the range from about 2.5:1 to 1:1.

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

The invention also relates to partially reduced activated anti-PDL1 antibodies in which at least one interchain disulfide bond in the activated antibody has been restored by a reducing agent without destroying any intrachain disulfide bonds in the activated antibody, where the activated antibody comprises the antibody or an antigen-binding fragment thereof (AB) that specifically binds to PDL1, a masking fragment (MM) that inhibits the binding of an AV activated antibody in an uncleaved state to a PDL1 target, and a cleavable fragment (CM) linked to AB, where CM is a polypeptide that functions in as a substrate for the protease. In some embodiments, the MM is connected to the AB via the CM. In some embodiments, one or more intrachain disulfide bonds of the activated antibody are not broken by the reducing agent. In some embodiments, one or more intrachain disulfide bonds (MM) in the activated antibody are not broken by the reducing agent. In some embodiments, the activated antibody in the uncleaved state has the following structural organization from the N-terminus to the C-terminus: MM-CM-AB or AB-CM-MM. In some embodiments, the reducing agent is TCEP.

The present invention also provides partially reduced activated antibodies wherein at least one interchain disulfide bond in the activated antibody has been reduced by a reducing agent without destroying any intrachain disulfide bonds in the activated antibody, wherein the activated antibody comprises an antibody or an antigen binding fragment (AB) that bind specifically to a target, for example, PDL1, a masking fragment (MM) that inhibits binding of an AV activated antibody in an uncleaved state to a target, and a cleavage fragment (CM) associated with an AB, where CM is a polypeptide that functions as a substrate for at least least for one protease. In some embodiments, the MM is connected to the AB via the CM. In some embodiments, one or more intrachain disulfide bonds of the activated antibody are not broken by the reducing agent. In some embodiments, one or more intrachain disulfide bonds (MM) in the activated antibody are not broken by the reducing agent. In some embodiments, the activated antibody in the uncleaved state has the following structural organization from the N-terminus to the C-terminus: MM-CM-AB or AB-SMMM. In some embodiments, the reducing agent is TCEP.

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

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

In some embodiments, the agent is a detectable group, such as

- 85 039736 as a label or other marker. For example, the agent is or includes a radiolabeled amino acid, one or more biotinyl groups that can be detected using labeled avidin (for example, streptavidin containing a fluorescent label or having an enzymatic activity that can be detected by optical or calorimetric methods), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzymatic labels, and/or one or more chemiluminescent agents. In some embodiments, the detection groups are attached via spacer molecules.

The invention also relates to immunoconjugates containing an antibody conjugated to a cytotoxic agent, such as a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof) or a radioactive isotope (ie, a radioconjugate). Suitable cytotoxic agents include, for example, dolastatins and their derivatives (eg, auristatin E, AFP, MMAF, MMAE, MMAD, DMAF, DMAE). For example, the agent is monomethylauristatin E (MMAE) or monomethylauristatin D (MMAD). In some embodiments, the agent is an agent selected from the group shown in Table. 11. In some embodiments, the agent is dolastatin. In some embodiments, the agent is auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is pyrrolobenzodiazepine. In some embodiments, the agent is a dimer of pyrrolobenzodiazepine.

In some embodiments, the agent binds to the AV using a caproyl-valine-citrulline maleimide linker or a PEG-valine-citrulline maleimide linker. In some embodiments, the agent binds to the AB using a caproyl-valinecitrulline maleimide linker. In some embodiments, the agent binds to the AV using a PEG-valine-citrulline maleimide linker. In some embodiments, the agent is AB-linked monomethylauristatin D (MMAD) using a PEGvaline-citrulline-aminobenzyloxycarbonyl maleimide linker, and this payload linker construct is referred to herein as vc-MMAD. In some embodiments, the agent is AB-linked monomethylauristatin E (MMAE) using a PEG-valinecitrulline-p-aminobenzyloxycarbonyl maleimide linker, and this payload linker construct is referred to herein as vc-MMAE. The vc-MMAD and vc-MMAE structures are shown below:

vc-MMAD

vc-MMAE

Kg:

....( O··'

The invention also provides conjugated activated antibodies that comprise an activated monomethylauristatin D (MMAD) payload-associated antibody, wherein the activated antibody comprises an antibody or an antigen-binding fragment (AB) thereof that specifically binds to a target, a masker (MM) that inhibits AB binding. an activated antibody in an uncleaved state with a target, and a cleavable fragment (CM) associated with AB, and CM is a polypeptide that functions as a substrate for at least one MMP protease.

In some embodiments, the MMAD-conjugated activated antibody may

- 86 039736 be conjugated using any of several methods of attachment of agents to AB: (a) attachment to the carbohydrate fragments of AB, or (b) attachment to the sulfhydryl groups of AB, or (c) attachment to the amino groups of AB, or (d) attachment to the carboxylate groups AB.

In some embodiments, the MMAD payload is conjugated to the AB via a linker. In some embodiments, the MMAD payload is conjugated to the cysteine in the AB via a linker. In some embodiments, the MMAD payload is conjugated to the lysine in the AB via a linker. In some embodiments, the MMAD payload is conjugated to another AB residue via a linker, such as those residues described herein. In some embodiments, the linker is a thiol-containing linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is selected from the group consisting of the linkers shown in Tables 13 and 14. In some embodiments, the activated antibody and the MMAD payload are linked via a caproyl-valinecitrulline maleimide linker. In some embodiments, the activated antibody and the MMAD payload are linked via a PEG-valine-citrulline maleimide linker. In some embodiments, the activated antibody and the MMAD payload are linked via a caproyl-valine-citrulline-para-aminobenzyloxycarbonyl maleimide linker. In some embodiments, the activated antibody and the MMAD payload are linked via a PEG-valinecitrulline-para-aminobenzyloxycarbonyl maleimide linker. In some embodiments, the MMAD payload is conjugated to the AB using the partial reduction and conjugation technology disclosed herein.

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

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

Enzymatically active toxins and their fragments that can be used include diphtheria toxin A chain, non-binding diphtheria toxin active fragments, exotoxin A chain (Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, proteins Aleurites fordii, diantin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Chinese bitter gourd (Momordica charantia) inhibitor, curcin, crotin, soapwort (Sapaonaria officinalis) inhibitor, gelonin, mitogellin, restrictocin, fenomycin, enomycin and trichothecenes. There are many available radionuclides for the preparation of radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ 1, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re.

Antibody-cytotoxic agent conjugates are prepared using a variety of bifunctional protein binding agents such as K-succinimidyl-3-(2pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyladipimidate HCL), active esters ( such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(pazidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(pdiazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene-2,6 -diisocyanate) and active fluorine bis compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetfa et al., (1987) Science, 238:1098. An exemplary chelating agent for conjugating a radionuclide to an antibody is carbon-14 labeled 1-isothiocyanatebenzyl-3methyldiethylenetriaminepentaacetic acid (MX-DTPA) (see WO 94/11026).

Table 11 lists some of the exemplary pharmaceutical agents that may be used in the disclosure described herein, but is by no means an exhaustive list.

Table 11 Exemplary Pharmaceutical Conjugators

Cytotoxic agents Auristatins Auristatin E Monomethylauristatin D (MMAD) Monomethylauristatin E (MMAE) Desmethylauristatin E (DMAE) Auristatin F Monomethylauristatin F (MMAF) Desmethylauristatin F (DMAF) Derivatives of auristatin, e.g. its amides Auristatin tyramine Turbostatin Fenstatins Hydroxifenstatin Spongistatin 5 Spongistatin 7 Galistatin 1 Galistatin 2 Galistatin 3 Modified bryostatins Halocomstatins Auristatin quinoline Dolastatin Dolastatin derivatives Dolastatin 16 DmJ Dolastatin 16 Dpv Maytansinoids, eg DM-1; DM-4 Maytansinoid derivatives Duocarmycin Duocarmycin derivatives Alpha-amanitin Anthracyclines Doxorubicin Daunorubicin Briostatins Camptothecin Captothecin derivatives 7-substituted camptothecin 10,11difluoromethylenedioxycamptothecin Combretastatins Debromaplysiatoxin Kagalalid-F Discodermolide Ecteinascidins Antivirals Acyclovir Vira A Symmetrel Antifungal drugs Pyrrolobenzimidazoles (PBI) Cybrostatin 6 Doxaliform Anthracycline analogues Cemadotin analog (CemCH2-SH) Pseudomonas toxin A variant (PE38) Pseudomonas toxin variant A (ZZPE38) ZJ-101 OSW-1 4-nitrobenzyloxycarbonyl derivatives of 06-benzylguanine Topoisomerase inhibitors Hemiasterlin Cephalotaxin Homogarringtonin Dimers of pyrrolobenzodiazepine (PBD) Pyrrolobenzodiazepenes Functionalized pyrrolobenzodiazepenes Dimers of functionalized pyrrolobenzodiazepenes Calicheamycins Podophyllotoxins Taxanes Vinca alkaloids Conjugated reagents for detection Fluorescein and its derivatives Fluorescein isothiocyanate (FITC) Radiopharmaceuticals Nystatin Additional anticancer drugs Adriamycin Cerubidin Bleomycin Alkeran Velban Oncovin Fluorouracil Methotrexate Thiope Bisanthren Novantron Thioguanine Procarbazine Cytarabine Antibacterial drugs 125j 131j 89 _Zr ^w 1p 123j 1311 mTc 201.jp ¹³³ Xe 11 _C ⁶² Cu 18р ⁶⁸ Ga ¹³ N ⁱ⁵ ₀ ³⁸ K ⁸² Rb mTc (technetium) Aminoglycosides Streptomycin Neomycin Kanamycin Amikacin Gentamicin

- 88 039736

Tobramycin Streptomycin B Spectinomycin Ampicillin Sulfonamide Polymyxin Chloramphenicol Heavy metals Barium Gold Platinum Antimycoplasma drugs Tylosin Spectinomycin

Those skilled in the art will recognize that a large number of possible molecules can be associated with the resulting antibodies of the invention (see, e.g., Conjugate Vaccines, Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, New York (1989), the contents of this source are incorporated here by reference in their entirety).

Coupling can be accomplished by any chemical reaction that will bind two molecules, provided that the antibody and the other molecule retain their respective activity. This bonding may involve a variety of chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordination bonding, and complexation. In some embodiments, the preferred bonding, however, is covalent bonding. Covalent bonding can be achieved by direct condensation of existing side chains or by the introduction of external bridging molecules. Many bivalent or polyvalent binding agents are useful for binding protein molecules, such as the antibodies of the present invention, to other molecules. For example, exemplary coupling agents may include organic compounds such as thioethers, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylenediamines. This list is not intended to be exhaustive of the various classes of binders known in the art, but rather is an example of the most commonly used binders (see Killen and Lindstrom, Jour. Immun., 133: 1335-2549 (1984) , Jansen et al., Immunological Reviews, 62: 185-216 (1982) and Vitetta et al., Science, 238: 1098 (1987).

In some embodiments, in addition to the compositions and methods provided herein, the conjugated activated antibody may also be modified for site-specific conjugation by modified amino acid sequences inserted or otherwise incorporated into the sequence of the activated antibody. Such modified amino acid sequences are designed to provide controlled placement and/or dosing of the conjugate agent in the conjugated, activated antibody. For example, an activated antibody can be designed to include cysteine substitutions at light and heavy chain positions that provide reactive thiol groups and do not adversely affect protein folding and assembly or alter antigen binding. In some embodiments, an activated antibody can be designed to incorporate or otherwise introduce one or more non-natural amino acid residues into the activated antibody to provide suitable sites for conjugation. In some embodiments, an activated antibody can be designed to incorporate or otherwise introduce enzymatically activated peptide sequences into the sequence of an activated antibody.

Suitable linkers are described in the literature (see, for example, Ramakrishnan S. et al., Cancer Res., 44: 201-208 (1984), which describes the use of MBS (m-maleimidobenzoyl-Nhydroxysuccinimide ester). See also patent No. 5,030,719, which describes the use of a halogenated acetylhydrazide derivative linked to an antibody via an oligopeptide linker In some embodiments, suitable linkers include: (i) EDC (1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride, (ii) SMPT (4 -succinimidyloxycarbonyl-alphamethyl-alpha-(2-pyridyldithio)toluene (Pierce Chem. Co., Cat (21558G), (iii) SPDP (succinimidyl-6-[3-(2pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat # 21651G); NHS (N-hydroxysulfosuccinimide: Pierce Chem Co., No. 24510) conjugated to EDC Additional linkers include, but are not limited to That is, SMCC, sulfo-SMCC, SPDB or sulfo-SPDB.

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

- 89 039736 bodiimides.

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

In the present invention, several methods of attachment of agents to AB are used: (a) attachment to the carbohydrate moieties of AB, or (b) attachment to the sulfhydryl groups of AB, or (c) attachment to the amino groups of AB, or (d) attachment to the carboxylate groups of AB. According to the present invention, the AB can be covalently attached to the agent through an intermediate linker containing at least two reactive groups, one of which reacts with AB and one reacts with the agent. The linker, which may include any compatible organic compound, may be chosen such that reaction with the AB (or agent) does not adversely affect the reactivity and selectivity of the AB. In addition, the attachment of the linker to the agent should not interfere with the activity of the agent. Suitable linkers for reaction with oxidized antibodies or oxidized antibody fragments include linkers that contain an amine selected from the group consisting of primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide, and thiosemicarbazide groups. Such reactive functional groups may exist as part of the linker structure or may be introduced by appropriate chemical modification of linkers that do not contain such groups.

According to the present invention, suitable linkers for attachment to reduced ABs include linkers that have certain reactive groups capable of interacting with the sulfhydryl group of the reduced antibody or fragment thereof. Such reactive groups include, but are not limited to: reactive haloalkyl groups (including, for example, haloacetyl groups), p-mercurbenzoate groups, and groups capable of Michael addition reactions (including, for example, maleimides and groups of the type described by Mitra and Lawton, 1979 , J. Amer. Chem. Soc., 101: 3097-3110).

According to the present invention, suitable linkers for attachment to Ab that are neither oxidized nor reduced include linkers that have certain functional groups capable of interacting with the primary amino groups present in the unmodified lysine residues in the Ab. Such reactive groups include, but are not limited to, NHS carboxylic acid esters or carboxylic esters, sulfo-NHS carboxylic acid esters or carboxylic esters, 4-nitrophenyl carboxylic acid esters or carboxylic esters, pentafluorophenyl carboxylic acid esters or carboxylic esters, acylimidazoles, isocyanates, and isothiocyanates. .

According to the present invention, suitable linkers for attachment to Ab that are neither oxidized nor reduced include linkers that have certain functional groups capable of interacting with carboxyl groups present on aspartate or glutamate residues in Ab that have been activated with suitable reagents. Suitable activating agents include EDC, with or without the addition of NHS or sulfo-NHS, and other dehydrating agents used to form carboxamide. In these instances, functional groups present on suitable linkers include primary and secondary amines, hydrazines, hydroxylamines, and hydrazides.

The agent may be attached to the linker before or after the linker is attached to the AB. In some applications, it may be desirable to first prepare an AB linker intermediate in which the linker does not contain a bound agent. Depending on the particular application, then a particular agent may be covalently attached to the linker. In some embodiments, the AB is first attached to the MM, SM, and related linkers and then attached to the linker for conjugation purposes.

branched linkers.

In particular embodiments, branched linkers are used that have multiple sites for agent attachment. For linkers with multiple sites, a single covalent attachment to AB will result in an ABlinker intermediate capable of binding the agent at multiple sites. The sites may represent aldehyde or sulfhydryl groups, or any chemical site to which agents may be attached.

In some embodiments, higher specific activity (or higher ratio of agents to AV) can be achieved by attaching a single site linker to multiple sites in the AV. This set of sites can be entered into AV in one of two ways. In the first way, it is possible to obtain several aldehyde groups and/or sulfhydryl groups in the same AB. In the second method, a branched linker having multiple functional sites can be attached to the aldehyde or sulfhydryl AB for subsequent attachment to the linkers. The functional sites of a branched or multi-site linker may be aldehyde or sulfhydryl groups, or may be any chemical site to which linkers may be attached. Even higher specific activities can be obtained by combining these two approaches, ie by adding linkers with multiple sites to multiple sites in the AB.

Cleavable Linkers.

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

Non-limiting examples of cleavable linker sequences are shown in table. 13.

Table 13. Exemplary linker sequences for conjugation

Types sequences fissile Amino acid subsequence sequences, fissile plasmin Prourokinase PRFKIIGG PRFRIIGG (SEQ (SEQ ID ID NO: NO: 396) 397) TGFP SSRHRRALD (SEQ ID NO: 398) Plasminogen RKS S111RMRDVVL ( SEQ ID NO: 399) Staphylokinase SSSFDKGKYKKGDDA (SEQ ID NO: 400) SSSFDKGKYKRGDDA (SEQ ID NO: 401) sequences, fissile IEGR (SEQ ID NO: 402) factor Ha IDGR(SEQ GGSIDGR( ID NO: SEQ ID 403) 40:404) sequences, fissile World Gelatinase A PLGLWA (SEQIE NO: 405) sequences, fissile collagenase Calf skin collagen (al(I) chain) GPQGIAGQ (SEQ ID NO: 406) Calf skin collagen (chain α2 (I)) GPQGLLGA (SEQ ID NO: 407) Calf cartilage collagen (al(II) chain) GIAGQ (SEQ ID) NO 408) Human liver collagen (al(II) chain) GPLGIAGI (SEQ ID NO: 409) Human a ₂ M GPEGLRVG (SEQ ID NO: 410) Human ΡΖΡ YGAGLGVV (SEQ ID NO: 411) AGLGVVER AGLGISST (SEQ (SEQ ID ID NO: NO: 412) 413) Rat ocM EPQALAMS QALAMSAI (SEQ (SEQ ID ID NO: NO: 414) 415) Rat a ₂ M AAYHLVSQ MDAFLESS (SEQ (SEQ ID ID NO: NO: 416) 417) Rat αχίβ (2J) ESLPVVAV (SEQ ID NO: 418) Rat a ₂ 1 ₃ (27J) SAPAVESE (SEQ ID NO: 419) human fibroblast collagenase DVAQFVLT (SEQ ID NO: 420) (autolytic cleavage) VAQFVLTE AQFVLTEG PVQPIGPQ (SEQ (SEQ (SEQ ID ID ID NO: NO: NO: 421) 422) 423)

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

Spacers and split elements.

In some embodiments, the linker may need to be designed to optimize the distance between the agent and the AB in the activated antibody. This can be achieved by using a linker of the following general structure:

where

W represents --NH-CH2-- or --CH2-;

Q represents an amino acid, a peptide; or n represents an integer from 0 to 20.

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

It should be clear from this discussion that the attachment of a linker to an agent (or a spacer element to a cleavable element, or a cleavable element to an agent) need not represent a specific mode of attachment or reaction. Any reaction that provides a product with suitable stability and biocompatibility is suitable.

Serum complement and linker selection.

According to one of the methods of the present invention, when release of an agent is required, an AB is used, which is an antibody of the class that can activate complement. The resulting conjugate retains the ability to bind the antigen and activate the complement cascade. Thus, according to this embodiment of the present invention, the agent is attached to one end of the cleavable linker or cleavable element, and the other end of the linker group is attached to a specific site in the AB. For example, if the agent contains a hydroxy group or an amino group, then it can be attached to the carboxyl end of the peptide, amino acid, or other appropriately selected linker via an ester or amide bond, respectively. For example, such agents can be attached to the linker peptide via a carbodimide reaction. If the agent contains functional groups that will interfere with attachment to the linker, then these interfering functional groups can be blocked before attachment and deblocked after the product, conjugate or intermediate has been formed. The opposite or amino terminus of the linker is then used either directly or after further modification to bind to an AB that is capable of activating complement.

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

Thus, when these conjugates bind to an antigen in the presence of complement, the amide or ester bond that attaches the agent to the linker will be cleaved, resulting in the release of the agent in its active form. These conjugates, when administered to a subject, will deliver and release the agent at the target site, and are particularly effective for in vivo delivery of pharmaceutical agents, antibiotics, antimetabolites, antiproliferative agents, and the like, as shown, without being limited to those in Table. eleven.

Linkers for release without complement activation.

In yet another application of targeted delivery, release of the agent without complement activation is desirable because activation of the complement cascade will eventually lyse the target cell. Therefore, this approach is useful when the delivery and release of the agent must be performed without death of the target cell. This is the goal when delivery of cellular mediators such as hormones, enzymes, corticosteroids, neurotransmitters, genes or enzymes to target cells is desired. These conjugates can be prepared by attaching an agent to an AB that is not capable of activating complement through a linker that is weakly sensitive to cleavage by serum proteases. When this conjugate is administered to an individual, antigen-antibody complexes will be rapidly formed, while cleavage of the agent will be slow, resulting in the release of the compound at the target site.

Biochemical cross-linkers.

In some embodiments, an activated antibody may be conjugated to one or more therapeutic agents using certain biochemical crosslinkers. Cross-linkers form molecular bridges that link the functional groups of two different molecules. For stepwise linking of two different proteins, hetero-bifunctional cross-linkers can be used, which eliminate undesirable homopolymer formation.

Also suitable are peptidyl linkers cleavable by lysosomal proteases, eg Val-Cit, Val-Ala or other dipeptides. In addition, acid-labile linkers cleavable in low pH lysosome media, such as bis-sialyl ester, can be used. Other suitable linkers include cathepsin-labile substrates, especially those that exhibit optimal function at an acidic pH.

Exemplary heterobifunctional crosslinkers are shown in Table 14.

Table 14 Exemplary Heterobifunctional Cross Linkers

Heterobifunctional cross-linkers Linker Reacts with Benefits and Applications Spacer arm length after cross-linking (angstroms) SMPT Primary amines Sulhydryls Higher stability 11.2 A SPDP 1primary amines Sulhydryls Thiolation Cleavable cross-linking 6.8 A LC-SPDP Primary amines Sulhydryls Extended spacer arm 15.6 A Sulfo-LC-SPDP Primary amines Sulhydryls Extended spacer arm 15.6 A

SMCC Primary amines Sulphhydryls stable reactive maleimide group Enzyme-antibody conjugation Hapten-carrier protein conjugation 11.6 A Sulfo-SMCC Primary amines Sulphhydryls Stable reactive group Enzyme-antibody conjugation 11.6 A MBS Primary amines Sulphhydryls Enzyme-antibody conjugation Hapten-carrier protein conjugation 9.9 A Sulfo-MBS Primary amines Sulphhydryls water soluble 9.9 A SI AB Primary amines Sulphhydryls Enzyme-antibody conjugation 10.6 A Sulfo-SIAB Primary amines Sulphhydryls water soluble 10.6 A SMPB Primary amines Sulphhydryls Extended spacer arm Enzyme-antibody conjugation 14.5 A Sulfo-SMPB Primary amines Sulphhydryls Extended spacer arm 14.5 A EDE/Sulfo-NHS Primary amines Carboxyl groups Hapten-carrier protein conjugation 0 ABH Carbohydrates Non-selective Reacts with sugar groups 11.9 A

Non-cleavable linkers or direct attachment.

In some embodiments, the conjugate may be designed such that the agent is delivered to the target but not released. This can be achieved by attaching the agent to the AB directly or via a non-cleavable linker.

These non-cleavable linkers may include amino acids, peptides, D-amino acids, or other organic compounds that can be modified to include functional groups that can then be used to attach to AB using the methods described herein.

- 93 039736 ways. The general formula of such an organic linker may be as follows:

W- (CH ₂ ) nQ where

W represents --NH-CH2-- or --CH ₂ -;

Q represents an amino acid, a peptide; or n represents an integer from 0 to 20.

non-cleavable conjugates.

In some embodiments, the implementation of the connection can be attached to the AB, which do not activate complement. When using ABs that are not capable of activating complement, such attachment can be accomplished using linkers that are sensitive to cleavage by activated complement, or using linkers that are not sensitive to cleavage by activated complement.

The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl Acad. sci. USA 77: 4030 (1980); and US Pat. Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are described in US Pat.

Particularly useful liposomes can be prepared by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters with a specific pore size to obtain liposomes with the desired diameter. Fab fragments of an antibody of the present invention can be conjugated to liposomes as described by Martin et al., J. Biol. Chern, 257: 286-288 (1982), via a disulfide exchange reaction.

Definitions

Unless otherwise indicated, scientific and technical terms used in this disclosure have the meanings commonly understood by those skilled in the art. The term a or an refers to one or more of these objects. For example, a connection refers to one or more connections. Thus, the terms a, an, one or more, and at least one may be used interchangeably. In addition, unless otherwise required by context, terms in the singular include terms in the plural, and terms in the plural include terms in the singular. Generally, the nomenclatures and methods used in connection with cell and tissue culture, molecular biology and protein, oligonucleotide or polynucleotide chemistry, and hybridization described herein are well known and commonly used in the art. Standard methods are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection). Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's specifications, or as they are usually performed in the art, or as described here. The above methods and procedures are generally performed in accordance with conventional methods well known in the art and described in various general and more specific references that are cited and discussed in the present description. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures used in connection with, and the laboratory procedures and methods of, analytical chemistry, synthetic organic chemistry, and drug and pharmaceutical chemistry are well known and commonly used in the art. Standard methods are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and for patient treatment.

As used herein in accordance with the present invention, the following terms, unless otherwise indicated, are understood to have the following meanings.

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

It is known that the basic structural unit of an antibody contains a tetramer. Each tetramer consists of two identical parts of polypeptide chains, each pair has one light (about 25 kD) and one heavy chain (about 50-70 kD). The amino-terminal region of each chain includes a variable region of about 100 to 110 or more amino acids in length, primarily responsible for the recognition

- 94 039736 antigen.

The carboxy-terminal region of each chain defines a constant region primarily responsible for effector function. In general, human-derived antibody molecules belong to any of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other in the nature of the heavy chain present in the molecule. Some classes also have subclasses such as IgG1, IgG2 and others. In addition, in humans, the light chain can be a kappa chain or a lambda chain.

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

The term antigen-binding site or binding fragment refers to the portion of an immunoglobulin molecule that is involved in antigen binding. The antigen-binding site is formed by the amino acid residues of the N-terminal variable (V) regions of the heavy (H) and light (L) chains.

The three highly divergent regions in the V regions of the heavy and light chains are referred to as hypervariable regions, which lie between more conserved flanking regions known as framework regions (FRs). As used herein, the term FR refers to amino acid sequences that are naturally found between and adjacent to hypervariable regions in immunoglobulins. In an antibody molecule, three hypervariable regions of the light chain and three hypervariable regions of the heavy chain are located relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as complementarity-determining regions or CDRs. The assignment of amino acids to each domain is as defined by Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987), or Chothia et al., Nature 342:878-883 (1989).

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

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

As used herein, the term isolated polynucleotide means a polynucleotide of genomic, cDNA, or polynucleotide of synthetic origin, or a combination thereof, and by virtue of its origin, an isolated polypeptide: 1) is not associated with all or part of the polynucleotide with which it is naturally associated; 2) is operably linked to a polynucleotide other than that to which it is naturally linked; 3) does not occur naturally as part of a larger sequence. The polynucleotides of the invention include nucleic acid molecules encoding immunoglobulin heavy chains shown here and nucleic acid molecules encoding immunoglobulin light chains shown here.

The term isolated protein as used herein means a protein from cDNA, recombinant RNA or nucleic acids of synthetic origin, or some combination thereof, and by virtue of its origin or source of origin, the isolated protein: (1) is not associated with proteins found in nature; (2) does not contain other proteins from the same source, eg does not contain murine proteins; (3) expressed by a cell of another species; or (4) does not occur naturally.

The term polypeptide is used here as a general term to refer to a native protein, fragments or analogues of a polypeptide sequence. Therefore, native protein fragments and analogs are species of the polypeptide genus. The polypeptides of the invention include the heavy chains of immunoglobulin molecules shown here and the light chains of immunoglobulin molecules shown here, as well as antibody molecules formed by combinations comprising heavy chains of immunoglobulin molecules with light chains of immunoglobulin molecules, such as the kappa light chain of immunoglobulin molecules and vice versa, as well as their fragments and analogues.

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

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

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

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

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

As used here, the twenty common amino acids and their abbreviations are used according to their traditional use. See Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Green, Eds., Sinauer Associates, Sunderland, Mass (1991). Stereoisomers (e.g., D-amino acids) of twenty common amino acids, unnatural amino acids such as α-, α -disubstituted amino acids, N-alkylamino acids, lactic acid, and other non-traditional amino acids may also be suitable components for the polypeptides of the present invention. , ε-N-acetylysine, Ophosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline) As used herein for a polypeptide, then the left hand direction is the amino terminus and the right hand direction is the carboxy terminus according to standard usage and convention.

Similarly, unless otherwise indicated, the left end of single stranded polynucleotide sequences is the 5' end, the left direction of double stranded polynucleotide sequences is indicated by the 5' direction. The 5' to 3' direction when adding emerging RNA transcripts refers to regions of the direction of the transcription sequence in a DNA strand having the same sequence as RNA and which represent the 5' to 5' end of the RNA transcript, refer to upstream sequences, sequence regions in a DNA strand that have the same sequence as RNA and that are 3' to the 3' end of the RNA transcript are called downstream sequences.

When applied to polypeptides, the term substantial identity means that two peptide sequences when optimally aligned, for example, using the GAP or BESTFIT programs using the default gap mass, have at least 80% sequence identity, in some embodiments at least 90% sequence identity. , in some embodiments at least 95% sequence identity, and in some embodiments at least 99% sequence identity.

In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

As discussed here, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are within the scope of the present invention, provided that the amino acid sequence changes are at least 75%, in some embodiments at least 80%, 90%, 95%, and in some options 99%. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are substitutions that occur within a family of amino acids that are similar in their side chains. Genetically encoded amino acids are usually divided into families: (1) acidic amino acids, which include aspartate, glutamate; (2) basic amino acids, which include lysine, arginine, histidine; (3) non-polar amino acids, which include alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids, which include glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include (i) serine and threonine, which are an aliphatic hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are an aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, it is reasonable to expect that a single substitution of leucine for isoleucine or valine, aspartate for glutamate, threonine for serine, or a similar substitution of an amino acid for a structurally related amino acid, would have little effect on the binding or properties of the resulting molecule, especially if the substitution does not include an amino acid in wireframe site. The extent to which a change in amino acid will result in a functional peptide can be easily determined by analyzing the specific activity of the polypeptide derivative. Such analyzes are described in detail here. Fragments or analogues of antibodies or immunoglobulin molecules can be easily obtained by experts in this field. Suitable amino-terminals and carboxy-terminals of fragments or analogs occur near the boundaries of the functional regions. Structural and functional regions can be established by comparing nucleotide and/or amino acid sequence data with known or proprietary sequence databases. In some embodiments, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods are known for identifying protein sequences that fit into a known three-dimensional structure. Bowie et al. Science 253: 164 (1991). Thus, the above examples demonstrate that those skilled in the art can determine motif sequences and structural conformations that can be used to determine the structural and functional domains of the invention.

Suitable amino acid substitutions are those that (1) desensitize proteolysis, (2) desensitize oxidation, (3) alter binding affinity to form protein complexes, (4) alter binding affinity, and (5) or modify other

- 97 039736 physico-chemical or functional properties of such analogues. The analogs may include different muteins of a sequence other than the naturally occurring peptide sequence. For example, one or more amino acid substitutions (eg, conservative amino acid substitutions) can be made in a naturally occurring sequence (eg, in a polypeptide fragment outside the junction-forming domain(s). A conservative amino acid substitution should not significantly change the structural characteristics of the original sequence (eg, the amino acid being changed should not break the helix that occurs in the original sequence, or break other types of secondary structure characteristic of the original sequence). Examples of secondary and tertiary polypeptide structures known in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et at., Nature 354:105 (1991).

As used herein, the term polypeptide fragment refers to a polypeptide containing a deletion in the amino-terminal or carboxy-terminal region, but in which the remainder of the amino acid sequence is identical to the corresponding regions of a naturally occurring sequence derived, for example, from a full-length cDNA sequence. Fragments are typically at least 5, 6, 8, or 10 amino acids in length, preferably at least 14 amino acids, more preferably at least 20 amino acids, typically at least 50 amino acids, and in some embodiments, at least 70 amino acids. As used herein, the term analog refers to polypeptides that consist of a segment of at least 25 amino acids that has a substantial identity with a fragment of a deduced amino acid sequence and that has specific binding to a target under suitable binding conditions. Typically, polypeptide analogs include a conservative amino acid substitution (or addition or deletion) relative to a naturally occurring sequence. Analogues are typically at least 20 amino acids in length, in some embodiments, at least 50 amino acids or more in length, and can often be the length of a full-length naturally occurring polypeptide.

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

As used herein, the terms labeled or labeled refer to the incorporation of a detectable label, such as the introduction of a radioactively labeled amino acid or the attachment to the polypeptide of biotinyl groups that can be detected by labeled avidin (for example, streptavidin containing a fluorescent label or having an enzymatic activity that can be detected by optical or calorimetric methods). In certain situations, the label or marker may also be therapeutic. Various methods for labeling polypeptides and glycoproteins are known and can be used in the art. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g. ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹ⁿ In, ¹²⁵ I, ¹³¹ I), , FITC, rhodamine, lanthanide phosphors), enzymatic labels (eg, horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotinyl groups, predefined polypeptide epitopes recognized by the secondary reporter (eg, leucine zipper sequences, binding sites secondary antibodies, metal binding regions, epitope tags). In some embodiments, the tags are attached using spacer arms of varying lengths to reduce potential dimensional hindrances. As used here, the term pharmaceutical agent or drug refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

Other chemical terms are used here in accordance with their usual use in the art, as shown in the McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

As used herein, the term substantially pure means that the entity molecule is the predominant molecule that is present (i.e., on a molar basis, it is more abundant than any other individual molecules in the composition), and in some embodiments, the substantially purified fraction is is a composition in which the object molecule is at least about 50% (on a molar basis) of all macromolecular species present.

Typically, a substantially pure composition will contain greater than 80% of all macromolecular species present in the composition, in some embodiments greater than 85%, 90%, 95%, and 99%. In some embodiments of the invention, the object molecule is purified to the required homogeneity (contaminant molecules cannot be detected in the composition by conventional methods of analysis), where the composition consists mainly of one kind of macromolecules.

The term patient includes human and veterinary subjects.

The antibodies and/or activated antibodies of the present invention specifically bind to a particular target, eg a human target protein such as human PDL1. Also

- 98 039736 the invention includes antibodies and/or activated antibodies that bind to the same epitope as the antibodies and/or activated antibodies described herein. The invention also includes antibodies and/or activated antibodies that compete with an anti-PDL1 antibody and/or an activated anti-PDL1 antibody described herein for binding to PDL1, eg human PDL1. The invention also includes antibodies and/or activated antibodies that cross-compete with an anti-PDL1 antibody and/or an activated anti-PDL1 antibody described herein for binding to PDL1, eg human PDL1.

Those skilled in the art will appreciate that without undue experimentation, it is possible to determine whether a monoclonal antibody (e.g., a mouse monoclonal or humanized antibody) has the same specificity as the monoclonal antibody used in the methods described herein by determining how much the first antibody interferes with the latter to contact the target. If the tested monoclonal antibody competes with the monoclonal antibody of the invention, as indicated by reduced binding by the monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same or similar epitope. An alternative way to determine if a monoclonal antibody has the specificity of a monoclonal antibody of the invention is to pre-incubate the monoclonal antibody of the invention with a target and then add the test monoclonal antibody to determine if the test monoclonal antibody's ability to bind to the target is inhibited. If a test monoclonal antibody is inhibited, it is likely to have the same or functionally equivalent epitopic specificity as the monoclonal antibody of the invention.

Polyspecific activated antibodies.

The invention also provides multispecific activated anti-PDL1 antibodies. The multispecific activated antibodies provided herein are multispecific antibodies that recognize PDL1 and at least one or more other antigens or epitopes and that include at least one masking fragment (MM) associated with at least one antigen-binding or epitope-binding site of the multispecific antibody thus, MM binding reduces the ability of the antigen-binding or epitope-binding site to bind to its target. In some embodiments, the MM is linked to an antigen-binding or epitope-binding site of a polyspecific antibody via a cleavable fragment (CM) that functions as a substrate for at least one protease. The activated polyspecific antibodies provided herein are stable in the circulatory system, activated at the intended sites of treatment and/or diagnosis, but not in the normal, i. healthy tissue and upon activation exhibit target binding that is at least comparable to that of the corresponding unmodified polyspecific antibody.

In some embodiments, multispecifically activated antibodies are designed to activate immune effector cells, also referred to herein as multispecifically activated antibodies, immune effector cell activators. In some embodiments, multispecifically activated antibodies are designed to activate leukocytes, also referred to herein as multispecifically activated antibodies, leukocyte activators. In some embodiments, polyspecifically activated antibodies are engineered to activate T cells, also referred to herein as multispecifically activated antibodies, T cell activators. In some embodiments, the polyspecific activated antibodies interact with a surface antigen on a leukocyte, such as a T cell, on a natural killer (NK) cell, on a myeloid mononuclear cell, on a macrophage, and/or on another immune effector cell. In some embodiments, the immune effector cell is a leukocyte. In some embodiments, the immune effector cell is a T cell. In some embodiments, the implementation of the immune effector cell is a NK cell. In some embodiments, the immune effector cell is a mononuclear cell, such as a myeloid mononuclear cell. In some embodiments, multispecific activated antibodies are designed to bind or otherwise interact with more than one target and or more than one epitope, also referred to here as activated antibodies that target multiple antigens. As used here, the terms target and antigen are used interchangeably.

In some embodiments, the multispecific activated antibodies, immune effector cell activators, include a targeting antibody or antigen-binding fragment thereof that binds to PDL1, and an antibody or antigen-binding site thereof, immune effector cell activators, wherein at least one of the targeting antibody or antigen-binding fragment thereof , and/or an antibody or antigen-binding fragment thereof, an immune effector cell activator, is masked. In some embodiments, the antibody or antigen-binding fragment thereof, an immune effector cell activator, includes a first antibody or antigen-binding fragment (AB1) that binds to a first target of the immune effector cell.

- 99 039736 cells, where AB1 is attached to a masking fragment (MM1) in such a way that MM1 binding reduces the ability of AB1 to bind to the first target. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which includes a second antibody or antigen-binding fragment thereof (AB2) that binds to PDL1, where AB2 is attached to a masking fragment (MM2) such that MM2 binding reduces the ability of AB2 to bind to PDL1. In some embodiments, the antibody or antigen-binding fragment thereof, immune effector cell activators, includes a first antibody or antigen-binding fragment (AB1) that binds to a first target of the immune effector cell, where AB1 is attached to a masking fragment (MM1) such that MM1 binding reduces the ability of AB1 to bind to the first target, and the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof, which includes a second antibody or antigen-binding fragment (AB2) that binds to PDL1, where AB2 is attached to a masking fragment (MM2), such in such a way that MM2 binding reduces the ability of AB2 to bind to PDL1. In some embodiments, the non-immune effector cell activating antibody is an antibody that targets a cancer. In some embodiments, the non-immune cellular effector antibody is IgG. In some embodiments, the immune effector cell activating antibody is scFv. In some embodiments, the PDL1-targeting antibody (eg, non-immune cellular effector antibody) is IgG and the immune effector cell activating antibody is scFv. In some embodiments, the immune effector cell is a leukocyte. In some embodiments, the immune effector cell is a T cell.

In some embodiments, the immune effector cell is an NK cell. In some embodiments, the immune effector cell is a myeloid mononuclear cell.

In some embodiments, the polyspecific activated antibodies, T cell activators of the invention, include an antibody targeting PDL1, or an antigen-binding fragment thereof, and an antibody, or antigen-binding fragment thereof, T-cell activators, wherein at least one of the antibody targeting PDL1, or its antigen-binding fragment, and/or the antibody or its antigen-binding fragment, T-cell activator, are masked. In some embodiments, the antibody or antigen-binding fragment thereof, a T cell activator, comprises a first antibody or antigen-binding fragment (AB1) thereof that binds to the first target of the T cell, where AB1 is attached to a masking fragment (MM1) such that the MM1 compound reduces the ability AB1 bind to the first target. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which includes a second antibody or antigen-binding fragment thereof (AB2) that binds to PDL1, where AB2 is attached to a masking fragment (MM2) such that the MM2 compound reduces the ability of AB2 to bind to PDL1. In some embodiments, the T cell activating antibody or antigen binding fragment thereof comprises a first antibody or antigen binding fragment (AB1) that binds to a first target of the T cell, where AB1 is attached to a masking fragment (MM1) such that the compound MM1 reduces the ability of AB1 to bind to the first target, and the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof, which includes a second antibody or antigen-binding fragment (AB2) that binds to PDL1, where AB2 is attached to a masking fragment (MM2) such in such a way that the MM2 compound reduces the ability of AB2 to bind to PDL1.

In some embodiments of a polyspecifically activated antibody, an immune effector cell activator, one antigen is PDL1 and the other antigen is typically an excitatory or inhibitory receptor present on the surface of a T cell, natural killer (NK) cell, myeloid mononuclear cell, macrophage, and/or another immune effector cell such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, Cd27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, 0X40, PD1, TIGIT, TIM3 or VISTA. In some embodiments, the antigen is a stimulatory receptor present on the surface of a T cell or NK cell; examples of such stimulatory receptors include, but are not limited to, CD3, CD27, CD28, CD137 (also referred to as 4-1BB), GITR, HVEM, ICOS, NKG2D, and 0X40. In some embodiments, the antigen is an inhibitory receptor present on the surface of a T cell; examples of such inhibitory receptors include, but are not limited to, BTLA, CTLA-4, LAG3, PD1, TIGIT, TIM3, and NK-expressed KIRs. The region of an antibody conferring specificity for a T cell surface antigen can also be replaced with a ligand or ligand domain that binds to a T cell receptor, NK cell receptor, macrophage receptor, and/or other immune effector cell receptor, such as, but not limited to these, B7-1, B7-2, B7H3, PDL1, PDL2 or TNFSF9.

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In some embodiments, a polyspecifically activated antibody, a T cell activator, comprises an anti-CD3 epsilon (CD3e, also refers here to CD3e and CD3) scFv, and a targeting antibody or antigen-binding fragment thereof, wherein at least one of an anti-CD3e scFv and/ or the targeting antibody or antigen-binding fragment thereof is masked. In some embodiments, the CD3e scFv comprises a first antibody or antigen-binding fragment (AB1) thereof that binds to CD3e, wherein the AB1 is attached to a masking fragment (MM1) such that the MM1 compound reduces the ability of AB1 to bind to CD3e. In some embodiments, the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof, which includes a second antibody or antigen-binding fragment (AB2) that binds to PDL1, where AB2 is attached to a masking fragment (MM2) in such a way that the connection of MM2 reduces the ability of AB2 to bind to PDL1. In some embodiments, the CD3s scFv comprises a first antibody or antigen-binding fragment (AB1) thereof that binds to CD3e, where AB1 is attached to a masking fragment (MM1) such that the MM1 compound reduces the ability of AB1 to bind to CD3e, and the targeting antibody or antigen-binding fragment thereof the fragment includes a second antibody or fragment thereof, which includes a second antibody or antigen-binding fragment (AB2) that binds to PDL1, where AB2 is attached to a masking fragment (MM2), so that the connection of MM2 reduces the ability of AB2 to bind to PDL1.

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

In some embodiments, a polyspecifically activated IgG antibody comprises masked IgG variable regions. In some embodiments, a polyspecifically activated antibody comprising an scFv contains masked regions of the scFv. In some embodiments, the implementation of the polyspecifically activated antibody includes IgG variable regions and scFv regions, where at least one of the IgG variable regions is associated with a masking fragment. In some embodiments, the multispecifically activated antibody comprises IgG variable regions and scFv regions, wherein at least one of the scFv regions is associated with a masking fragment. In some embodiments, the multispecific activated antibody comprises IgG variable regions and scFv regions, wherein at least one of the IgG variable regions is associated with a masking fragment and at least one of the scFv regions is associated with a masking fragment. In some embodiments, the implementation of the polyspecifically activated antibody includes IgG variable regions and scFv regions, where each of the IgG variable regions and each of the scFv regions is associated with their own masking fragment. In some embodiments, one region of the polyspecifically activated antibody is specific for the target antigen and another region of the antibody is specific for the T cell surface antigen. In some embodiments, one region of the multispecific activated antibody has specificity for a target antigen and another region of the antibody has specificity for a different target antigen. In some embodiments, one region of the multispecific activated antibody has specificity for a target antigen epitope and another region of the antibody has specificity for a different target antigen epitope.

In a polyspecific activated antibody, the scFv may be fused to the carboxyl terminus of the heavy chain of an activated IgG antibody, to the carboxyl terminus of the light chain of an activated IgG antibody, or to the carboxyl termini of both the heavy and light chains of an activated IgG antibody. In a polyspecific activated antibody, the scFv may be fused to the amino terminus of the heavy chain of an activated IgG antibody, to the amino terminus of the light chain of an activated IgG antibody, or to the amino termini of both the heavy and light chains of an activated IgG antibody. In a polyspecific activated antibody, the scFv may be fused to any combination of one or more carboxyl termini and one or more amino termini of an activated IgG antibody. In some embodiments, a masking fragment (MM) associated with a cleavable fragment (CM) is attached to and masks an IgG antigen-binding site. In some embodiments, a masking fragment (MM) associated with a cleavable fragment (CM) is attached to and masks the antigen-binding site of at least one scFv. In some embodiments, the masking fragment

- 101 039736 (MM) associated with a cleavable fragment (CM) is attached and masks the antigen-binding site of IgG, and a masking fragment (MM) associated with a cleavable fragment (CM) is attached and masks the antigen-binding site of at least one scFv.

The invention provides exemplary structures of multispecifically activated antibodies, which include, but are not limited to, the following:

(VL-CL) ₂ : (VH-CH1-CH2-CH3-L4-VH*L3-VL*-L2-CM-L1-MM) ₂ ; (VL-CL) ₂ : (VH-CH1-CH2-CH3-L4-VL*-L3-VH*-L2CM-L1-MM) ₂ ; (MM-L1-CM-L2-VL-CL) ₂ : (VH-CH1-CH2-CH3-L4-VH*-L3-VL*) ₂ ;

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

(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3) ₂ ; (VL-CL) ₂ : (MM-L1-CML2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3) ₂ ; (MM-L1-CM-L2-VL-CL) ₂ : (VL*-L3VH*-L4-VH-CH1-CH2-CH3) ₂ ; (MM-L1-CM-L2-VL-CL) ₂ : (VH*-L3-VL*-L4-VHCH1-CH2-CH3) ₂ ; (VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM) ₂ : (VH-CH1-CH2CH3) ₂ ; (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)2 : (VH-CH1-CH2-CH3)2; (MML1-CM-L2-VL*-L3-VH*-L4-VL-CL)2 : (VH-CH1-CH2-CH3) ₂ ; (MM-L1-CM-L2VH*-L3-VL*-L4-VL-CL) ₂ : (VH-CH1-CH2-CH3) ₂ ; (VL-CL-L4-VH*-L3-VL*-L2CM-L1-MM) ₂ : (MM-Ll-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3) 2; (VL-CLL4-VH*-L3-VL*-L2-CM-L1-MM) ₂ : (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1CH2-CH3) ₂ ; (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM) ₂ : (MM-L1-CM-L2-VL*L3-VH*-L4-VH-CH1-CH2-CH3 ) ₂ ; (VL-CL-L4-VL*-L3-VH*-L2-CM-L1MM) ₂ : (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3) 2; (VL-CL-L4-VH*L3-VL*)2 : (MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3) ₂ ; (VL-CL-L4VH*-L3-VL*)2 : (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3) ₂ ; (VL-CLL4-VL*-L3-VH*) ₂ : (MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3) ₂ ; (VLCL-L4-VL*-L3-VH*) ₂ : (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;

(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM) ₂ : (VL*-L3-VH*-L4-VH-CH1-CH2CH3) ₂ ; (VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM) ₂ : (VH*-L3-VL*-L4-VH-CH1CH2-CH3) ₂ ; (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM) ₂ : (VL*-L3-VH*-L4-VHCH1-CH2-CH3)2; OR (VL- CL-L4-VL*-L3-VH*-L2-CM-L1-MM) ₂ : (VH*-L3VL*-L4-VH-CH1-CH2-CH3) ₂ where VL and VH represent variable light and heavy chain regions of the first specificity that are part of IgG; VL* and VH* represent the variable regions of the light and heavy chains of the second specificity that are part of the scFv; L1 is a linker peptide connecting the masking fragment (MM) and the cleavable fragment (CM); L2 is a linker peptide connecting the cleavable fragment (CM) and the antibody; L3 is a linker peptide that connects scFv variable regions; L4 is a linker peptide connecting an antibody of the first specificity with an antibody of the second specificity; CL represents the light chain constant region; and CH1, CH2, CH3 are heavy chain constant regions. The first and second specificities may refer to any antigen or epitope.

In some embodiments of a polyspecific activated antibody, a T cell activator, one antigen is PDL1 and the other antigen is typically a stimulatory (also referred to herein as an activating) or inhibitory receptor present on the surface of a T cell, natural killer (NK) cell, myeloid mononuclear cell, macrophage and/or other immune effector cell such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137 (also refers here to TNFRSF9), CTLA -4, GITR, HVEM, ICOS, LAG3, NKG2D, 0X40, PD1, TIGIT, TIM3, or VISTA. The antibody region conferring specificity for T cell surface antigen can also be replaced with a ligand or ligand region that binds to a T cell receptor, NK cell receptor, macrophage receptor, and/or other immune effector cell receptor, such as, not limited to this,

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PDL1.

In some embodiments, the targeting antibody is an anti-PDL1 antibody disclosed herein. In some embodiments, the targeting antibody may be in the form of an activated antibody. In some embodiments, the scFv(s) may be in pro-scFv form (see, for example, international applications WO 2009/025846, WO 2010/081173).

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

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

GGGSGGGGSGSGGGSGGGGSGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQA

PRLLIYGASSRATGIPDRFSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTTFGGGTKVEIKRSG GSTITSYNVYYTKLSSSGTQVQLVQTGGGWQPGRSLRLSCAASGTFSSYAMSWVRQAPGKGLEWV SAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATNSLYWYFDLWGRGTLVT

VSSAS (SEQ ID NO: 424)

In some embodiments, the anti-CTLA-4 scFv comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 424.

In some embodiments, the anti-CD3ε scFv comprises the amino acid sequence:

GGGSGGGGSGSGGGSGGGGSGGGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQG

LEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQ

GTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPK

RWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 425)

In some embodiments, the anti-CD3ε scFv comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 425.

In some embodiments, the scFv is specific for binding to one or more T cells, one or more NK cells, and/or one or more macrophages. In some embodiments, the scFv is specific to bind to a target selected from the group consisting of B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM , ICOS, LAG3, NKG2D, 0X40, PD1, TIGIT, TIM3 or VISTA.

In some embodiments, the multispecific activated antibody also includes an AB conjugated agent. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is an antineoplastic agent. In some embodiments, the agent is a toxin or fragment thereof. In some embodiments, the agent is conjugated to a polyspecifically activated antibody via a linker. In some embodiments, the agent is conjugated to the AB via a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylator or a DNA intercalator, or another DNA damaging agent. In some embodiments, the linker is a cleavable linker. In some embodiments, the implementation of the agent is an agent selected from the group shown in table. 11. In some embodiments, the agent is dolastatin. In some embodiments, the agent is auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is pyrrolobenzodiazepine. In some embodiments, the agent is a dimer of pyrrolobenzodiazepine.

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In some embodiments, the multispecifically activated antibody also includes a detectable label. In some embodiments, the detectable label is a diagnostic agent.

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

The present invention also provides an isolated nucleic acid molecule encoding a polyspecifically activated antibody described herein, as well as vectors that include these isolated nucleic acid sequences. The present disclosure provides methods for producing a polyspecifically activated antibody by culturing a cell under conditions that result in the expression of an activated antibody, wherein the cell contains such a nucleic acid molecule. In some embodiments, the implementation of the cell contains such a vector.

The present invention also provides a method for producing multispecifically activated antibodies of the present disclosure by (a) culturing a cell containing a nucleic acid construct that encodes a multispecifically activated antibody under conditions that result in expression of the multispecifically activated antibody, and (b) isolating the polyspecifically activated antibody. Suitable AB, MM and/or CM include any of the AB, MM and/or CM disclosed herein.

The present invention also provides polyspecifically activated antibodies and/or compositions of polyspecifically activated antibodies that comprise at least a first antibody or antigen-binding fragment (AB1) thereof that specifically binds to a first target or first epitope and a second antibody or antigen-binding fragment thereof ( AB2) that bind to a second target or second epitope, where at least AB1 is connected or otherwise attached to a masking fragment (MM1) in such a way that the connection of MM1 reduces the ability of AB1 to bind to its target. In some embodiments, MM1 is linked to AB1 via a first cleavable fragment (CM1) sequence that includes a substrate for a protease, e.g., a protease that is co-localized with an AB1 target at a site being treated or diagnosed in a subject. The multispecific activated antibodies provided here are stable in the circulatory system, activated at the intended sites of treatment and/or diagnosis, but not in the normal, i. healthy tissue and, when activated, exhibit binding to the AB1 target that is at least comparable to that of the corresponding, unmodified polyspecific antibody. Suitable AB, MM and/or CM include any of the AB, MM and/or CM disclosed herein.

The present invention also provides compositions and methods that include a polyspecifically activated antibody that includes at least a first antibody or antibody fragment (AB1) that specifically binds to a target and a second antibody or antibody fragment (AB2) wherein at least At least the first AB in a polyspecifically activated antibody is coupled to a masking fragment (MM1) that reduces the ability of AB1 to bind to its target. In some embodiments, each AV is associated with an MM that reduces the ability of its corresponding AV to bind to each target. For example, in embodiments of a bispecific activated antibody, AB1 is coupled to a first masking fragment (MM1) that reduces the ability of AB1 to bind to its target, and AB2 is coupled to a second masking fragment (MM2) that reduces the ability of AB2 to bind to its target. In some embodiments, the multispecifically activated antibody comprises more than two AB regions; in such embodiments, AB1 is associated with a first masking fragment (MM1) that reduces the ability of AB1 to bind to its target, AB2 is connected to a second masking fragment (MM2) that reduces the ability of AB2 to bind to its target, AB3 is associated with a third masking fragment (MM3 ), which reduces the ability of AB3 to bind to its target, and so on for each AB in the polyspecific activated antibody. Suitable AB, MM and/or CM include any of the AB, MM and/or CM disclosed herein.

In some embodiments, the multispecifically activated antibody further comprises at least one cleavable fragment (CM) that is a substrate for a protease, wherein the CM binds MM to AB. For example, in some embodiments, a polyspecifically activated antibody comprises at least a first antibody or antibody fragment (AB1) that specifically binds to a target and a second antibody or antibody fragment (AB2) wherein at least the first AB in the polyspecific The activated antibody is linked via a first cleavable fragment (CM1) to a masking fragment (MM1) that reduces the ability of AB1 to bind to its target. In some embodiments of bispecific activated antibodies, AB1 is linked via CM1 to MM1, and AB2 is linked via a second cleavable fragment (CM2) to a second masking fragment (MM2) that reduces the ability of AB2 to bind to its target. In some embodiments, the multispecifically activated antibody comprises more than two AB regions; in some of these embodiments, AB1 is connected via

- 104 039736

CM1 is connected to MM1, AB2 is connected via CM2 to MM2, and AB3 is connected via a third cleavage fragment (CM3) to a third masking fragment (MM3), which reduces the ability of AB3 to bind to its target, and so on. for each AB in the polyspecific activated antibody. Suitable

AB, MM and/or CM include any of the AB, MM and/or CM disclosed here.

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

The present invention also provides activated antibodies that include non-binding steric groups (NB) or binding partners (BP) for non-binding steric groups, where the BP recruits or otherwise attracts the NB to the activated antibody. The activated antibodies provided herein include, for example, an activated antibody that contains a non-binding steric group (NB), a cleavable linker (CL), and an antibody or antibody fragment (AB) that binds to a target; an activated antibody that includes a binding partner for a non-binding steric group (BP), CL, and AB; and an activated antibody that includes BP to which NB is recruited, CL and AB that binds to the target. Activated antibodies in which NBs are covalently linked to CL and AB of an activated antibody, or are linked by interaction with a BP that is covalently linked to CL and AB of an activated antibody, are referred to herein as NB-containing activated antibodies. Activated or switchable means that the activated antibody exhibits a first level of target binding when the activated antibody is in an inhibited, masked, or non-cleaved state (i.e., the first conformation), and a second level of target binding when the activated antibody is in an uninhibited, unmasked state. and/or a cleaved state (ie a second conformation, ie an activated antibody) wherein the second level of target binding is higher than the first level of target binding. Activated antibody compositions may exhibit increased bioavailability and more favorable biodistribution compared to conventional therapeutic antibodies.

In some embodiments, activated antibodies provide reduced toxicity and/or reduced adverse side effects that might otherwise result from binding at sites not requiring treatment and/or diagnosis if the AV had not been masked or otherwise inhibited in relation to association with such a place.

Activated anti-PDL1 antibodies that include a non-binding steric group (NB) can be prepared using the methods described in PCT International Application Publication WO 2013/192546, the contents of which are incorporated herein by reference in its entirety.

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

Obviously it is clear that the introduction of therapeutic agents of the present invention will be carried out with suitable carriers, excipients and other agents that are included in the composition to provide improved transfer, delivery, tolerability and the like. Many suitable compositions can be found in the pharmaceutical handbook known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing, Easton, PA (1975)), in particular in chapter 87, Blaug, Seymour . Such compositions include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (such as lipofectin), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in emulsions. - oils, carb wax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing carb wax. Any of the above mixtures may be suitable in the treatments and formulations of the present invention, provided that the active ingredient in the composition is not inactivated by the composition and the composition is physiologically compatible and tolerated by the particular route of administration. See also Baldrick P., Pharmaceutical excipient development: the need for preclinical guidance, Regul. Toxicol Pharmacol., 32(2):210-8 (2000), Wang W., Lyophilization and development of solid protein Pharmaceuticals, Int. J. Pharm., 203(1-2): 1-60 (2000), Charman W.N., Lipids, lipophilic drugs and oral drug delivery-some emerging concepts J. Pharm. Sci., 89(8):967-78 (2000), Powell et al., Compendium of excipients for parenteral formulations, PDA J. Pharm. sci. Technol., 52:238-311 (1998) and references therein for additional information relating to compositions, excipients and carriers well known to pharmaceutical chemists.

Therapeutic compositions of the present invention, which comprise an anti-PDL1 antibody and/or an anti-PDL1-activated antibody, for example, as a non-limiting example, an antibody, a conjugated antibody, an activated antibody, and/or a conjugated activated antibody, are used to prevent, treat, or otherwise attenuating a disease or disorder associated with aberrant expression and/or target activity. For example, therapeutic compositions of the present invention, which include an antibody, a conjugated antibody, an activated antibody, and/or a conjugated activated antibody, are used to treat or otherwise ameliorate cancer or other neoplastic disease, inflammation, inflammatory disorder, and/or auto-immune disease. In some embodiments, the cancer is a solid tumor or a hematological malignancy in which the target is expressed. In some embodiments, the cancer is a solid tumor in which the target is expressed. In some embodiments, the cancer is a hematological malignancy in which the target is expressed. In some embodiments, the target is expressed in the parenchyma (eg, in cancer, in a region of an organ or tissue that often performs the function(s) of an organ or tissue). In some embodiments, the target is expressed in a cell, tissue, or organ. In some embodiments, the target is expressed in the stroma (ie, the connective support scaffold of a cell, tissue, or organ). In some embodiments, the target is expressed in an osteoblast. In some embodiments, the target is expressed in the endothelium (vasculature). In some embodiments, the target is expressed in a cancer stem cell. In some embodiments, the agent to which the antibody and/or the activated antibody is conjugated is a microtubule inhibitor. In some embodiments, the agent to which the antibody and/or the activated antibody is conjugated is a nucleic acid damaging agent.

The effectiveness of prevention, reduction or treatment is determined using any known method for diagnosing or treating a disease or disorder associated with the expression and/or activity of the target, such as aberrant expression and/or activity of the target. Prolongation of a subject's survival, or otherwise slowing the progression of a disease or disorder associated with target expression and/or activity, e.g., aberrant target expression and/or activity, in a subject is indicative that the antibody, conjugated antibody, activated antibody, and/or conjugated activated antibody antibody, confers clinical benefit.

The antibody, conjugated antibody, activated antibody, and/or conjugated activated antibody may be administered in the form of pharmaceutical compositions. The principles and discussion associated with the preparation of such compositions, as well as guidance on the selection of ingredients, are provided in detail, for example, in Remington:

The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton,

Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne,

Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

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

The active ingredients can also be incorporated into microcapsules prepared, for example, by coacervation or interfacial polymerization methods, such as hydroxymethyl cellulose microcapsules, or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, and in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.

Compositions used for in vivo administration must be sterile. This is easily achieved by filtration through sterile membrane filters.

Sustained release formulations can be prepared. Suitable examples of sustained release compositions include semi-permeable matrices of solid hydrophobic polymers containing the antibody, where the matrices are in the form of molded articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g. poly(2-hydroxyethyl methacrylate) or polyvinyl alcohol), polylactides (US Pat. No. 3,777,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LU-106 039736

PRON DEPORT™ (injectable microspheres consisting of lactic acid-glycolic acid copolymer), and poly-O-(-)-3-hydroxybutyric acid. Although polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow molecules to be released for over 100 days, some hydrogels release proteins for shorter periods of time.

In some embodiments, the antibody, conjugated antibody, activated antibody, and/or conjugated activated antibody contains a detectable label. An intact antibody or fragment thereof is used (eg Fab, scFv or F(ab)2). The term labeled in relation to a probe or antibody is intended to include direct labeling of a probe or antibody by coupling (i.e., physical coupling) of the substance to be detected to the probe or antibody, as well as indirect labeling of the probe or antibody by reaction with another directly labeled reagent. . Examples of mediated labeling include detection of the primary antibody using a fluorescently labeled secondary antibody and end labeling of the probe DNA with biotin so that it can be detected using fluorescently labeled streptavidin. The term biological sample is intended to include tissues, cells, and body fluids isolated from the subject's body, as well as tissues, cells, and fluids present in the subject's body. Thus, the term biological sample includes blood and a fraction or component of blood, including blood serum, blood plasma or lymph. That is, the detection method of the present invention can be used to detect an mRNA, protein or genomic DNA analyte in a biological sample in vitro as well as in vivo. For example, in vitro methods for detecting an mRNA analyte include Northern blotting and in situ hybridization. In vitro methods for detecting a protein analyte include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, immunochemical staining, and immunofluorescence. In vitro methods for detecting a genomic DNA analyte include Southern blotting. Procedures for conducting immunoassays are described, for example, in ELISA: Theory and Practice: Methods in Molecular Biology, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; Immunoassay, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and Practice and Theory of Enzyme Immunoassays, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. In addition, in vivo methods for detecting a protein analyte involve administering a labeled antibody to the protein of interest to the subject. For example, an antibody can be labeled with a radioactive label, the presence and location of which in a subject can be detected by standard imaging techniques.

The antibodies, conjugated antibodies, activated antibodies and/or conjugated activated antibodies of the present invention are also suitable for various diagnostic and prophylactic compositions. In one embodiment, patients at risk of developing one or more of the above disorders are administered with an antibody, a conjugated antibody, an activated antibody, and/or a conjugated activated antibody. The predisposition of a patient or organ to one or more of the above disorders can be determined using genotypic, serological, or biochemical markers.

In some embodiments of the present invention, the antibody, conjugated antibody, activated antibody, and/or conjugated activated antibody is administered to humans diagnosed with a clinical indication associated with one or more of the above disorders. When making a diagnosis, an antibody, a conjugated antibody, an activated antibody, and/or a conjugated activated antibody is administered to attenuate or reverse the effects of the clinical indication.

The antibody, conjugated antibody, activated antibody and/or conjugated activated antibody of the invention are also useful for detecting a target in patient samples and are accordingly suitable for use as diagnostics. For example, the antibodies and/or activated antibodies and conjugated variants thereof of the present invention are used in in vitro assays, such as ELISA, to determine target levels in a patient sample.

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

The wells are then treated with a test sample suspected of containing the antigen or with a solution containing a standard amount of antigen. Such a sample is, for example, a blood serum sample from a subject suspected of having levels of circulating antigen that are considered diagnostic of the pathology. After washing the test sample or standard, the solid carrier is treated with a second antibody that contains a detectable label. The labeled second antibody serves as a detection antibody. Measure the level of detectable

- 107 039736 labels and determine the concentration of the target antigen in the test sample by comparison with a standard curve built on standard samples.

Obviously it is clear that based on the results obtained using antibodies and activated antibodies of the present invention, and their conjugated variants, in an in vitro diagnostic assay, it is possible to determine the stage of the disease in a subject based on the expression levels of the target antigen. For a particular disease, blood samples are taken from subjects diagnosed as being at different stages of disease progression and/or at different points in the therapeutic treatment of the disease. Using a sample population that provides statistically significant results for each stage of progression or treatment, a range of antigen concentrations is drawn up that can be considered representative of each stage.

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

For example, SM can be selected as a substrate for at least one protease found at the site of a tumor, at the site of a viral or bacterial infection at a biologically restricted site (eg, such as an abscess, organ, etc.), and the like. AB may be a molecule that binds to a target antigen. Using the methods disclosed herein or, when appropriate, methods known to one of skill in the art, a detectable label (e.g., a fluorescent label or a radioactive label or a radiotracer) can be conjugated to an AB or other region of an antibody and/or an activated antibody. Suitable detectable labels are discussed in the context of the above screening methods, and further specific examples are given below. By using an AB specific for a disease marker protein or peptide, together with at least one protease whose activity is elevated in the diseased tissue of interest, activated antibodies will exhibit an increased rate of binding to diseased tissues compared to tissues lacking the enzyme at a detectable level. or is present at a lower level than in the diseased tissue of the disease, or is inactive (for example, is in the form of a zymogen or in combination with an inhibitor). Since small molecular weight proteins and peptides are rapidly cleared from the blood by the renal filtration system, and because the enzyme specific for SM is absent at a detectable level (or present at lower levels in healthy tissues or present in an inactive conformation), the accumulation of activated antibodies in the patient tissue is enhanced compared to healthy tissues.

In yet another example, activated antibodies can be used to detect the presence or absence of a degradable agent in a sample. For example, when activated antibodies contain a SM that is sensitive to enzyme digestion, then the activated antibodies can be used to determine (qualitatively or quantitatively) the presence of the enzyme in a sample. In yet another example, when activated antibodies contain a CM that is sensitive to cleavage by a reducing agent, then the activated antibodies can be used to determine (qualitatively or quantitatively) the presence of reducing conditions in a sample. To facilitate analysis in these methods, the activated antibodies may be labeled with a detectable label and may be associated with a carrier (eg, a solid support such as glass or bead). The detectable label may be located in a region of the activated antibody that is not released after cleavage, for example, the detectable label may be a quenched fluorescent label or other label that is not detected until cleavage occurs. The assay can be performed, for example, by contacting immobilized, detectable, labeled, activated antibodies with a sample suspected of containing an enzyme and/or reducing agent for a time sufficient to digest, then washing to remove excess sample and contaminants. The presence or absence of a cleaving agent (e.g., enzyme or reducing agent) in the sample is then assessed by the change in the detectable signal of activated antibodies prior to contact with the sample, for example, by the presence and/or increase in the detected signal due to cleavage

- 108 039736 activated antibodies under the action of a cleaving agent in the sample.

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

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

These methods are suitable for use with any frozen cells or tissues obtained from a diseased site (eg, tumor tissue) or healthy tissue. Such methods are also suitable for fresh cell or tissue samples.

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

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

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

These methods are also useful in kits and/or as reagents for detecting, identifying, or characterizing protease activity in various cells, tissues, and organisms based on protease-specific SM in an activated antibody.

The present invention provides methods for using antibodies and/or activated antibodies for various diagnostic and/or prophylactic indications. For example, the present invention provides methods for detecting the presence or absence of a cleaving agent and a target of interest in a subject or sample by (i) contacting the subject or sample with an activated antibody, wherein the activated antibody contains a masking moiety (e.g., MM) that cleaves the moiety (CM), which is cleaved by a cleaving agent, such as a protease, and an antigen-binding site or fragment thereof (AB), which specifically binds to the target of interest, where the activated antibody in an uncleaved, inactive state has the following structural organization from the N-terminus to the C-terminus: MM-CM-AB or AB-CM-MM; (a) where MM is a peptide that inhibits AV binding to a target, and where MM does not have the amino acid sequence of a natural AB binding partner and is not a modified form of a natural AB binding partner; and (b) where, in an uncleaved inactive state, MM interferes with specific binding of AB to a target, and in a cleaved, activated state, MM does not interfere with or compete for specific binding of AB to a target; and (ii) measuring the level of activated activated antibody in the subject or sample, wherein the detectable level of activated activated antibody in the subject or sample indicates that the degraded

- 109 039736 the cleaving agent and the target are present in the subject or in the sample, and where an undetectable level of activated activated antibody in the subject or in the sample indicates that the cleaving agent, the target, or both the cleaving agent and the target are absent and/or insufficiently present in the subject or in the sample . In some embodiments, the activated antibody is an activated antibody to which the therapeutic agent is conjugated. In some embodiments, the activated antibody is not conjugated to an agent. In some embodiments, the implementation of the activated antibody contains a detectable label. In some embodiments, the detectable label is located in the AB. In some embodiments, measurement of the level of an activated antibody in a subject or sample is performed using a secondary reagent that specifically binds to the activated antibody, where the reagent contains a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

The present invention also provides methods for detecting the presence or absence of a cleaving agent in a subject or sample by (i) contacting the subject or sample with an activated antibody in the presence of a target of interest, such as a target, where the activated antibody contains a masking fragment (MM), a cleavable fragment (CM) , which is cleaved by a cleaving agent, such as a protease, and an antigen-binding site or fragment thereof (AB), which specifically binds to the target of interest, where the activated antibody in the uncleaved, non-activated state has the following structural organization from the N-terminus to the C-terminus: MM-CM-AB or AB-SM-MM; (a) where MM is a peptide that inhibits AV binding to a target, and where MM does not have the amino acid sequence of a natural AB binding partner and is not a modified form of a natural AB binding partner; and (b) where, in a non-cleaved, non-activated state, MM interferes with specific binding of AB to a target, and in a cleaved, activated state, MM does not interfere with or compete for specific binding of AB to a target; and (ii) measuring the level of activated activated antibody in the subject or sample, wherein the detectable level of activated activated antibody in the subject or sample indicates that the degrading agent is present in the subject or sample, and the undetectable level of activated activated antibody in the subject or sample indicates that the cleavage agent is absent and/or insufficiently present in the subject or in the sample. In some embodiments, the activated antibody is an activated antibody to which the therapeutic agent is conjugated. In some embodiments, the activated antibody is not conjugated to an agent. In some embodiments, the implementation of the activated antibody contains a detectable label. In some embodiments, the detectable label is located in the AB. In some embodiments, measurement of the level of an activated antibody in a subject or sample is performed using a secondary reagent that specifically binds to the activated antibody, where the reagent contains a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

The present invention also provides kits for use in methods for detecting the presence or absence of a cleaving agent and a target in a subject or sample, wherein the kits comprise at least an activated antibody that contains a masking fragment (MM), a cleavable fragment (CM), which is cleaved a cleaving agent, such as a protease, and an antigen-binding site or fragment thereof (AB), which specifically binds to the target of interest, where the activated antibody in the uncleaved, non-activated state has the following structural organization from the N-terminus to the C-terminus: MM-CM-AB or AB -SM-MM; (a) where MM is a peptide that inhibits AV binding to a target, and where MM does not have the amino acid sequence of a natural AB binding partner and is not a modified form of a natural AB binding partner; and (b) where, in a non-cleaved, non-activated state, MM interferes with specific binding of AB to a target, and in a cleaved, activated state, MM does not interfere with or compete for specific binding of AB to a target; and (ii) measuring the level of activated activated antibody in the subject or sample, where the detectable level of activated activated antibody in the subject or sample indicates that a degrading agent is present in the subject or sample, and where the undetectable level of activated activated antibody in the subject or sample indicates that the cleavage agent is absent and/or insufficiently present in the subject or in the sample. In some embodiments, the activated antibody is an activated antibody to which the therapeutic agent is conjugated. In some embodiments, the activated antibody is not conjugated to an agent. In some embodiments, the implementation of the activated antibody contains a detectable label. In some embodiments, the detectable label is located in the AB. In some embodiments, measurement of the level of an activated antibody in a subject or sample is performed using a secondary reagent that specifically binds to the activated antibody, where the reagent contains a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

- 110 039736

The present invention also provides methods for detecting the presence or absence of a cleaving agent in a subject or sample by (i) contacting the subject or sample with an activated antibody, wherein the activated antibody comprises a masking fragment (MM), a cleavable fragment (CM), which is cleaved by a cleaving agent, e.g. a protease, an antigen-binding site (AB) that specifically binds to the target, and a detectable label, where the activated antibody in the uncleaved, non-activated state has the following structural organization from the N-terminus to the C-terminus: MM-CM-AB or AB-CM-MM; where MM is a peptide that inhibits AV binding to a target, and where MM does not have the amino acid sequence of a natural AB binding partner and is not a modified form of a natural AB binding partner; where in a non-cleaved, non-activated state, MM prevents specific binding of AB to a target, and in a split, activated state, MM does not prevent or compete for specific binding of AB to a target; and where the detectable label is located in the area of the activated antibody, which is released after cleavage of the CM; and (ii) measuring the level of detectable label in the subject or sample, where the detectable level of detectable label in the subject or sample indicates that the cleavage agent is absent and/or insufficiently present in the subject or sample, and where the undetectable level of detectable label in the subject or in the sample indicates that the disintegrating agent is present in the subject or in the sample. In some embodiments, the activated antibody is an activated antibody to which the therapeutic agent is conjugated. In some embodiments, the activated antibody is not conjugated to an agent. In some embodiments, the implementation of the activated antibody contains a detectable label. In some embodiments, the detectable label is located in the AB. In some embodiments, measurement of the level of an activated antibody in a subject or sample is performed using a secondary reagent that specifically binds to the activated antibody, where the reagent contains a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

The present invention also provides kits for use in methods for detecting the presence or absence of a cleavage agent and a target in a subject or sample, wherein the kits comprise at least an activated antibody and/or a conjugated activated antibody (e.g., an activated antibody to which the therapeutic agent is conjugated ) described herein for use in contacting a subject or biological sample and a means for detecting a level of activated activated antibody and/or a conjugated activated antibody in the subject or biological sample, wherein the detected level of activated activated antibody in the subject or biological sample indicates that that the cleaving agent and the target are present in the subject or in the biological sample, and where an undetectable level of activated activated antibody in the subject or in the biological sample indicates that the cleaving agent, the target, or both the cleaving agent and the target are are absent and/or insufficiently present in the subject or in the biological sample such that target binding and/or protease cleavage of the activated antibody cannot be detected in the subject or in the biological sample.

The present invention also provides methods for detecting the presence or absence of a cleavage agent in a subject or sample by (i) contacting the subject or biological sample with an activated antibody in the presence of the target, and (ii) measuring the level of activated activated antibody in the subject or biological sample, where the detectable the level of activated activated antibody in the subject or in the biological sample indicates that the cleavage agent is present in the subject or in the biological sample, and where an undetectable level of activated activable antibody in the subject or in the biological sample indicates that the cleavage agent is absent and/or insufficiently present in the subject or in the biological sample at a detectable level, such that protease cleavage of the activated antibody cannot be detected in the subject or in the biological sample. Such an activated antibody includes a masking fragment (MM), a cleavable fragment (CM), which is cleaved by a cleaving agent, such as a protease, and an antigen-binding site or fragment (AB), which specifically binds to a target, where the activated antibody in the non-cleaved (i.e. inactivated) state has the following structural organization from the N-terminus to the C-terminus: MM-CM-AB or AB-CM-MM; (a) where MM is a peptide that inhibits AV binding to a target, and where MM does not have the amino acid sequence of a natural AV binding partner; and (b) where the MW of the activated antibody in the uncleaved state interferes with the specific binding of the AB to the target, and where the MW of the activated antibody in the cleaved (ie, activated) state does not interfere with or compete for the specific binding of the AB to the target. In some embodiments, the activated antibody is an activated antibody to which the therapeutic agent is conjugated. In some embodiments, the activated antibody is not conjugated to an agent. In some embodiments, a detectable label is attached to a masking fragment. In some embodiments, a detectable label is attached to the N-terminus of the cleavage fragment to a protease cleavage site. In some embodiments, one antigen binding site of AB is masked. In some embodiments, where an antibody of the present disclosure has at least two antigen binding sites, at least one antigen binding site is masked and at least one antigen binding site is not masked. In some embodiments, all antigen binding sites are masked. In some embodiments, the measurement step includes the use of a secondary reagent containing a detectable label.

The present invention also provides kits for use in methods for detecting the presence or absence of a cleavage agent and a target in a subject or sample, wherein the kits comprise at least an activated antibody and/or a conjugated activated antibody described herein, for use in contacting a subject or biological sample with an activated antibody in the presence of a target and measuring the level of activated activated antibody in the subject or in the biological sample, where the detectable level of activated activated antibody in the subject or in the biological sample indicates that the degrading agent is present in the subject or in the biological sample and where the undetectable level of activated activated antibody antibodies in the subject or in the biological sample indicates that the cleavage agent is absent and/or insufficiently present in the subject or in the biological sample at a detectable level such that protease cleavage of the activated antibody and cannot be detected in a subject or in a biological sample. Such an activated antibody includes a masking fragment (MM), a cleavable fragment (CM), which is cleaved by a cleaving agent, such as a protease, and an antigen-binding site or fragment (AB), which specifically binds to a target, where the activated antibody in the non-cleaved (i.e. inactivated) state has the following structural organization from the N-terminus to the C-terminus: MM-CM-AB or AB-CM-MM; (a) where MM is a peptide that inhibits AV binding to a target, and where MM does not have the amino acid sequence of a natural AV binding partner; and (b) where the MW of the activated antibody in the uncleaved state interferes with the specific binding of the AB to the target, and where the MW of the activated antibody in the cleaved (ie, activated) state does not interfere with or compete for the specific binding of the AB to the target. In some embodiments, the activated antibody is an activated antibody to which the therapeutic agent is conjugated. In some embodiments, the activated antibody is not conjugated to an agent. In some embodiments, a detectable label is attached to a masking fragment. In some embodiments, a detectable label is attached to the N-terminus of the cleavable fragment to a protease cleavage site. In some embodiments, one AB antigen-binding site is masked. In some embodiments, where an antibody of the present disclosure has at least two antigen-binding sites, at least one antigen-binding site is masked and at least one antigen-binding site is not masked. In some embodiments, all antigen binding sites are masked. In some embodiments, the measurement step includes the use of a secondary reagent containing a detectable label.

The present invention also provides kits for use in methods for detecting the presence or absence of a cleaving agent in a subject or sample, wherein the kits comprise at least an activated antibody and/or a conjugated activated antibody described herein, for use in contacting a subject or biological sample, and means for detecting the level of an activated activated antibody and/or a conjugated activated antibody in a subject or in a biological sample, where the activated antibody includes a detectable label, which is located on the site of the activated antibody, which is released after cleavage of SM, where the detectable level of activated activated antibody in the subject or in biological sample indicates that the cleavage agent is absent and/or insufficiently present in the subject or in the biological sample such that target binding and/or protease cleavage of the activated antibody cannot be detected in the subject or biological sample, and where a non-detectable level of activated activated antibody in the subject or biological sample indicates that a degrading agent is present in the subject or biological sample at a detectable level.

The present invention provides methods for detecting the presence or absence of a cleavage agent and a target in a subject or sample by (i) contacting the subject or biological sample with an activated antibody, wherein the activated antibody includes a detectable label that is located in a region of the activated antibody that is released after CM cleavage, and (ii) measuring the level of activated activated antibody in the subject or biological sample, where the detectable level of activated activated antibody in the subject or biological sample indicates that the cleavage agent, the target, or both the cleavage agent and the target are absent and/or insufficiently present in the subject, or in a biological sample such that target binding and/or protease cleavage of the activated antibody cannot be detected in the subject or in the biological sample, and where a reduced detectable level of activated activated antibody in the subject or biological in a biological sample indicates that the cleavage agent and target are present in the subject or in the biological sample. A reduced level of detectable label is, for example, a decrease of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90 %, about 95% and/or about 100%. Such an activated antibody includes a masking fragment (MM), a cleavable fragment (CM), which is cleaved by a cleaving agent, and an antigen-binding site or fragment (AB) that specifically binds to a target, where the activated antibody is in an uncleaved (i.e., non-activated) state has the following structural organization from the N-end to the End: MM-CM-AB or AB-CM-MM; (a) where MM is a peptide that inhibits AV binding to a target, and where MM does not have the amino acid sequence of a natural AV binding partner; and (b) where the MW of the activated antibody in the uncleaved state interferes with the specific binding of the AB to the target, and where the MW of the activated antibody in the cleaved (ie, activated) state does not interfere with or compete for the specific binding of the AB to the target. In some embodiments, the activated antibody is an activated antibody to which the therapeutic agent is conjugated. In some embodiments, the activated antibody is not conjugated to an agent. In some embodiments, the implementation of the activated antibody contains a detectable label. In some embodiments, the implementation of the detectable label is located in the AB. In some embodiments, measurement of the level of an activated antibody in a subject or sample is performed using a secondary reagent that specifically binds to the activated antibody, where the reagent contains a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

The present invention also provides kits for use in methods for detecting the presence or absence of a cleavage agent and a target in a subject or sample, wherein the kits comprise at least an activated antibody and/or a conjugated activated antibody described herein for use in contacting a subject or biological sample and means for detecting the level of activated activated antibody and/or conjugated activated antibody in the subject or in the biological sample, where the detected level of activated activated antibody in the subject or in the biological sample indicates that the cleavage agent, the target, or both the cleavage agent and the target are absent and/ or are not sufficiently present in the subject or in the biological sample such that target binding and/or protease cleavage of the activated antibody cannot be detected in the subject or in the biological sample, and where a reduced detectable level of activated The presence of an antibody in a subject or biological sample indicates that the cleavage agent and target are present in the subject or biological sample. A reduced level of detectable label is, for example, a decrease of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% about 50% about 55% about 60% about 65% about 70% about 75% about 80% about 85% about 90% about 95% and/or about 100%.

The present invention also provides methods for detecting the presence or absence of a cleavage agent in a subject or sample by (i) contacting the subject or biological sample with an activated antibody, wherein the activated antibody includes a detectable label that is located in a region of the activated antibody that is released after CM cleavage; and (ii) measuring the level of a detectable label in the subject or in the biological sample, where the detectable level of the detectable label in the subject or in the biological sample indicates that the cleavage agent is absent and/or insufficiently present in the subject or in the biological sample at a detectable level, such that cleavage a protease-activated antibody cannot be detected in the subject or in the biological sample, and wherein a reduced detectable level of a detectable label in the subject or in the biological sample indicates that a degrading agent is present in the subject or in the biological sample. A reduced level of detectable label is, for example, a decrease of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%. Such an activated antibody includes a masking fragment (MM), a cleavable fragment (CM), which is cleaved by a cleaving agent, and an antigen-binding site or fragment (AB) that specifically binds to a target, where the activated antibody is in an uncleaved (i.e., non-activated) state has the following structural organization from the N-terminus to the C-terminus: MM-CM-AB or AB-CM-MM; (a) where MM is a peptide that inhibits AV binding to a target, and where MM does not have the amino acid sequence of a natural AV binding partner; and (b) where the MW of the activated antibody in the uncleaved state interferes with the specific binding of the AB to the target, and where the MW of the activated antibody in the cleaved (ie, activated) state does not interfere with or compete for the specific binding of the AB to the target. In some embodiments

- 113 039736 an activated antibody is an activated antibody to which a therapeutic agent is conjugated. In some embodiments, the activated antibody is not conjugated to an agent. In some embodiments, the implementation of the activated antibody contains a detectable label. In some embodiments, the implementation of the detectable label is located in the AB. In some embodiments, measurement of the level of an activated antibody in a subject or sample is performed using a secondary reagent that specifically binds to the activated antibody, where the reagent contains a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

The present invention also provides kits for use in methods for detecting the presence or absence of a cleavage agent of interest in a subject or sample, wherein the kits comprise at least an activated antibody and/or a conjugated activated antibody described herein for use in contacting a subject or a biological sample and means for determining the level of an activated activated antibody and/or a conjugated activated antibody in a subject or in a biological sample, where the activated antibody includes a detectable label that is located in the site of the activated antibody that is released after SM cleavage, where the detectable level of the detectable label is at subject or in a biological sample indicates that the cleavage agent, the target, or both the cleavage agent and the target are absent and/or insufficiently present in the subject or in the biological sample such that binding to the target and/or cleavage by the protease an activated antibody cannot be detected in the subject or biological sample, and wherein a reduced detectable level of detectable label in the subject or biological sample indicates that the cleavage agent and target are present in the subject or biological sample. A reduced level of detectable label is, for example, a decrease of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% about 50% about 55% about 60% about 65% about 70% about 75% about 80% about 85% about 90% about 95% and/or about 100%.

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

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

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

In some embodiments, in situ imaging and/or in vivo imaging is useful in methods for identifying patients to be treated. For example, in situ imaging, activated antibodies are used to screen patient samples to identify patients having the appropriate protease(s) and target(s) at the appropriate location, such as a tumor site.

In some embodiments, in situ imaging is used to identify or otherwise refine a patient population suitable for treatment with an activated antibody of the present disclosure. For example, patients who test positive for one

- 114 039736 transiently to the target (eg, target) and to a protease that cleaves the substrate in the cleavable fragment (CM) of the activated antibody being tested (eg, accumulating activated antibodies at the disease site) are identified as suitable candidates for treatment with such an activated antibody containing such a SM. Similarly, patients who test negative for one or both of a target (e.g., a target) and a protease that cleaves a substrate in the SM in an activated antibody that is tested using these methods may be identified as suitable candidates for another therapy. In some embodiments, such patients who test negative for the first activated antibody may be tested with other activated antibodies containing different CMs until a suitable activated antibody for treatment is identified (e.g., an activated antibody containing SM, which is cleaved by the patient at the site of the disease). In some embodiments, the patient is then administered a therapeutically effective amount of an activated antibody for which the patient has tested positive.

In some embodiments, in vivo imaging is used to identify or otherwise refine a patient population suitable for treatment with an activated antibody of the present invention. For example, patients who test positive both for the target (e.g., target) and for a protease that cleaves the substrate in the cleavable fragment (CM) of the activated antibody that is being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates. for treatment with such an activated antibody containing such SM. Similarly, patients who test negative may be identified as suitable candidates for another type of therapy. In some embodiments, such patients who test negative for the first activated antibody may be tested with other activated antibodies containing different CMs until a suitable activated antibody for treatment is identified (e.g., an activated antibody containing SM, which is cleaved by the patient at the site of the disease). In some embodiments, the patient is then administered a therapeutically effective amount of an activated antibody for which the patient has tested positive.

In some embodiments of the methods and kits, the method or kit is used to identify or otherwise refine a patient population suitable for treatment with an activated antibody of the present invention. For example, patients who test positive both for the target (e.g., target) and for the protease that cleaves the substrate in the cleavable fragment (CM) of the activated antibody that is tested in these methods are identified as suitable candidates for treatment with such an activated antibody. containing such a SM. Similarly, patients who test negative for both a target (eg, a target) and a protease that cleaves a substrate in the SM in an activated antibody that is tested using these methods may be identified as suitable candidates for another therapy. In some embodiments, such patients may be tested with other activated antibodies until a suitable activated antibody for treatment (eg, an activated antibody containing CM that is cleaved by the patient at the disease site) is identified. In some embodiments, patients who test negative for any target (eg, target) are identified as suitable candidates for treatment with such an activated antibody containing such a CM. In some embodiments, patients who test negative for any target (eg, target) are identified as unsuitable candidates for treatment with such an activated antibody containing such a SM. In some embodiments, such patients may be tested with other activated antibodies until a suitable activated antibody for treatment is identified (eg, an activated antibody containing CM that is cleaved by the patient at the disease site). In some embodiments, the activated antibody is an activated antibody to which the therapeutic agent is conjugated. In some embodiments, the activated antibody is not conjugated to an agent. In some embodiments, the implementation of the activated antibody contains a detectable label. In some embodiments, the detectable label is located in the AB. In some embodiments, measurement of the level of an activated antibody in a subject or sample is performed using a secondary reagent that specifically binds to the activated antibody, where the reagent contains a detectable label. In some embodiments, the secondary reagent is an antibody containing a detectable label.

In some embodiments, the method or kit is used to identify or otherwise refine a population of patients suitable for treatment with an activated antibody and/or a conjugated target activated antibody (e.g., an activated antibody conjugated to a therapeutic agent) of the present invention, followed by treatment with the administration of the present invention.

- 115 039736 an activated antibody and/or a conjugated activated antibody to a subject in need thereof. For example, patients who, when tested, test positive for both the target (e.g., target) and the protease that cleaves the substrate in the cleavable fragment (CM) of the activated antibody and/or the conjugated activated antibody that is tested in these methods are identified as suitable candidates. for treatment with such antibody and/or such conjugated activated antibody containing such SM, and the patient is then administered a therapeutically effective amount of the activated antibody and/or conjugated activated antibody that has been tested. Similarly, patients who test negative for one or both of the target (eg, target) and the protease that cleaves the substrate in the SM in the activated antibody that is tested using these methods may be identified as suitable candidates for another therapy. In some embodiments, such patients may be tested with another antibody and/or conjugated activated antibody until a suitable antibody and/or conjugated activated antibody for treatment (e.g., an activated antibody and/or a conjugated activated antibody containing CM) is identified. , which is cleaved by the patient at the site of the disease). In some embodiments, the patient is then administered a therapeutically effective amount of an activated antibody and/or a conjugated activated antibody for which the patient has tested positive.

In some embodiments of these methods and kits, MM is a peptide having a length of about 4 to 40 amino acids. In some embodiments of these methods and kits, the activated antibody comprises a linker peptide, wherein the linker peptide is located between the MM and the SM. In some embodiments of these methods and kits, the activated antibody comprises a linker peptide, wherein the linker peptide is located between AB and CM. In some embodiments of these methods and kits, the activated antibody comprises a first linker peptide (L1) and a second linker peptide (L2), wherein the first linker peptide is located between MM and CM and the second linker peptide is located between AB and CM. In some embodiments of these methods and kits, each of L1 and L2 is a peptide of 1 to 20 amino acids in length, and wherein each of LI and L2 need not be the same linker. In some embodiments of these methods and kits, one or both of L1 and L2 comprise a glycine-serine polymer. In some embodiments of these methods and kits, at least one of L1 and L2 contains an amino acid sequence selected from the group consisting of (GS) _n , (GSGGS) _n (SEQ ID NO: 191) and (GGGS) _n (SEQ ID NO: ID NO: 192), where n is an integer, at least one. In some embodiments of these methods and kits, at least one of L1 and L2 contains an amino acid sequence having the formula (GGS) _n , where n is an integer, at least one. In some embodiments of these methods and kits, at least one of L1 and L2 contains an amino acid sequence selected from the group consisting of

Gly-Gly-Ser-Gly (SEQ ID NO: 193) , Gly-GlyISer-Gly-Gly (SEQ ID NO: 194), Gly-Ser-Gly-Ser-Gly (SEQ ID NO:

195), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 196), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 197), and Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 198).

In some embodiments of these methods and kits, the AB comprises an antibody or antibody fragment selected from the cross-reactive antibody sequences provided herein. In some embodiments of these methods and kits, the AB comprises a Fab fragment, scFv, or a single chain antibody (scAb).

In some embodiments of these methods and kits, the cleaving agent is a protease that colocates with a target in a subject or sample, and the CM is a polypeptide that functions as a substrate for the protease, wherein the protease cleaves the CM in the activated antibody when the activated antibody is exposed to proteases. In some embodiments of these methods and kits, the CM is a polypeptide up to 15 amino acids in length. In some embodiments of these methods and kits, the SM is linked to the N-terminus of the AB. In some embodiments of these methods and kits, the CM is linked to the C-terminus of the AB. In some embodiments of these methods and kits, the CM is linked to the N-terminus of the VL AB chain.

The antibodies, conjugated antibodies, activated antibodies and/or conjugated activated antibodies of the present invention are used in diagnostic and prophylactic compositions. In one embodiment, an activated antibody is administered to patients who are at risk for developing one or more of the above inflammation, inflammatory diseases, cancer, or other disorders.

The predisposition of a patient or organ to one or more of the above disorders can be determined using genotypic, serological, or biochemical markers.

In some embodiments, the antibody, conjugated antibody, activated antibody, and/or conjugated activated antibody is administered to a person diagnosed with a clinical symptom associated with one or more of the above disorders. After diagnosis, the antibody, conjugated antibody, activated antibody, and/or conjugated activated antibody is administered to alleviate or reverse the effects of the clinical disorder.

The antibodies, conjugated antibodies, activated antibodies and/or conjugated activated antibodies of the invention are also suitable for target detection in patient samples and accordingly they are suitable for use as diagnostics. For example, the antibodies, conjugated antibodies, activated antibodies, and conjugated activated antibodies of the present invention are used in in vitro assays, such as ELISA, to determine target levels in a patient sample.

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

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

Obviously, based on the results obtained using antibodies and activated antibodies of the present invention in an in vitro diagnostic assay, it is possible to determine the stage of the disease in a subject based on the expression levels of the target antigen. For a given disease, blood samples are taken from subjects diagnosed as being at different stages of disease progression and/or at different points in the therapeutic treatment of the disease. Using a sample population that provides statistically significant results for each stage of progression or treatment, a range of antigen concentrations is drawn up that can be considered representative of each stage.

Antibodies, conjugated antibodies, activated antibodies and/or conjugated activated antibodies can also be used in diagnostic and/or imaging techniques. In some embodiments, such methods are in vitro methods. In some embodiments, such methods are in vivo methods. In some embodiments, such methods are in situ methods. In some embodiments, such methods are ex vivo methods. For example, activated antibodies having enzymatically cleavable CM can be used to detect the presence or absence of an enzyme that is capable of cleaving CM. Such activated antibodies can be used in diagnostics, which can include in vivo detection (e.g., qualitatively or quantitatively) of enzyme activity (or, in some embodiments, an environment with an increased reduction potential, such as an environment that can provide disulfide bond reduction) by measured accumulation of activated antibodies (i.e., antibodies resulting from cleavage of an activated antibody) in a given cell or tissue of a given host organism. This accumulation of activated antibodies not only indicates that the tissue is expressing enzymatic activity (or increased repair potential depending on the nature of the SM), but also that the tissue is expressing a target to which the activated antibody binds.

For example, CM can be selected as a substrate for a protease found at a tumor site, at a viral or bacterial infection site at a biologically restricted site (eg, such as an abscess, organ, etc.), and the like. AB can be a molecule that binds to a target antigen. Using methods known to the person skilled in the art, a detectable label (eg, a fluorescent label, or a radioactive label, or a radiotracer) can be conjugated to the AB or other region of the antibody and/or the antibody to be activated. Suitable detectable labels are discussed in the context of the above screening methods and further specific examples are provided below. By using an AB specific for a disease marker protein or peptide, together with at least one protease whose activity is elevated in the diseased tissue of interest, activated antibodies will exhibit an increased rate of binding to diseased tissues compared to tissues lacking the enzyme at a detectable level. or is present at a lower level than in the diseased tissue of the disease, or is inactive (for example, is in the form of a zymogen or in combination with an inhibitor). Since low molecular weight

- 117 039736 proteins and peptides are rapidly cleared from the blood by the renal filtration system, and because the enzyme specific for SM is absent at a detectable level (or present at lower levels in healthy tissues or present in an inactive conformation), the accumulation of activated antibodies in diseased tissue is enhanced compared to healthy tissues.

In yet another example, activated antibodies can be used to detect the presence or absence of a degradable agent in a sample. For example, when activated antibodies contain a SM that is sensitive to enzyme digestion, then the activated antibodies can be used to determine (qualitatively or quantitatively) the presence of the enzyme in a sample. In yet another example, when activated antibodies contain a CM that is sensitive to cleavage by a reducing agent, then the activated antibodies can be used to determine (qualitatively or quantitatively) the presence of reducing conditions in a sample. To facilitate analysis in these methods, the activated antibodies may be labeled with a detectable label and may be associated with a carrier (eg, a solid support such as glass or bead). The detectable label may be located in a region of the activated antibody that is not released after cleavage, for example, the detectable label may be a quenched fluorescent label or other label that is not detected until cleavage occurs. The assay can be performed, for example, by contacting immobilized, detectable, labeled, activated antibodies with a sample suspected of containing an enzyme and/or reducing agent for a time sufficient to digest, then washing to remove excess sample and contaminants. The presence or absence of a cleaving agent (e.g., an enzyme or a reducing agent) in the sample is then assessed by a change in the detectable signal of activated antibodies prior to contact with the sample, for example, by the presence and/or increase in the detected signal due to cleavage of the activated antibody by the cleaving agent in the sample.

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

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

These methods are suitable for use with any frozen cells or tissues obtained from a disease site (eg, tumor tissue) or healthy tissue. Such methods are also suitable for fresh cell or tissue samples.

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

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

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

These methods are also useful in kits and/or as reagents for detecting, identifying, or characterizing protease activity in various cells, tissues, and organisms based on protease-specific SM in an activated antibody.

In some embodiments, in situ imaging and/or in vivo imaging is useful in methods for identifying patients to be treated. For example, in situ imaging, activated antibodies are used to screen patient samples to identify patients having the appropriate protease(s) and target(s) at the appropriate location, such as a tumor site.

In some embodiments, in situ imaging is used to identify or otherwise refine a patient population suitable for treatment with an activated antibody of the present disclosure. For example, patients who test positive both for the target and for a protease that cleaves the substrate in the cleavable fragment (CM) of the activated antibody being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates for treatment with such an activated antibody containing such a CM. Similarly, patients who test negative for one or both of the target and the protease that cleaves the substrate in the SM in the activated antibody that is tested using these methods are identified as eligible candidates for another therapy (i.e., unsuitable for treatment). the activated antibody being tested). In some embodiments, such patients who test negative for the first activated antibody may be tested with other activated antibodies containing different CMs until a suitable activated antibody for treatment is identified (e.g., an activated antibody containing SM, which is cleaved by the patient at the site of the disease).

In some embodiments, in vivo imaging is used to identify or otherwise refine a patient population suitable for treatment with an activated antibody of the present invention. For example, patients who test positive for both the target and the protease that cleaves the substrate in the cleavable fragment (CM) of the activated antibody that is being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates for treatment with that activated antibody. containing such a SM. Similarly, patients who test negative are identified as suitable candidates for another type of therapy (ie, unsuitable for treatment with the activated antibody that is being tested). In some embodiments, those patients who test negative for the first activated antibody may be tested with other activated antibodies containing different SMs until a suitable activated antibody for treatment is identified (i.e. activated antibody containing SM, which is cleaved by the patient at the site of the disease).

Pharmaceutical compositions

Antibodies, conjugated antibodies, activated antibodies and/or conjugated activated antibodies of the present invention (also referred to herein as active compounds) and derivatives, fragments, analogs and homologues thereof may be included in pharmaceutical compositions suitable for administration. Such compositions typically include an antibody, a conjugated antibody, an activated antibody and/or a conjugated activated antibody, and a pharmaceutically acceptable carrier. As used herein, the term pharmaceutically acceptable carrier is intended to include any and all diluents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference guide in the art, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, Ringer's saline solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except when any conventional media or agent is incompatible with the active compound, their use in the compositions is contemplated. Additional active compounds may also be included in the compositions.

The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and agents for the regulation of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The parenteral preparation may be enclosed in ampoules, disposable syringes or multi-dose glass or plastic vials.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (when soluble in water) or dispersions and sterile powders for the preparation ex tempore of sterile injectable solutions or dispersions. For intravenous administration, suitable vehicles include saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that it can be easily drawn into a syringe. It must be stable under the conditions of production and storage and must be stored without contamination by microorganisms such as bacteria and fungi. The carrier can serve as a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, maintaining the required particle size in the case of a dispersion, and using surfactants. Prevention of the action of microorganisms can be achieved with various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, it will be desirable to include isotonic agents, eg sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by the inclusion of an agent that delays absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or more of the ingredients listed above, followed by filtered sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile filtered solution.

Compositions for oral administration typically include an inert diluent or edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For purposes of oral therapeutic administration, the active compound may be administered with excipients and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash, wherein the compound in the liquid carrier is applied orally and rinsed, spit out or swallowed. Pharmaceutically compatible binding agents and/or auxiliary materials may be included in the composition. Tablets, pills, capsules, lozenges and the like may contain any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a lubricant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavor such as peppermint, methyl salicylate, or orange flavor.

For inhalation administration, the compounds are delivered as an aerosol spray from a pressurized container or dispenser that contains a suitable propellant, for example a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be carried out using transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants corresponding to the permeable barrier are used in the compositions. Such penetrants are commonly known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be carried out using nasal sprays or suppositories. For transdermal administration, the active compounds are formulated as ointments, salves, gels or creams, as is known in the art.

The compounds may also be formulated as suppositories (eg, with common suppository bases such as cocoa butter and other glycerides) or retention enemas for

- 120 039736 rectal delivery.

In one embodiment, active compounds are formulated with carriers that protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Those skilled in the art will recognize methods for preparing such formulations. Materials may also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes directed to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. They can be prepared by methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

It is particularly advantageous to formulate oral or parenteral compositions in unit dosage form for ease of administration and dosage uniformity. As used here, a single dosage form refers to physically discrete units suitable as unit doses for a subject to be treated; where each unit contains a predetermined amount of the active compound, calculated to obtain the desired therapeutic effect in combination with the required pharmaceutical carrier. The specification for unit dosage forms is disclosed and is directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved and the limitations in formulating such an active compound for the treatment of patients.

Pharmaceutical compositions may be included in a container, pack or dispenser along with instructions for administration.

The invention will be further described in the following examples, which do not limit the scope of the invention as set forth in the claims.

Examples

Example 1 Selection of embodiments of human scFvs that bind to human and/or mouse PDL1.

This example shows that scFvs (single chain variable fragments) that bind to PDL1 can be selected from an scFv phage display library with different CDR sequences, and that such binding can inhibit PDL1 binding to PD1 and B7-1.

scFvs were selected from a fully human ScFv library exposed to bacteriophage M13; selection of scFv-bearing phages was performed under contract with Creative Biolabs, Shirley, NY. A fusion protein consisting of the extracellular domain (ECD) of human PDL1 or mouse PDL1 with a His6 carboxy-terminal tag (Sino Biological, Cat. No. 10084-H02H-200) was used as antigen in three alternating rounds of selection for scFvs exposed to M13 bacteriophage, that bind to PDL1. In the first round, bound phage was released by trypsin digestion, and in subsequent rounds, phage was eluted with human PD1 fusion protein (R&D Systems, Cat. No. 1080PD-050). Five (5) unique scFvs have been isolated that bind to PDL1. Table 1 lists 5 scFvs and SEQ ID NOs of their respective nucleic acid and amino acid sequences.

Table 1. SEQ ID NO of selected scFvs

scFv Acid sequence nucleic Amino acid sequence PDL1 c8 SEQID NO: one SEQ ID NO: 2 PDL1 cl2 SEQID NO: 3 SEQ ID NO: 4 PDL1 cl6 SEQID NO: 5 SEQ ID NO: 6 PDL1 c20 SEQID NO: 7 SEQ ID NO: 8 PDL1 c60 SEQID NO: 9 SEQ ID NO: 10

The nucleic acid and amino acid sequences of each of the anti-PDL1 scFvs (with underlined CDRs) are shown below.

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SEQ ID NO: 1

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAGATATTACTGCGTCGGGTTAGAGGACAACGTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAGATCGAAGATTGCTTTTGACTACTGGGGCCAGGGAACCCTGGT CACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATC CAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGG CAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCT GATCTATAAGGCATCCCGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACA GATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGCGTG CGCTTAAGCCTGTGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGG

SEQ ID NO: 2

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDITASGQRTTYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCARSKIAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDI QMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQRALKPVTFGQGTKVEIKR

SEQ ID NO: 3

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTATCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAAGTATTAATAAGGATGGTCATTATACAAGTTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAAAATCTTGATGAGTTTGACTACTGGGGCCAGGGAACCCTGGT CACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATC CAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGG CAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCT GATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACA GATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTT ACAGTACCCCTAATACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGG

SEQ ID NO: 4

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSINKDGHYTSYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNLDEFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDI QMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR

SEQ ID NO: 5

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GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCATCTATTATGGCTACTGGTGCTGGTACATTGTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAAGATGGTGCGGGGTTTGACTACTGGGGCCAGGGAACCCTGGT CACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATC CAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGG CAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCT GATCTATTCTGCATCCCAGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACA GATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGCGA ATTCGCGGCCTTCTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGG

SEQ ID NO: 6

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIMATGAGTLYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKpGAGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDI QMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASQLQSGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQANSRPSTFGQGTKVEIKR

SEQ ID NO: 7

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GTAGTGGGTCTCAACTATTACTTCTTCTGGTGCTGCTACATATTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAAAATTATACTGGTTTTGACTACTGGGGCCAGGGAACCCTGGT

CACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATC

CAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGG CAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCT GATCTATAATGCATCCTCCTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACA GATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATA CTTATGGTCCTGGTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGG

SEQ ID NO: 8

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLQWVSTITSSGAATYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNYTGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDI QMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYNASSLQSGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQYTYGPGTFGQGTKVEIKR

SEQ ID NO: 9

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT

CTCCTGTG

- 123 039736

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG

CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT

GGAGTGGGTCTCAAGTATTTATTCTACTGGTGGTGCTACAGCTTACGCAGACTCCGTGAAGGGCCGG

TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG

ACACGGCCGTATATTACTGTGCGAAATCTTCTGCTGGTTTTGACTACTGGGGCCAGGGAACCCTGGT

CACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATC

CAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGG

CAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCT

GATCTATTATGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACA

GATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGATA

ATGGTTATCCTTCTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGG

SEQ ID NO: 10

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIYSTGGATAYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDI QMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYASTLQSGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQDNGYPSTFGQGTKVEIKR

In FIG. 1 shows the results of an ELISA-based binding assay showing that c60 scFv phage PDL1 binds specifically to human and mouse PDL1. Briefly, human PDL1, mouse PDL1, human PD1, mouse PD1, human 41BBLig or Notchl (R&D Systems) were adsorbed into individual wells of a 96-well ELISA plate. The phage was introduced into the wells of the tablet and left for binding. Bound phage was visualized using an anti-M13-HRP conjugate (GE Healthcare, Piscataway, NY) and developed using tetramethylbenzidine (TMB) chromogenic substrate (Thermo Scientific, Rockford, IL).

In FIG. 2 shows that PD1 incorporation can inhibit scFv phage PDL1 binding to PDL1 in an ELISA-based binding assay. Briefly, human PDL1 or mouse PDL1 (R&D Systems) were adsorbed into individual wells of a 96-well ELISA plate. The phage was introduced into the wells of the plate in the presence and absence of human and mouse PD1-Fc (R&D Systems) and allowed to bind. Bound phage was visualized using an anti-M13-HRP conjugate (GE Healthcare, Piscataway, NY) and developed using tetramethylbenzidine (TMB) chromogenic substrate (Thermo Scientific, Rockford, IL). Phage binding was reduced in the presence of PD1-Fc, demonstrating that the phage binding epitope overlapped with that for PD1 binding.

Example 2 Preparation and testing of fully human anti-PDL1 c60 IgG antibodies.

This example shows that PDL1 c60 scFv phage that binds to human and mouse PDL1 can be converted into fully human IgG antibodies that retain the ability to bind to human and mouse PDL1.

Obtaining fully human IgG containing PDL1 c60 variable regions was carried out using methods similar to those described in the publication of the international application PCT WO 2010/081173. DNA molecules encoding scFv phage c60 PDL1 variable regions were cloned into expression vectors for the expression of fully human IgG. Light chain variable regions (Lc) were amplified from scFv arrays. The vector (LcpOP (modified from pCDNA3, Invitrogen, Carlsbad, CA)) and amplified DNA were digested overnight with BsiWI and EcoRI, ligated together, and transformed into E. coli MC1061 cells. Heavy chain variable regions (Hc) were amplified from scFv templates. Fully human anti-PDL1 c60 IgG was expressed from transiently transfected HEK-293 cells and purified from the culture supernatant by protein A chromatography.

In FIG. 3 shows that fully human anti-PDL1 c60 IgG binds to human and mouse PDL1 with an affinity of 1 nM for human PDL1 and 30 nM for mouse PDL1. Briefly, human PDL1-Fc or mouse PDL1-Fc (R&D Systems, Minneapolis, MN) was adsorbed into the wells of a 96-well ELISA plate. Purified anti-PDL1 c60 was added to the wells of the plate and allowed to bind. Bound antibody was visualized using an anti-human Fab-specific IgG-HRP conjugate (Sigma, St. Louis, MO, Cat # A0293-1ML) and developed using TMB chromogenic substrate.

Example 3 Affinity maturation of anti-PDL1 antibody embodiments.

This example shows the isolation of embodiments of antibodies with improved binding kinetics and an improved production manufacturability profile.

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Anti-PDL1 antibodies were isolated from CDR libraries modified from anti-PDLl c60. These libraries were constructed as shown in Tables 2 and 3. Four Fab phage libraries based on the c60 anti-PDL1 sequence were constructed using degenerate synthetic DNA. The residues were changed by randomization at each indicated nucleotide. In addition, additional randomized residues to the heavy chain CDR3 were added to libraries 3 and 6. Libraries were transfected into E. coli TGI strain and phage was obtained after superinfection with M13KO7 (Invitrogen).

Performed three rounds of selection for each library under increasing stringency. Three alternating rounds of screening were performed by mixing biotinylated human PDL1 or biotinylated mouse PDL1 with phage, the mixture was blocked with 100 μg/ml pooled human IgG (hulgG or hlgG) and 2% skimmed milk (NFDM) in Tris-buffered saline (TBS, 40 mM Tris, 129 mM NaCl, pH 7.4). After incubation at room temperature, phage-bound Bio-PDL1 was captured on streptavidin-coated magnetic beads, which were washed vigorously at 37°C, and the remaining bound phage was eluted with 100 mM triethanolamine (TEA) (Sigma, St. Louis, MO). and produced in TGI E. coli. Selected Lc libraries were combined with Hc libraries and each was subjected to an additional seven rounds of selection. Table 2. CDR sequences for affinity matured heavy chain variable region libraries

Table 3. CDR sequences for affinity matured light chain variable region libraries

ID NO:

(SEQID NO: 179) (SEQID NO: 179) (SEQID NO: 179) (SEQID NO: 179) (SEQID NO: 179) ., .. (SEQID NO: 179) L2nnk+L3walk (SEQ ID NO: 179) (SEQID NO: 179) (SEQID NO: 179) (SEQID NO: 179) (SEQID NO: 179) (SEQID NO: 179) X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X

1.26E+07

Forty-seven (47) individual isolates were selected from the final pool. Phage was obtained from each isolate and analyzed for binding to antigens, human PDL1-Fc, human B7-1-FC and human CD28-Fc (R&D Systems). Briefly, antigens were adsorbed into the wells of a 96-well ELISA plate with each ligand in a separate plate. The phage was introduced into the appropriate wells of each plate and left to bind. Bound phage was visualized using an anti-M13-HRP conjugate and developed using the TMB chromogenic substrate. Based on the ELISA results and DNA sequence, 6 unique clones with mature heavy chain variable CDR3s and 10 heavy chain variable CDR2s were selected for further study. 13 phage clones, 10 CDR2 and 3 CDR3, were analyzed for their method

- 125 039736 binding to mouse and human PDL1, and the results of the phage ELISA are shown in FIG. 4A and B. All phage clones bind to human PDL1 but not B7-1, in addition CDR2 clones C05 and G12 show strong binding to mouse PDL1 and CDR3 clone H9 shows weak binding to mouse PDL1. Table 4 lists the heavy chain variable regions encoded by the clones and SEQ ID NOs for the nucleic acid and amino acid sequences of their respective heavy chains.

The light chain variable regions for each are identical to the c60 domains (SEQ ID NOS 11 and 12). SEQ ID NO: 11

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTT

GCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAA

GCTCCTGATCTATTATGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCT GGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAAC AGGATAATGGTTATCCTTCTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGG

SEQ ID NO: 12

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGCARKLLIYYASTLQSGVPSRFSGSGS

GTDFTLTIS SLQPEDFATYYCQQDNGYPSTFGQGTKVEIKR

Table 4

vh Nucleic acid sequence Amino acid sequence PDL1 cl SEQ ID NO 13 SEQ ID NO: fourteen PDL1 dl SEQ ID NO fifteen SEQ ID NO: 16 PDL1 g7 SEQ ID NO 17 SEQ ID NO: eighteen PDL1 h9 SEQ ID NO 19 SEQ ID NO: twenty PDL1 NO SEQ ID NO 21 SEQ ID NO: 22 PDL1 EY SEQ ID NO 23 SEQ ID NO: 24 PDL1 AO 5 SEQ ID NO 25 SEQ ID NO: 26 PDL1 C05 SEQ ID NO 27 SEQ ID NO: 28 PDL1 CIO SEQ ID NO 29 SEQ ID NO: thirty

- 126 039736

PDL1 D08 SEQ ID NO: 31 SEQ ID NO: 32 PDL1 G09 SEQ ID NO: 33 SEQ ID NO: 34 PDL1 GIO SEQ ID NO: 35 SEQ ID NO: 36 PDL1 G12 SEQ ID NO: 37 SEQ ID NO: 38 PDL1 El SEQ ID NO: 39 SEQ ID NO: 40 PDL1 DOI SEQ ID NO: 41 SEQ ID NO: 42 PDL1 H06 SEQ ID NO: 43 SEQ ID NO: 44

SEQ ID NO: 13

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAAGTATTTATTCTACTGGTGGTGCTACAGCTTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAATCTTCTGCTGGTAGTCGGCCGGGTTTTGACTACTGGGGCCA GGGAACCCTGGTCACCGTCTCGAGC

SEQ ID NO: 14

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIYSTGGATAYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGQSRPGFDYWGQGTLVTVSS

SEQ ID NO: 15

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAAGTATTTATTCTACTGGTGGTGCTACAGCTTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAATCTTCTGCTGGTTCGTGGCCGGGTTTTGACTACTGGGGCCA GGGAACCCTGGTCACCGTCTCGAGC

SEQ ID NO: 16

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIYSTGGATAYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGQSWPGFDYWGQGTLVTVSS

SEQ ID NO: 17

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAAGTATTTATTCTACTGGTGGTGCTACAGCTTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAATCTTCTGCTGGTCAGTCGTTTCCGGGTTTTGACTACTGGGG CCAGGGAACCCTGGTCACCGTCTCGAGC

SEQ ID NO: 18

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIYSTGGATAYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGQSFPGFDYWGQGTLVTVSS

SEQ ID NO: 19

- 127 039736

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAAGTATTTATTCTACTGGTGGTGCTACAGCTTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAATGGTCTGCTGCTTTTGACTACTGGGGCCAGGGAACCCTGGT CACCGTCTCGAGC

SEQ ID NO: 20

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIYSTGGATAYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDYWGQGTLVTVSS

SEQ ID NO: 21

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAAGTATTTATTCTACTGGTGGTGCTACAGCTTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAATGGTCTGCTGCTTTTGACTACTGGGGCCAGGGAACCCTGGT CACCGTCTCGAGC

SEQ ID NO: 22

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIYSTGGATAYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAGYDYWGQGTLVTVSS

SEQ ID NO: 23

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAAGTATTTATTCTACTGGTGGTGCTACAGCTTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAATGGTCTAAGGGTTTTGACTACTGGGGCCAGGGAACCCTGGT CACCGTCTCGAGC

SEQ ID NO: 24

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIYSTGGATAYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSKGFDYWGQGTLVTVSS

SEQ ID NO: 25

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAAGTATTTGGAAGTAGGGTATTGTGACAGTGAGCTTACGCAGACTCCGTGAAGGG CCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC GAGGACACGGCCGTATATTACTGTGCGAAATCTTCTGCTGGTTTTGACTACTGGGGCCAGGGAACCC TGGTCACCGTCTCGAGC

SEQ ID NO: 26

- 128 039736

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWKQGIVTVYDSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTV

SEQ ID NO: 27

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAAGTATTTGGCGGAATGGTATTGTTACAGTTAGCTTACGCAGACTCCGTGAAGGG CCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC GAGGACACGGCCGTATATTACTGTGCGAAATCTTCTGCTGGTTTTGACTACTGGGGCCAGGGAACCC TGGTCACCGTCTCGAGC

SEQ ID NO: 28

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYDSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS

SEQ ID NO: 29

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAGATATTTGGAAGTAGGGTATGGTTACAGTGAGCTTACGCAGACTCCGTGAAGGG CCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC GAGGACACGGCCGTATATTACTGTGCGAAATCTTCTGCTGGTTTTGACTACTGGGGCCAGGGAACCC TGGTCACCGTCTCGAGC

SEQ ID NO: 30

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDIWKQGMVTVYDSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS

SEQ ID NO: 31

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCATCGATTTGGAGGTAGGGTCTGGCGACAGCGAGCTTACGCAGACTCCGTGAAGGG CCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC GAGGACACGGCCGTATATTACTGTGCGAAATCTTCTGCTGGTTTTGACTACTGGGGCCAGGGAACCC TGGTCACCGTCTCGAGC

SEQ ID NO: 32

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRQGLATAYDSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS

SEQ ID NO: 33

- 129 039736

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAGAGATTGTGGCTACTGGTATTTTGACAAGTAGCTTACGCAGACTCCGTGAAGGG CCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC GAGGACACGGCCGTATATTACTGTGCGAAATCTTCTGCTGGTTTTGACTACTGGGGCCAGGGAACCC TGGTCACCGTCTCGAGC

SEQ ID NO: 34

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSEIVATGILTSYDSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS

SEQ ID NO: 35

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCATCGATTGGTCGGTAGGGTTTGATTACAGTTAGCTTACGCAGACTCCGTGAAGGG CCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC GAGGACACGGCCGTATATTACTGTGCGAAATCTTCTGCTGGTTTTGACTACTGGGGCCAGGGAACCC TGGTCACCGTCTCGAGC

SEQ ID NO: 36

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIGRQGLITVYDSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS

SEQ ID NO: 37

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCATCTATTTGGTATTAGGGTCTGGTGACAGTTAGCTTACGCAGACTCCGTGAAGGG CCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC GAGGACACGGCCGTATATTACTGTGCGAAATCTTCTGCTGGTTTTGACTACTGGGGCCAGGGAACCC TGGTCACCGTCTCGAGC

SEQ ID NO: 38

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWYQGLVTVYDSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS

SEQ ID NO: 39

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAGATATTTGGAAGTAGGGTTTTGCTACAGCGAGCTTACGCAGACTCCGTGAAGGG CCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC GAGGACACGGCCGTATATTACTGTGCGAAATCTTCTGCTGGTTTTGACTACTGGGGCCAGGGAACCC TGGTCACCGTCTCGAGC

SEQ ID NO: 40

- 130 039736

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDIWKQGFATADSVKGRFT

ISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAGFDYWGQGTLVTVSS

SEQ ID NO: 41

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT

GGAGTGGGTCTCAAGTATTTGGAAGTAGGGTATTGTGACAGTGAGCTTACGCAGACTCCGTGAAGGG CCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC GAGGACACGGCCGTATATTACTGTGCGAAATCTTCTGCTGGTTTTGACTACTGGGCCAGGGAACCC TGGTCACCGTCTCGAGC

SEQ ID NO: 42

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWKQGIVTVYDSVKGRF

TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS

SEQ ID NO: 43

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCATCGATTTGGAGGTAGGGTCTGGCGACAGCGAGCTTACGCAGACTCCGTGAAGGG CCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC GAGGACACGGCCGTATATTACTGTGCGAAATCTTCTGCTGGTTTTGACTACTGGGGCCAGGGAACCC TGGTCACCGTCTCGAGC

SEQ ID NO: 44

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRQGLATAYDSVKGRF

TISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS

Example 4 Isolation and Testing of Embodiments of Affinity Mature Anti-PDLl Antibodies.

This example shows that mature PDL1 Fab phage that binds to human and mouse PDL1 can be converted into fully human IgG antibodies that retain binding to human PDL1 and show enhanced binding to mouse PDL1.

Obtaining fully human IgG containing the variable regions of Mature clones was carried out using methods similar to those described in the publication of international application PCT WO 2010/081173. DNA encoding the variable regions of the anti-PDL1 Fab-phage clones C1, D1, G7, C05, G12 and a combination of the CDR3 of the heavy chain variable regions of the anti-PDL1 Fab-phage of clones C05 or G12 with the CDR2 of the light chain variable region of the anti-PDL1 Fab-phage clones B10, E10 or H9 were cloned into expression vectors for the expression of fully human IgG. The light chain variable regions for each are identical to the c60 domains (SEQ ID NO: 10). Light chain variable regions (Lc) were amplified from Fab templates. The vector (LcpOP (modified from pCDNA3, Invitrogen, Carlsbad, CA)) and amplified DNA were digested overnight with BsiWl and EcoRI, ligated and transfected into E. coli MC1061 cells. Heavy chain variable regions (Hc) were amplified from Fab templates. Fully human IgG anti-PDL1 antibodies were expressed from transiently transfected HEK-293 cells and isolated from the culture supernatant by protein A chromatography.

Affinity matured anti-PDL1 antibodies were analyzed for their ability to bind to human and mouse PDL1. Briefly, human PDL1-Fc or mouse PDL1-Fc (R&D Systems, Minneapolis, MN) was adsorbed into the wells of a 96-well ELISA plate. Purified anti-PDL1 antibodies were added to the wells of the plate and left for binding. Bound antibody was visualized with a Fab-specific anti-human IgG-HRP conjugate (Sigma, St. Louis, MO, Cat # A0293-1ML) and developed using TMB chromogenic substrate. In FIG. 5 shows that combining mature heavy chain variable region CDR3 with heavy chain variable region CDR2 results in antibodies with increased affinity for human PDL1. In addition, the combination of the CDR2 of the mature C05 heavy chain variable regions with the CDR3 of the H9 heavy chain variable regions results in an antibody with high affinity for human and mouse PDL1. In table. 5 shows SEQ ID NOs for the nucleic acid and amino acid sequences of the corresponding heavy chain variable regions.

- 131 039736

The anti-PDL1 antibodies C5H9, C5B10 and C5E10 were then characterized by their species specificity in relation to binding to PDL1 and other related proteins. Briefly, human PDL1-Fc, mouse PDL1-Fc (R&D Systems, Minneapolis, MN), or cynomolgus monkey PDL1-Fc (Sino biological) were adsorbed into the wells of a 96-well ELISA plate. Purified anti-PDL1 antibodies were added to the wells of the plate and left for binding. Bound antibody was visualized with a Fab-specific anti-human IgG-HRP conjugate (Sigma, St. Louis, MO, Cat # A0293-1ML) and developed using TMB chromogenic substrate. In FIG. 6A and B show that C5H9 binds to human and mouse PDL1 with almost equal affinity, and C5B10 and C5E10 only bind to human PDL1. None of the antibodies tested bind to human B7-1 (FIG. 6D) or human CD28 (FIG. 6C), two closely related proteins. In FIG. 7 shows that the anti-PDL1 antibody C5H9 binds with high and equal affinity to human, mouse, and cynomolgus PDL1, but not to human B7H3 (also referred to here as B7-H3) (R&D Systems, Minneapolis, MN).

Table 5. Heavy chain variable regions of anti-PDL1 antibody embodiments

vh H7Kleic acid sequence Amino acid sequence PDL1 C5H9 SEQ ID NO 45 SEQ ID NO: 46 PDL1 C5B10 SEQ ID NO 47 SEQ ID NO: 48 PDL1 C5E10 SEQ ID NO 49 SEQ ID NO: fifty PDL1 G12H9 SEQ ID NO 51 SEQ ID NO: 52 PDL1 G12B10 SEQ ID NO 53 SEQ ID NO: 54 PDL1 G12E10 SEQ ID NO 55 SEQ ID NO: 56

SEQ ID NO: 45

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAAGTATTTGGCGGAATGGTATTGTTACAGTTTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAATGGTCTGCTGCTTTTGACTACTGGGGCCAGGGAACCCTGGT CACCGTCTCGAGC

SEQ ID NO: 46

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDYWGQGTLVTVSS

- 132 039736

SEQ ID NO: 47

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG

CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT

GGAGTGGGTCTCAAGTATTTGGCGGAATGGTATTGTTACAGTTTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACGCTGTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAATGGTCTGCTGGTTATGACTACTGGGCCAGGGAACCCTGGT CACCGTCTCGAGC

SEQ ID NO: 48

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAGYDYWGQGTLVTVSS

SEQ ID NO: 49

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG

CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT

GGAGTGGGTCTCAAGTATTTGGCGGAATGGTATTGTTACAGTTTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACGCTGTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAATGGTCTAAGGGTTTTGACTACTGGGCCAGGGAACCCTGGT CACCGTCTCGAGC

SEQ ID NO: 50

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSKGFDYWGQGTLVTVSS

SEQ ID NO: 51

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG

CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT

GGAGTGGGTCTCATCTATTTGGTATCAGGGTCTGGTGACAGTTTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACGCTGTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAATGGTCTGCTGCTTTTGACTACTACTGGGCCAGGGAACCCTGGT CACCGTCTCGAGC

SEQ ID NO:52

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWYQGLVTVYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDYWGQGTLVTVSS

- 133 039736

SEQ ID NO: 53

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG

CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAAGTATTTGGCGGAATGGTATTGTTACAGTTTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAATGGTCTGCTGGTTATGACTACTGGGGCCAGGGAACCCTGGT CACCGTCTCGAGC

SEQ ID NO: 54

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWYQGLVTVYADSVKGR

FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAGYDYWGQGTLVTVSS

SEQ ID NO: 55

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG

CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAAGTATTTGGCGGAATGGTATTGTTACAGTTTACGCAGACTCCGTGAAGGGCCGG TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCCGTATATTACTGTGCGAAATGGTCTAAGGGTTTTGACTACTGGGGCCAGGGAACCCTGGT CACCGTCTCGAGC

SEQ ID NO: 56

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWYQGLVTVYADSVKGR

FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSKGFDYWGQGTLVTVSS

Example 5 Germline modifications in the light chain variable region of the C5H9 anti-PDL1 antibody increase manufacturability and reduce potential immunogenicity.

This example describes an additional embodiment of an anti-PDL1 antibody that exhibits reduced predicted immunogenicity, reduced aggregation, and increased expression.

In silico immunogenicity prediction using ProPred, a strong potential epitope was found in the CDR2 of the C5H9 light chain variable region; anti-PDL1 antibody C5H9, also referred to herein as C5H9, includes a light chain containing SEQ ID NO: 12 and a heavy chain containing SEQ ID NO: 46. Alignment of the light chain variable region of the C5H9 antibody with human germline antibodies revealed two amino acids in CDR2 that, when modified into the germline, eliminated the predicted epitope. The residues are underlined and in bold in the sequence shown below in SEQ ID NO: 12.

In addition, light chain framework 4 of the C5H9 antibody was modified as indicated in bold italics in SEQ ID NO: 58, replacing glycine (G) with glutamine (Q) in the J region. This modification was aimed at increasing the flexibility of the connection between the variable and constant regions, which potentially reduced the tendency of C5H9 to form aggregates. The resulting DNA sequences and amino acid sequences are shown in SEQ ID NO: 57 and SEQ ID NO: 58, respectively. The resulting anti-PDL1 antibody, also referred to herein as anti-PDL1 antibody C5H9v2, also referred to here as C5H9v2, includes a light chain containing SEQ ID NO: 58 and a heavy chain containing SEQ ID NO: 46.

- 134 039736

SEQ ID NO: 12

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYASTLQSGVPSRFSGSGS

GTDFTLTISSLQPEDFATYYCQQDNGYPSTFGQGTKVEIKR

SEQ ID NO: 57

GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCGATCGCGTGACCATTACCT GCCGCGCGAGCCAGAGCATTAGCAGCTATCTGAACTGGTATCAGCAGAAACCGGGCAAAGCGCCGAA ACTGCTGATTTATGCGGCGAGCAGCCTGCAGAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGCAGC

GGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGACCTATTATTGCCAGC

AGGATAACGGCTATCCGAGCACCTTTGGCGGCGGCACCAAAAGTGGAAATTAAACGC

SEQ ID NO: 58

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS

GTDFTLTISSLQPEDFATYYCQQDNGYPSTFGGGTKVEIKR

In FIG. 8A and B show that the germline modifications described in SEQ ID NO: 58 reduce predicted immunogenicity (upper panel) and aggregation potential and increase expression levels by a factor of three.

Example 6 In vitro characterization of anti-PDL1 antibodies C5H9 and C5H9v2.

This example shows the binding specificity and biological activity of anti-PDL1 antibodies of the invention.

Obtaining fully human IgG containing the variable regions of Mature clones was carried out using methods similar to those described in the publication of international application PCT WO 2010/081173. DNA encoding the heavy chain variable regions of the anti-PDL1 antibody C5H9 (nucleic acid sequence of SEQ ID NO: 45, amino acid sequence of SEQ ID NO: 46) was amplified and cloned into vectors for the expression of fully human IgG. DNA encoding the light chain variable regions of the anti-PDL1 antibody C5H9 (nucleic acid sequence of SEQ ID NO: 11, amino acid sequence of SEQ ID NO: 12) was amplified and cloned into vectors for the expression of fully human IgG. Similarly, DNA encoding the heavy chain variable regions of the anti-PDL1 antibody C5H9 (nucleic acid sequence of SEQ ID NO: 45, amino acid sequence of SEQ ID NO: 46) was amplified and cloned into vectors for the expression of chimeric mouse IgG2a. DNA encoding the light chain variable regions of the anti-PDL1 antibody C5H9v2 (nucleic acid sequence of SEQ ID NO: 57, amino acid sequence of SEQ ID NO: 58) was amplified and cloned into vectors for the expression of chimeric mouse IgG2a. Fully human IgG or chimeric mouse IgG2a anti-PDL1 antibodies were expressed from transiently transfected HEK-293 cells and isolated from the culture supernatant by protein A chromatography. and a light chain comprising a light chain variable region comprising SEQ ID NO: 12. The C5H9v2 anti-PDL1 IgG antibody comprises a heavy chain comprising a heavy chain variable region comprising SEQ ID NO: 46 and a light chain comprising a light chain variable region containing SEQ ID NO: 58.

The anti-PDL1 antibody C5H9v2 was characterized for species-specific binding to PDL1 and how much the antibody would bind to a panel of human proteins. Briefly, human PDL1-Fc, mouse PDL1-Fc (R&D Systems, Minneapolis, MN), or cynomolgus monkey PDL1-Fc (Sino biological) were adsorbed into the wells of a 96-well ELISA plate. Purified anti-PDL1 antibodies C5H9 and C5H9v2 were added to the wells of the tablet and left for binding. Bound antibody was visualized with a Fab-specific anti-human IgG-HRP conjugate (Sigma, St Louis, MO, Cat # A0293-1ML) and developed using TMB chromogenic substrate. In FIG. 9 shows that both anti-PDL1 antibodies C5H9 and C5H9v2 bind to human, mouse PDL1 and cynomolgus PDL1 with almost the same affinity. EC ₅₀ values for anti-PDL1 antibody C5H9v2 to human PDL1, cynomolgus monkey PDL1, rat and mouse PDL1 also had almost the same values: 0.25 nM, 0.28 nM, 0.31 nM and 0.30 nM, respectively.

In a similar experiment, anti-PDL1 antibody C5H9 was evaluated for binding to a panel of human and mouse proteins. Briefly, each protein shown in FIG. 10 were absorbed into the wells of the ELISA plate. Purified anti-PDL1 antibodies C5H9 and C5H9v2 were added to the wells of the tablet and left for binding. Bound antibody was visualized with an anti-human Fab-specific IgG-HRP conjugate (Sigma, St Louis, MO, Cat # A0293-1ML) and developed using TMB chromogenic substrate. Anti-PDL1 antibody C5H9v2 binds only to human PDL1 and

- 135 039736 mice, showing specificity for PDL1.

The biological activity of PDL1 is mediated by binding to PD1 and/or B7-1. Blocking binding is a desirable characteristic for a therapeutic antibody; therefore, the effectiveness of blocking the interaction of PDL1 with PD1 and B7-1 was evaluated. Briefly, human, mouse PDL1 or Cynomolgus PDL1 were adsorbed into the wells of an ELISA plate. Biotinylated human, mouse or monkey cynomolgus or B7-1 PDL1 was added to the wells in the absence or presence of increasing concentrations of C5H9 anti-PDL1 antibody or C5H9v2 anti-PDL1 antibody and allowed to bind. Bound biotinylated PD1 or B7-1 was visualized with a streptavidin-HRP conjugate (Thermo Scientific, Cat. No. 21126) and developed using the TMB chromogenic substrate. In FIG. 11 shows that both C5H9 anti-PDL1 antibody and C5H9v2 anti-PDL1 antibody are strong blockers of B7-1 or PD1 binding to PDL1 and that blocking includes all three human, cynomolgus monkey and mouse PDL1 species with similar EC ₅₀ values at the nM level.

Example 7 An anti-PDL1 antibody of the invention accelerates the induction of diabetes in NOD mice.

In this example, anti-PDL1 antibody C5H9 was analyzed for the ability to induce diabetes in NOD mice.

NOD mice, subsp. NOD/ShiLtJ, were obtained from the Jackson Laboratory nursery at 6 weeks of age and acclimatized in situ for 1 week. After 7 weeks, mice were checked for diabetes prior to enrollment, grouping and dosing as shown in Table 6.

Table 6. Groups and doses in NOD mice

Group Number of mice Type of processing Dose (mg/kg) Load/Regular Volumetric dose (ml/kg) Scheme Route of administration one 6 control 20/10 ten q2dx6 i/b 2 6 anti-PDL1 antibody (C5H9) 20/10 ten q2dx6 i/b 3 6 anti-PDLl antibody (10F9G2) 20/10 ten q2dx6 i/b

In FIG. 12, which plots % of diabetic mice versus number of days after initial dose, showing that anti-PDL1 antibody C5H9 (Gr2-C5H9) induces diabetes in NOD mice, similar to anti-PDL1 antibody 10F9G2, which was used as positive control. Control rat IgG2b did not induce diabetes in NOD mice.

Anti-PDL1 antibody C5H9v2 was also tested for the ability to induce diabetes in NOD mice in a dose dependent manner using a method similar to that described above in 9 week old animals using the groups and doses shown in Table 7.

Table 7. Groups and doses in a dose-dependent study in NOD mice

Group Number of mice Floor Type of processing Dose (mg/kg) Volumetric dose (ml/kg) Scheme Route of administration one eight females mlgG2a (Cl.18.4) 25 ten single dose i/b 2 eight females anti- PDL1- antibody (C5H9v2) 25 ten single dose i/b 3 eight females antiPDL1 antibody (C5H9v2) 5 ten single dose i/b four eight females antiPDL1 antibody (C5H9v2) one ten single dose i/b 5 eight females antiPDL1 antibody (C5H9v2) 0.2 ten single dose i/b

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In FIG. 13, which plots % of diabetic mice versus number of days after initial dose, showing that anti-PDL1 antibody C5H9v2 induces diabetes in mice.

NOD by dose.

Example 8 Embodiments of anti-PDL1 antibodies suppress the growth of MC38 tumors in mice.

In this example, the anti-PDL1 antibody C5H9v2 was analyzed for its ability to inhibit the growth of syngeneic MC38 tumors.

The murine colon carcinoma cell line MC38 was obtained from ATCC (American Type Culture Collection, Manassas, VA). MC38 cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37° C. in an atmosphere with 5% CO ₂ in air. Cells were harvested during log phase growth, resuspended in PBS and stored on ice to induce tumors.

Each mouse was inoculated subcutaneously on the right flank with 1 x ^{10 6} MC38 cells in PBS for tumor growth. Treatments were started when the average size of the tumors reached about 100-200 mm ³ (no more than 200 mm ³ ). The sizes of the tumors were determined twice a week in two dimensions using a caliper, and the volume was expressed in mm ³ using the formula: V=0.5 aχb ² where a and b are the long and short diameters of the tumor, respectively.

Mice were divided into groups and injected antibodies, as indicated in table.8.

Table 8. Groups and doses for the MC38 sign tumor experiment

Group Number of mice Type of processing Dose (mg/kg) Volumetric dose (ml/kg) Scheme Route of administration one ten anti-PDLl antibody (10F9G2) ten ten t.i.w. within 2 weeks (MWF) i/b 2 ten anti-PDLl antibody (C5H9v2) ten ten t.i.w. within 2 weeks (MWF) i/b 3 ten mlgG2a+lgGl hamster ten ten t.i.w. within 2 weeks (MWF) i/b

In FIG. 14, which plots tumor volume versus number of days after initial dose, shows that anti-PDL1 antibody C5H9v2 inhibits the growth of syngeneic MC38 tumors, similar to anti-PDL1 antibody 10F9G2, which was used as a positive control.

Example 9 Masking Fragments of C5H9v2 Activated Anti-PDL1 Antibody.

This example describes the identification of masking fragments (MMs) to reduce binding of activated C5H9v2 anti-PDL1 antibodies to their target.

Anti-PDL1 antibody C5H9v2 and Fab were used to screen libraries using a method similar to that described in PCT International Application WO 2010/081173, published July 15, 2010. The screening consisted of one round of MACS and three rounds of FACS sorting. Initial MACS was performed using Dynabeads magnetic beads with protein A (Invitrogen) and C5H9v2 anti-PDL1 antibody at 100 nM. For MACS, approximately 1 x 10 ¹¹ cells were screened for binding and 6 x 10 ⁶ cells were harvested. Anti-PDL1 antibodies C5H9v2 directly labeled with Alexafluor-488 were used as a probe for all FACS variants. In the first round of FACS, cells were labeled with 100 nM AlexaFluor anti-PDL1 antibody C5H9v2. In the second round of FACS, cells were labeled with 10 nM AlexaFluor-anti-PDL1 antibody C5H9v2. In the third round of FACS, cells were labeled with 1 nM AlexaFluor-anti-PDL1 antibody C5H9v2. It was confirmed that the positive population from the third round of FACS is inhibited by recombinant human PDL1 protein in relation to binding to anti-PDL1 antibody C5H9v2 and Fab. Individual peptide clones were identified by sequence analysis and then tested for their ability to bind to anti-PDL1 antibody C5H9v2 and Fab.

The sequences of the masking fragments of the anti-PDL1 antibody C5H9v2 are shown in Table 9.

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Table 9. Masking fragments (MM) of aHTu-PDL1 antibody C5H9v2

mm Amino acid sequence SEQ ID NO PL01 YCEVSELFVLPWCMG SEQ ID NO: 208 PL02 SCLMHPHYAHDYCYV SEQ ID NO: 426 PL03 LCEVLMLLQHPWCMG SEQ ID NO 59 PL04 IACRHFMEQLPFCHH SEQ ID NO 60 PL05 FGPRCGEASTCVPYE SEQ ID NO 61 PL06 ILYCDSWGAGCLTRP SEQ ID NO 62 PL07 GIALCPSHFCQLPQT SEQ ID NO 63 PL08 DGPRCFVSGECS PIG SEQ ID NO 64 PL09 LCYKLDYDDRSYCHI SEQ ID NO 65 PL10 PCHPHPYDARPYCNV SEQ ID NO 66 PL11 PCYWHPFFAYRYCNT SEQ ID NO 67 PL12 VCYYMDWLGRNWCSS SEQ ID NO 68 PL13 LCDLFKLREFPYCMG SEQ ID NO 69 PL14 YLPCHFVPIGACNNK SEQ ID NO 70 PL15 IFCHMGWVPQCANY SEQ ID NO 71 PL16 ACHPHPYDARPYCNV SEQ ID NO 72 PL17 PCHPAPYDARPYCNV SEQ ID NO 73 PL18 PCHPHAYDARPYCNV SEQ ID NO 74 PL19 PCHPHPADARPYCNV SEQ ID NO 75 PL20 PCHPHPYAARPYCNV SEQ ID NO 76

PL21 PCHPHPYDAAPYCNV SEQ ID NO: 77 PL22 PCHPHPYDARPACNV SEQ ID NO: 78 PL23 PCHPHPYDARPYCAV SEQ ID NO: 79 PL24 PCHAHPYDARPYCNV SEQ ID NO: 80 PL25 PCHPHPYDARAYCNV SEQ ID NO: 81

Example 10 Activated Antibodies Including Anti-PDL1 Antibody C5H9v2.

This example describes examples of activated antibodies of the invention, including the anti-PDL1 antibody C5H9v2.

Activated anti-PDL1 antibodies, comprising an anti-PDL1 antibody masking fragment, a cleavage fragment, and an anti-PDL1 antibody C5H9v2, were prepared by methods similar to those described in PCT International Application Publications WO 2009/025846 and WO 2010/081173. Quality control of the obtained activated antibodies showed that most of them contain at least 95% of the monomer. Below are the amino acid and nucleic acid sequences of several activated antibodies, including the anti-PDL1 antibody C5H9v2. Each activated antibody comprises a C5H9v2 anti-PDL1 antibody having a heavy chain variable region amino acid sequence shown in SEQ ID NO: 46 and a light chain amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953,, 959 , 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1144-119 , 1200 and 1201 and below.

Although the sequences shown below include a spacer follower

- 138 039736 SEQ ID NO: 923, those skilled in the art will appreciate that the activated anti-PDL1 antibodies of the invention may include any suitable spacer sequence, such as, for example, a spacer sequence selected from the group consisting of QGQSGS ( SEQ ID NO: 923); GQSGS (SEQ ID NO: 1192); QSGS (SEQ ID NO: 1193); SGS (SEQ ID NO: 1194); GS (SEQ ID NO: 1195); S; QGQSGQG (SEQ ID NO: 924); GQSGQG (SEQ ID NO: 395); QSGQG (SEQ ID NO: 925) ; SGQG (SEQ ID NO: 926); GQG (SEQ ID NO: 927); QG (SEQ ID NO: 928); G; QGQSGQ (SEQ ID NO: 1196); GQSGQ (SEQ ID NO: 1197); QSGQ (SEQ ID NO: 1198); SGQ (SEQ ID NO: 616); GQ (SEQ ID NO: 1199); Q. Although the sequences shown below include the spacer sequence of SEQ ID NO: 923, those skilled in the art will also appreciate that the activated anti-PDL1 antibodies of the invention do not include a spacer sequence in some embodiments.

Amino acid sequence of VH C5H9v2.

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYADSVKGR

FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDYWGQGTLVTVSS (SEQ ID NO: 46)

[Spacer (SEQ ID NO: 929)] [PL01-0003 LC (SEQ ID NO: 930)]

[CAAGGTCAGTCTGGATCC][TATTGCGAGGTTAGTGAGCTGTTTGTTCTTCCTTGGTGCATGGGTG

GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 82)

[Spacer (SEQ ID NO: 923)] [PL01-0003 LC (SEQ ID NO: 931)]

[QGQSGS][YCEVSELFVLPWCMGGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 83)

Nucleotide sequence of PL01-0003 LC

- 139 039736

TATTGCGAGGTTAGTGAGCTGTTTGTTCTTCCTTGGTGCATGGGTGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 930)

Amino acid sequence PL01-0003 LC

YCEVSELFVLPWCMGGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 931)

[Spacer (SEQ ID NO: 929)] [PL02-0003 LC (SEQ ID NO: 932)]

[CAAGGTCAGTCTGGATCC][TCTTGCCTTATGCATCCGCATTATGCTCATGATTATTGCTATGTTG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 84)

[Spacer (SEQ ID NO: 923)] [PL02-0003 LC (SEQ ID NO: 933)]

[QGQSGS][SCLMHPHYAHDYCYVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 85)

Nucleotide sequence of PL02-0003 LC

TCTTGCCTTATGCATCCGCATTATGCTCATGATTATTGCTATGTTGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 932)

Amino acid sequence PL02-0003 LC

SCLMHPHYAHDYCYVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 933)

- 140 039736

[Spacer (SEQ ID NO: 929)] [PL03-0003 LC (SEQ ID NO: 934)]

[CAAGGTCAGTCTGGATCC][TTGTGCGAGGTTTTGATGTTGTTGCAGCATCCGTGGTGCATGGGGG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 86)

[Spacer (SEQ ID NO: 923)] [PL03-0003 LC (SEQ ID NO: 935)]

[QGQSGS][LCEVLMLLQHPWCMGGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 87)

Nucleotide sequence of PL03-0003 LC

TTGTGCGAGGTTTTGATGTTGTTGCAGCATCCGTGGTGCATGGGGGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 934)

Amino acid sequence PL03-0003 LC

LCEVLMLLQHPWCMGGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 935)

[Spacer (SEQ ID NO: 929)] [PL04-0003 LC (SEQ ID NO: 936)]

[CAAGGTCAGTCTGGATCC][ATTGCGTGCCGGCATTTTATGGAGCAGTTGCCGTTTTGCCATCATG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 88)

[Spacer (SEQ ID NO: 923)] [PL04-0003 LC (SEQ ID NO: 937)]

- 141 039736

[QGQSGS][IACRHFMEQLPFCHHGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 89)

Nucleotide sequence of PL04-0003 LC

ATTGCGTGCCGGCATTTTATGGAGCAGTTGCCGTTTTGCCATCATGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 936)

Amino acid sequence PL04-0003 LC

IACRHFMEQLPFCHHGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 937)

[Spacer (SEQ ID NO: 929)] [PL05-0003 LC (SEQ ID NO: 938)]

[CAAGGTCAGTCTGGATCC][TTTGGTCCTAGGTGCGGTGAGGCTTCTACTTGCGTTCCGTATGAGG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 90)

[Spacer (SEQ ID NO: 923)] [PL05-0003 LC (SEQ ID NO: 939)]

[QGQSGS][FGPRCGEASTCVPYEGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 91)

Nucleotide sequence of PL05-0003 LC

- 142 039736

TTTGGTCCTAGGTGCGGTGAGGCTTCTACTTGCGTTCCGTATGAGGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 938) Аминокислотная последовательность PL05-0003 LC

FGPRCGEASTCVPYEGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 939)

[Spacer (SEQ ID NO: 929)] [PL06-0003 LC (SEQ ID NO: 940)] [CAAGGTCAGTCTGGATCC][ATTCTTTATTGCGATAGTTGGGGGGCGGGTGCTTGACGCGGCCGG

GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 92)

[Spacer (SEQ ID NO: 923)] [PL06-0003 LC (SEQ ID NO: 941)]

[QGQSGS][ILYCDSWGAGCLTRPGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 93)

Nucleotide sequence of PL06-0003 LC

ATTCTTTATTGCGATAGTTGGGGGGCGGGGTGCTTGACGCGGCCGGGAGGTGGCTCGAGCGGTGGCA

GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 940)

Amino acid sequence PL06-0003 LC

- 143 039736

ILYCDSWGAGCLTRPGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR

ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD

NGYPSTFGGGTKVEIKR (SEQ ID NO: 941)

[Spacer (SEQ ID NO: 929)] [PL07-0003 LC (SEQ ID NO: 942)]

[CAAGGTCAGTCTGGATCC][GGGATTGCGTTGTGCCCGTCTCATTTTTGCCAGCTGCCTCAGACTG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 94)

[Spacer (SEQ ID NO: 923)] [PL07-0003 LC (SEQ ID NO: 943)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 95)

Nucleotide sequence of PL07-0003 LC

GGGATTGCGTTGTGCCCGTCTCATTTTTGCCAGCTGCCTCAGACTGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 942)

Amino acid sequence PL07-0003 LC

GIALCPSHFCQLPQTGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 943)

[Spacer (SEQ ID NO: 929)] [PL08-0003 LC (SEQ ID NO: 944)]

- 144 039736

[CAAGGTCAGTCTGGATCC][GATGGGCCGCGTTGCTTTGTGTCGGGGGAGTGCTCTCCGATTGGTG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 96)

[Spacer (SEQ ID NO: 923)] [PL08-0003 LC (SEQ ID NO: 945)]

[QGQSGS][DGPRCFVSGECSPIGGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 97)

Nucleotide sequence of PL08-0003 LC

GATGGGCCGCGTTGCTTTGTGTCGGGGGAGTGCTCTCCGATTGGTGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 944)

Amino acid sequence PL08-0003 LC

DGPRCFVSGECSPIGGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 945)

[Spacer (SEQ ID NO: 929)] [PL09-0003 LC (SEQ ID NO: 946)]

[CAAGGTCAGTCTGGATCC][TTGTGCTATAAGCTGGATTATGATGATAGGTCTTATTGCCATATTG

GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 98)

[Spacer (SEQ ID NO: 923)] [PL09-0003 LC (SEQ ID NO: 947)]

- 145 039736

[QGQSGS][LCYKLDYDDRSYCHIGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV

GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED

FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 99)

Nucleotide sequence of PL09-0003 LC

TTGTGCTATAAGCTGGATTATGATGATAGGTCTTATTGCCATATTGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC

CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC

AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 946)

Amino acid sequence PL09-0003 LC

LCYKLDYDDRSYCHIGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 947)

[Spacer (SEQ ID NO: 929)] [PL10-0003 LC (SEQ ID NO: 948)]

[CAAGGTCAGTCTGGATCC][CCGTGCCATCCGCATCCTTATGATGCTCGTCCTTATTGCAATGTGG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 100)

[Spacer (SEQ ID NO: 923)] [PL10-0003 LC (SEQ ID NO: 949)]

[QGQSGS][PCHPHPYDARPYCNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV

GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR](SEQ ID NO: 101)

Nucleotide sequence of PL10-0003 LC

- 146 039736

CCGTGCCATCCGCATCCTTATGATGCTCGTCCTTATTGCAATGTGGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 948)

Amino acid sequence of PL10-0003 LC

PCHPHPYDARPYCNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR(SEQ ID NO: 949)

[Spacer (SEQ ID NO: 929)] [PL11-0003 LC (SEQ ID NO: 950)]

[CAAGGTCAGTCTGGATCC][CCTTGCTATTGGCATCCTTTTTTTGCGTATAGGTATTGCAATACTG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG

T] (SEQ ID NO: 102)

[Spacer (SEQ ID NO: 923)] [PL11-0003 LC (SEQ ID NO: 951)]

[QGQSGS][PCYWHPFFAYRYCNTGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 103)

Nucleotide sequence of PL11-0003 LC

CCTTGCTATTGGCATCCTTTTTTTGCGTATAGGTATTGCAATACTGGAGGTGGCTCGAGCGGTGGCA

GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT

CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA

GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC

TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC

CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC

AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 950)

Amino acid sequence of PL11-0003 LC

- 147 039736

PCYWHPFFAYRYCNTGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 951)

[Spacer (SEQ ID NO: 929)] [PL12-0003 LC (SEQ ID NO: 952)]

[CAAGGTCAGTCTGGATCC][GTTTGCTATTATATGGATTGGTTGGGGCGGAATTGGTGCTCTTCGG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 104)

[Spacer (SEQ ID NO: 923) ] [PL12-0003 LC (SEQ ID NO: 953) ]

[QGQSGS][VCYYMDWLGRNWCSSGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 105)

Nucleotide sequence of PL12-0003 LC

GTTTGCTATTATATGGATTGGTTGGGGCGGAATTGGTGCTCTTCGGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 952)

Amino acid sequence of PL12-0003 LC

VCYYMDWLGRNWCSSGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 953)

[Spacer (SEQ ID NO: 929)] [PL13-0003 LC (SEQ ID NO: 954)]

[CAAGGTCAGTCTGGATCC][CTGTGCGATCTGTTTAAGTTGCGTGAGTTTCCTTATTGCATGGGGG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT

- 148 039736

TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 106)

[Spacer (SEQ ID NO: 923)] [PL13-0003 LC (SEQ ID NO: 955)]

[QGQSGS][LCDLFKLREFPYCMGGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 107)

Nucleotide sequence of PL13-0003 LC

CTGTGCGATCTGTTTAAGTTGCGTGAGTTTCCTTATTGCATGGGGGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 954)

Amino acid sequence of PL13-0003 LC

LCDLFKLREFPYCMGGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 955)

[Spacer (SEQ ID NO: 929)] [PL14-0003 LC (SEQ ID NO: 956)]

[CAAGGTCAGTCTGGATCC][TATCTTCCGTGCCATTTTGTTCCGATTGGGGCTTGCAATAATAAGG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 108)

[Spacer (SEQ ID NO: 923)] [PL14-0003 LC (SEQ ID NO: 957)]

[QGQSGS][YLPCHFVPIGACNNKGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 109)

Nucleotide sequence of PL14-0003 LC

- 149 039736

TATCTTCCGTGCCATTTTGTTCCGATTGGGGCTTGCAATAATAAGGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 956)

Amino acid sequence of PL14-0003 LC

YLPCHFVPIGACNNKGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 957)

[Spacer (SEQ ID NO: 929)] [PL15-0003 LC (SEQ ID NO: 958)]

[CAAGGTCAGTCTGGATCC][ATTTTTTGCCATATGGGTGTTGTGGTTCCTCAGTGCGCGAATTATG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 110)

[Spacer (SEQ ID NO: 923)] [PL15-0003 LC (SEQ ID NO: 959)]

[QGQSGS] [ IFCHMGVWPQCANYGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 111)

Нуклеотидная последовательность PL15-0003 LC _{ATTTTTTGCCATATGGGTGTTGTGGTTCCTCAGTGCGCGAATTATGGAGGTGGCTCGAGCGGTGGCA} GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 958)

Amino acid sequence of PL15-0003 LC

- 150 039736

(SEQ ID NO: 959)

[Spacer (SEQ ID NO: 929)] [PL16-0003 LC (SEQ ID NO: 960)]

[CAAGGTCAGTCTGGATCC][GCGTGCCATCCGCATCCTTATGATGCTCGTCCTTATTGCAATGTGG

GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 112)

[Spacer (SEQ ID NO: 923)] [PL16-0003 LC (SEQ ID NO: 961)]

[QGQSGS][ACHPHPYDARPYCNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 113)

Nucleotide sequence of PL16-0003 LC

GCGTGCCATCCGCATCCTTATGATGCTCGTCCTTATTGCAATGTGGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC

CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 960)

Amino acid sequence of PL16-0003 LC

(SEQ ID NO: 961)

[Spacer (SEQ ID NO: 929)] [PL17-0003 LC (SEQ ID NO: 962)]

- 151 039736

[CAAGGTCAGTCTGGATCC][CCGTGCCATCCGGCTCCTTATGATGCTCGTCCTTATTGCAATGTGG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 114)

[Spacer (SEQ ID NO: 923)] [PL17-0003 LC (SEQ ID NO: 963)]

[QGQSGS][PCHPAPYDARPYCNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 115)

Nucleotide sequence of PL17-0003 LC

CCGTGCCATCCGGCTCCTTATGATGCTCGTCCTTATTGCAATGTGGGAGGTGGCTCGAGCGGTGGCA

GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT

CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 962)

Amino acid sequence of PL17-0003 LC

PCHPAPYDARPYCNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 963)

[Spacer (SEQ ID NO: 929)] [PL18-0003 LC (SEQ ID NO: 964)]

[CAAGGTCAGTCTGGATCC][CCGTGCCATCCGCATGCTTATGATGCTCGTCCTTATTGCAATGTGG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG

T] (SEQ ID NO: 116)

[Spacer (SEQ ID NO: 923)] [PL18-0003 LC (SEQ ID NO: 965)]

- 152 039736

[QGQSGS][PCHPHAYDARPYCNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV

GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED

FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 117)

Nucleotide sequence of PL18-0003 LC

CCGTGCCATCCGCATGCTTATGATGCTCGTCCTTATTGCAATGTGGGAGGTGGCTCGAGCGGTGGCA

GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT

CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA

GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC

TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC

CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC

AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 964)

Amino acid sequence of PL18-0003 LC

PCHPHAYDARPYCNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD

NGYPSTFGGGTKVEIKR (SEQ ID NO: 965)

[Spacer (SEQ ID NO: 929)] [PL19-0003 LC (SEQ ID NO: 966)]

[CAAGGTCAGTCTGGATCC][CCGTGCCATCCGCATCCTGCTGATGCTCGTCCTTATTGCAATGTGG

GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC

ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC

AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC

CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT

TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC

ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA

AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG

T] (SEQ ID NO: 118)

[Spacer (SEQ ID NO: 923)] [PL19-0003 LC (SEQ ID NO: 967)]

[QGQSGS][PCHPHPADARPYCNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED

FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 119)

Nucleotide sequence of PL19-0003 LC

- 153 039736

CCGTGCCATCCGCATCCTGCTGATGCTCGTCCTTATTGCAATGTGGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 966)

Amino acid sequence of PL19-0003 LC

PCHPHPADARPYCNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 967)

[Spacer (SEQ ID NO: 929)] [PL20-0003 LC (SEQ ID NO: 968)]

[CAAGGTCAGTCTGGATCC][CCGTGCCATCCGCATCCTTATGCTGCTCGTCCTTATTGCAATGTGG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT

TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC

ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA

AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG

T] (SEQ ID NO: 120)

[Spacer (SEQ ID NO: 923)] [PL20-0003 LC (SEQ ID NO: 969)]

[QGQSGS][PCHPHPYAARPYCNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 121)

Nucleotide sequence of PL20-0003 LC

CCGTGCCATCCGCATCCTTATGCTGCTCGTCCTTATTGCAATGTGGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA

AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 968)

Amino acid sequence of PL20-0003 LC

- 154 039736

PCHPHPYAARPYCNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR

ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD

NGYPSTFGGGTKVEIKR (SEQ ID NO: 969)

[Spacer (SEQ ID NO: 929)] [PL21-0003 LC (SEQ ID NO: 970)]

[CAAGGTCAGTCTGGATCC][CCGTGCCATCCGCATCCTTATGATGCTGCTCCTTATTGCAATGTGG

GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 122)

[Spacer (SEQ ID NO: 923)] [PL21-0003 LC (SEQ ID NO: 971)]

[QGQSGS][PCHPHPYDAAPYCNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 123)

Nucleotide sequence of PL21-0003 LC

CCGTGCCATCCGCATCCTTATGATGCTGCTCCTTATTGCAATGTGGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 970)

Amino acid sequence of PL21-0003 LC

(SEQ ID NO: 971)

[Spacer (SEQ ID NO: 929)] [PL22-0003 LC (SEQ ID NO: 972)]

- 155 039736

[CAAGGTCAGTCTGGATCC][CCGTGCCATCCGCATCCTTATGATGCTCGTCCTGCTTGCAATGTGG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 124)

[Спейсер (SEQ ID NO: 923)] [PL22-0003 LC (SEQ ID NO: 973)] [QGQSGS][PCHPHPYDARPACNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 125)

Nucleotide sequence of PL22-0003 LC

CCGTGCCATCCGCATCCTTATGATGCTCGTCCTGCTTGCAATGTGGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 972)

Amino acid sequence of PL22-0003 LC

PCHPHPYDARPACNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 973)

[Спейсер (SEQ ID NO: 929)] [PL23-0003 LC (SEQ ID NO: 974)] [CAAGGTCAGTCTGGATCC][CCGTGCCATCCGCATCCTTATGATGCTCGTCCTTATTGCGCTGTGG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 126)

[Spacer (SEQ ID NO: 923)] [PL23-0003 LC (SEQ ID NO: 975)]

- 156 039736

[QGQSGS][PCHPHPYDARPYCAVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV

GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED

FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 127)

Nucleotide sequence of PL23-0003 LC

CCGTGCCATCCGCATCCTTATGATGCTCGTCCTTATTGCGCTGTGGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 974)

Amino acid sequence of PL23-0003 LC

PCHPHPYDARPYCAVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 975)

[Spacer (SEQ ID NO: 929)] [PL24-0003 LC (SEQ ID NO: 976)]

[CAAGGTCAGTCTGGATCC][CCGTGCCATGCGCATCCTTATGATGCTCGTCCTTATTGCAATGTGG GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 128)

[Spacer (SEQ ID NO: 923)] [PL24-0003 LC (SEQ ID NO: 977)]

[QGQSGS][PCHAHPYDARPYCNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 129)

Nucleotide sequence of PL24-0003 LC

- 157 039736

CCGTGCCATGCGCATCCTTATGATGCTCGTCCTTATTGCAATGTGGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 976)

Amino acid sequence of PL24-0003 LC

PCHAHPYDARPYCNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD NGYPSTFGGGTKVEIKR (SEQ ID NO: 977)

[Spacer (SEQ ID NO: 929)] [PL25-0003 LC (SEQ ID NO: 978)]

[CAAGGTCAGTCTGGATCC][CCGTGCCATCCGCATCCTTATGATGCTCGTGCTTATTGCAATGTGG

GAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCC ACGTGGCGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGC CCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATT TTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCA AGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTG T] (SEQ ID NO: 130)

[Spacer (SEQ ID NO: 923)] [PL25-0003 LC (SEQ ID NO: 979)]

[QGQSGS][PCHPHPYDARAYCNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 131)

Nucleotide sequence of PL25-0003 LC

CCGTGCCATCCGCATCCTTATGATGCTCGTGCTTATTGCAATGTGGGAGGTGGCTCGAGCGGTGGCA GCGGTGGCTCTGGTGGTACTAGCACCTCTGGTCGTTCCGCTAACCCACGTGGCGGCGGTTCTGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGC TGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCAC CGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGAC AACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 978)

Amino acid sequence of PL25-0003 LC

- 158 039736

PCHPHPYDARAYCNVGGGSSGGSGGSGGTSTSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTITCR

ASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQD

NGYPSTFGGGTKVEIKR (SEQ ID NO: 979)

[Spacer (SEQ ID NO: 929)] [PL03-2001 LC (SEQ ID NO: 952)]

[CAAGGTCAGTCTGGATCC][GGAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATTAGCA GTGGTCTGTTAAGCGGTCGTAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAG CAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGC TACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTC TGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAG CTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTT GGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTG TCAAGAGCTTCAACAGGAATGAGTGT] (SEQ ID NO: 132)

[Spacer (SEQ ID NO: 923)] [PL03-2001 LC (SEQ ID NO: 981)]

[QGQSGS][LCEVLMLLQHPWCMGGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 133)

Nucleotide sequence of PL03-2001 LC

GGAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATTAGCAGTGGTCTGTTAAGCGGTCGTA GCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGG CGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAG AAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCA GATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTT CGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAA ATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATG AGTGT (SEQ ID NO: 980)

Amino acid sequence of PL03-2001 LC

(SEQ ID NO: 981)

[Spacer (SEQ ID NO: 923) ] [PL04-2001 LC (SEQ ID NO: 1183) ]

[QGQSGS][IACRHFMEQLPFCHHGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1182)

Amino acid sequence of PL04-2001 LC

IACRHFMEQLPFCHHGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSASVGDRVTITC RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKR (SEQ ID NO: 1183)

- 159 039736

[Spacer (SEQ ID NO: 929) ] [PL06-2001-mk LC (SEQ ID NO

982)]

[CAAGGTCAGTCTGGATCC][GGAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATTAGCA GTGGTCTGTTAAGCGGTCGTAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAG CAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGC TACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTC TGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAG CTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTT GGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTG TCAAGAGCTTCAACAGGAATGAGTGT] (SEQ ID NO: 134)

[Spacer (SEQ ID NO: 923) ] [PL06-2001-mk LC (SEQ ID NO 983) ]

[QGQSGS][ILYCDSWGAGCLTRPGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 135)

Nucleotide sequence of PL06-2001-mk LC

GGAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATTAGCAGTGGTCTGTTAAGCGGTCGTA GCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGG CGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAG AAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCA GATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTT CGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAA ATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATG AGTGT (SEQ ID NO: 982)

Amino acid sequence PL06-2001-mk LC

ILYCDSWGAGCLTRPGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSASVGDRVTITC RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKR (SEQ ID NO: 983)

[Spacer (SEQ ID NO: 929)] [PL07-2001 LC (SEQ ID NO: 984)]

[CAAGGTCAGTCTGGATCC][GGAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATTAGCA GTGGTCTGTTAAGCGGTCGTAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAG CAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGC TACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTC TGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAG CTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTT GGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTG TCAAGAGCTTCAACAGGAATGAGTGT] (SEQ ID NO: 136)

- 160 039736

[Spacer (SEQ ID NO: 923)] [PL07-2001 LC (SEQ ID NO: 985)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 137)

Nucleotide sequence of PL07-2001 LC

GGAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATTAGCAGTGGTCTGTTAAGCGGTCGTA GCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGG CGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAG AAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCA GATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTT CGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAA ATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATG AGTGT (SEQ ID NO: 984)

Amino acid sequence of PL07-2001 LC

GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSASVGDRVTITC RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKR (SEQ ID NO: 985)

[Spacer (SEQ ID NO: 929)] [PL10-2001 LC (SEQ ID NO: 986)]

[CAAGGTCAGTCTGGATCC][GGAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATTAGCA GTGGTCTGTTAAGCGGTCGTAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAG CAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGC TACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTC TGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAG CTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTT

GGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTG TCAAGAGCTTCAACAGGAATGAGTGT] (SEQ ID NO: 138)

[Spacer (SEQ ID NO: 923)] [PL10-2001 LC (SEQ ID NO: 987)]

[QGQSGS][PCHPHPYDARPYCNVGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 139)

Nucleotide sequence of PL10-2001 LC

- 161 039736

GGAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATTAGCAGTGGTCTGTTAAGCGGTCGTA GCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGG CGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAG AAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCA GATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTT CGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAA ATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATG AGTGT (SEQ ID NO: 986)

Amino acid sequence of PL06-2001 LC

(SEQ ID NO: 987)

[Spacer (SEQ ID NO: 923)] [PL11-2001 LC (SEQ ID NO: 1185)]

[QGQSGS][PCYWHPFFAYRYCNTGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1184)

Amino acid sequence of PL11-2001 LC

PCYWHPFFAYRYCNTGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSASVGDRVTITC RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKR (SEQ ID NO: 1185)

[Spacer (SEQ ID NO: 923)] [PL12-2001 LC (SEQ ID NO: 1187)]

[QGQSGS] [VCYYMDWLGRNWCSSSGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1186)

Amino acid sequence of PL12-2001 LC

VCYYMDWLGRNWCSSGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSASVGDRVTITC RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKR (SEQ ID NO: 1187)

[Spacer (SEQ ID NO: 929)] [PL14-2001 LC (SEQ ID NO: 988)]

[CAAGGTCAGTCTGGATCC][GGAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATTAGCA GTGGTCTGTTAAGCGGTCGTAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAG CAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGC TACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTC TGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAG CTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTT GGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTG TCAAGAGCTTCAACAGGAATGAGTGT] (SEQ ID NO: 140)

- 162 039736

[Spacer (SEQ ID NO: 923)] [PL14-2001 LC (SEQ ID NO: 989)]

[QGQSGS][YLPCHFVPIGACNNKGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 141)

Nucleotide sequence of PL14-2001 LC

GGAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATTAGCAGTGGTCTGTTAAGCGGTCGTA GCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGG CGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAG AAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCA GATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTT CGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAA ATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATG AGTGT (SEQ ID NO: 988)

Amino acid sequence of PL14-2001 LC

YLPCHFVPIGACNNKGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSASVGDRVTITC RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKR (SEQ ID NO: 989)

[Spacer (SEQ ID NO: 929)] [PL15-2001 LC (SEQ ID NO: 990)]

[CAAGGTCAGTCTGGATCC][GGAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATTAGCA

GTGGTCTGTTAAGCGGTCGTAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAG

CAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGC

TACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTC

TGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAG

CTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTT

GGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTG TCAAGAGCTTCAACAGGAATGAGTGT] (SEQ ID NO: 142)

[Spacer (SEQ ID NO: 923)] [PL15-2001 LC (SEQ ID NO: 991)]

[QGQSGS] [ IFCHMGVWPQCANYGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 143)

Nucleotide sequence of PL15-2001 LC

- 163 039736

GGAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATTAGCAGTGGTCTGTTAAGCGGTCGTA GCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGG CGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAG AAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCA GATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTT CGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAA ATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATG AGTGT (SEQ ID NO: 990)

Amino acid sequence of PL15-2001 LC

(SEQ ID NO: 991)

[Spacer (SEQ ID NO: 929)] [PL18-2001 LC (SEQ ID NO: 992)]

[CAAGGTCAGTCTGGATCC][GGAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATTAGCA GTGGTCTGTTAAGCGGTCGTAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAG CAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGC TACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTC TGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAG CTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTT

GGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTG TCAAGAGCTTCAACAGGAATGAGTGT] (SEQ ID NO: 144)

[Spacer (SEQ ID NO: 923) ] [PL18-2001 LC (SEQ ID NO: 993) ]

[QGQSGS][PCHPHAYDARPYCNVGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 145)

Nucleotide sequence of PL18-2001 LC

GGAGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATTAGCAGTGGTCTGTTAAGCGGTCGTA GCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGTGGG CGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAG AAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCAGCA GATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTT CGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTGGAA ATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATG AGTGT (SEQ ID NO: 992)

Amino acid sequence of PL18-2001 LC

(SEQ ID NO: 993)

- 164 039736

[Spacer (SEQ ID NO: 923)] [PL19-2001 LC (SEQ ID NO: 1189)]

[QGQSGS][PCHPHPADARPYCNVGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSAS

VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE

DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1188)

Amino acid sequence of PL19-2001 LC

PCHPHPADARPYCNVGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSASVGDRVTITC

RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ

DNGYPSTFGGGTKVEIKR (SEQ ID NO: 1189)

[Spacer (SEQ ID NO: 923)] [PL20-2001 LC (SEQ ID NO: 1191)]

[QGQSGS][PCHPHPYAARPYCNVGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSAS

VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE

DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1190)

Amino acid sequence of PL20-2001 LC

PCHPHPYAARPYCNVGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSASVGDRVTITC

RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ

DNGYPSTFGGGTKVEIKR (SEQ ID NO: 1191)

[Spacer (SEQ ID NO: 929) ] [PL03-1004/GG/0001 (also refers to PL03-3001 here) LC (SEQ ID NO: 994)]

[CAAGGTCAGTCTGGATCC][GGAGGTGGCTCGAGCGGTGGCAGCGGTGCTGTGGGTCTCCTGGCTC

CCCCGGGCGGCCTGTCCGGCCGCAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCC

CAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGC

AGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCT

CTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCAT

CAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACC

TTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCA

TTGTCAAGAGCTTCAACAGGAATGAGTGT] (SEQ ID NO: 146)

[Spacer (SEQ ID NO: 923) ] [PL03-1004/GG/0001 LC (SEQ ID

NO: 995)]

[QGQSGS][LCEVLMLLQHPWCMGGGGSSGGSGAVGLLAPPGGLSGRSDNHGGSDIQMTQSPSSLSA

SVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQP

EDFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 147)

Nucleotide sequence PL03-1004/GG/0001 LC

- 165 039736

GGAGGTGGCTCGAGCGGTGGCAGCGGTGCTGTGGGTCTCCTGGCTCCCCCGGGCGGCCTGTCCGGCC GCAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGT GGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAG CAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCA GCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGA CTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTG GAAATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGA ATGAGTGT (SEQ ID NO: 994)

Amino acid sequence PL03-1004/GG/0001 LC

(SEQ ID NO: 995)

[Spacer (SEQ ID NO: 929)] [PL06-1004/GG/0001 (also refers to PL06-3001 here) LC (SEQ ID NO: 996)]

[CAAGGTCAGTCTGGATCC][GGAGGTGGCTCGAGCGGTGGCAGCGGTGCTGTGGGTCTCCTGGCTC CCCCGGGCGGCCTGTCCGGCCGCAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCC CAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGC AGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCT CTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCAT CAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACC TTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCG TCACAAAGAGCTTCAACAGGGGAGAGTGT] (SEQ ID NO: 148)

[Spacer (SEQ ID NO: 923) ] [PL06-1004/GG/0001 LC (SEQ ID

NO: 997)]

[QGQSGS][ILYCDSWGAGCLTRPGGGSSGGSGAVGLLAPPGGLSGRSDNHGGSDIQMTQSPSSLSA SVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 149)

Nucleotide sequence PL06-1004/GG/0001 LC

GGAGGTGGCTCGAGCGGTGGCAGCGGTGCTGTGGGTCTCCTGGCTCCCCCGGGCGGCCTGTCCGGCC

GCAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGT

GGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAG

CAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCA

GCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGA

CTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTG

GAAATCAAGCGTTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGT (SEQ ID NO: 996)

Amino acid sequence PL06-1004/GG/0001 LC

- 166 039736

(SEQ ID NO: 997)

[Spacer (SEQ ID NO: 929) ] [PL07-1004/GG/0001 (also refers to PL07-3001 here) LC (SEQ ID NO: 998)]

[CAAGGTCAGTCTGGATCC][GGAGGTGGCTCGAGCGGTGGCAGCGGTGCTGTGGGTCCTGGCTC CCCCGGGCGGCCTGTCCGGCCGCAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCC CAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGC AGCTACCTAGCGAGCCATCGACTCGTAGCAGCAGACCCC

CTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCAT

CAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACC TTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCG TCACAAAAGGCTTCAACAGGGGAGAGTGT] (SEQ ID NO: 150)

[Spacer (SEQ ID NO: 923) ] [PL01-1004/GG/0001 LC (SEQ ID

NO: 999)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGAVGLLAPPGGLSGRSDNHGGSDIQMTQSPSSLSA SVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 151)

Nucleotide sequence PL07-1004/GG/0001 LC

GGAGGTGGCTCGAGCGGTGGCAGCGGTGCTGTGGGTCTCCTGGCTCCCCCGGGCGGCCTGTCCGGCC

GCAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGT GGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAG CAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCA GCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGA CTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTG GAAATCAAGCGTTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGT (SEQ ID NO: 998)

Amino acid sequence PL07-1004/GG/0001 LC

GIALCPSHFCQLPQTGGGSSGGSGAVGLLAPPGGLSGRSDNHGGSDIQMTQSPSSLSASVGDRVTIT CRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QDNGYPSTFGGGTKVEIKR (SEQ ID NO: 999)

[Spacer (SEQ ID NO: 929) ] [PL14-1004/GG/0001 (also refers to PL14-3001 here) LC (SEQ ID NO: 1000)]

- 167 039736

[CAAGGTCAGTCTGGATCC][GGAGGTGGCTCGAGCGGTGGCAGCGGTGCTGTGGGTCTCCTGGCTC

CCCCGGGCGGCCTGTCCGGCCGCAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCC

CAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGC

AGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCT

CTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCAT

CAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACC

TTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCG

TCACAAAGAGCTTCAACAGGGGAGAGTGT] (SEQ ID NO: 152)

[Spacer (SEQ ID NO: 923) ] [PL14-1004/GG/0001 LC (SEQ ID

NO: 1001)]

[QGQSGS][YLPCHFVPIGACNNKGGGSSGGSGAVGLLAPPGGLSGRSDNHGGSDIQMTQSPSSLSA

SVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQP

EDFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 153)

Nucleotide sequence PL14-1004/GG/0001 LC

GGAGGTGGCTCGAGCGGTGGCAGCGGTGCTGTGGGTCTCCTGGCTCCCCCGGGCGGCCTGTCCGGCC

GCAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGT

GGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAG

CAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCA

GCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGA

CTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTG

GAAATCAAGCGTTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGT (SEQ ID NO: 1000)

Amino acid sequence PL14-1004/GG/0001 LC

YLPCHFVPIGACNNKGGGSSGGSGAVGLLAPPGGLSGRSDNHGGSDIQMTQSPSSLSASVGDRVTIT

CRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QDNGYPSTFGGGTKVEIKR (SEQ ID NO: 1001)

[Spacer (SEQ ID NO: 929) ] [PL15-1004/GG/0001 (also refers to PL15-3001 here) LC (SEQ ID NO: 1002)]

[CAAGGTCAGTCTGGATCC][GGAGGTGGCTCGAGCGGTGGCAGCGGTGCTGTGGGTCTCCTGGCTC

CCCCGGGCGGCCTGTCCGGCCGCAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCC

CAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGC

AGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCT

CTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCAT

CAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACC

TTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCA

TTGTCAAGAGCTTCAACAGGAATGAGTGT] (SEQ ID NO: 154)

[Spacer (SEQ ID NO: 923) ] [PL15-1004/GG/0001 LC (SEQ ID

- 168 039736

NO: 1003)]

[QGQSGS][IFCHMGVWPQCANYGGGSSGGSGAVGLLAPPGGLSGRSDNHGGSDIQMTQSPSSLSA

SVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 155)

Nucleotide sequence PL15-1004/GG/0001 LC

GGAGGTGGCTCGAGCGGTGGCAGCGGTGCTGTGGGTCTCCTGGCTCCCCCGGGCGGCCTGTCCGGCC GCAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGT GGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAG CAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCA GCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGA CTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTG GAAATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGA ATGAGTGT (SEQ ID NO: 1002)

Amino acid sequence PL15-1004/GG/0001 LC

(SEQ ID NO: 1003)

[Spacer (SEQ ID NO: 929)] [PL18-1004/GG/0001 (also refers here to PL18-3001) LC (SEQ ID NO: 1004)]

[CAAGGTCAGTCTGGATCC][GGAGGTGGCTCGAGCGGTGGCAGCGGTGCTGTGGGTCTCCTGGCTC CCCCGGGCGGCCTGTCCGGCCGCAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCC CAGCAGCCTGTCTGCTAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGC AGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCT CTCTGCAGTCTGGCGTGCCCAGCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCAT CAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACC TTTGGCGGAGGTACCAAGGTGGAAATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCA TTGTCAAGAGCTTCAACAGGAATGAGTGT] (SEQ ID NO: 156)

[Spacer (SEQ ID NO: 923) ] [PL18-1004/GG/0001 LC (SEQ ID

NO: 1005)]

[QGQSGS][PCHPHAYDARPYCNVGGGSSGGSGAVGLLAPPGGLSGRSDNHGGSDIQMTQSPSSLSA

SVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 157)

Nucleotide sequence PL18-1004/GG/0001 LC

- 169 039736

GGAGGTGGCTCGAGCGGTGGCAGCGGTGCTGTGGGTCTCCTGGCTCCCCCGGGCGGCCTGTCCGGCC GCAGCGATAATCATGGCGGTTCTGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCTAGCGT GGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAG CAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCTCTCTGCAGTCTGGCGTGCCCA GCAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGA CTTCGCCACCTACTACTGCCAGCAGGACAACGGCTACCCCAGCACCTTTGGCGGAGGTACCAAGGTG GAAATCAAGCGTTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGA ATGAGTGT (SEQ ID NO: 1004)

Amino acid sequence PL18-1004/GG/0001 LC

PCHPHAYDARPYCNVGGGSSGGSGAVGLLAPPGGLSGRSDNHGGSDIQMTQSPSSLSASVGDRVTIT CRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QDNGYPSTFGGGTKVEIKR (SEQ ID NO: 1005)

PL07-0001

[Spacer (SEQ ID NO: 923)] [PL07-1001 LC (SEQ ID NO: 1145)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGLSGRSDNHGGSDIQMTQSPSSLSASVGDRV

TITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1144)

Amino acid sequence PL07-1001

GIALCPSHFCQLPQTGGGSSGGSGGSGGLSGRSDNHGGSDIQMTQSPSSLSASVGDRVTITCRASQS ISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDNGYP STFGGGTKVEIKR (SEQ ID NO: 1145)

PL07-0002

[Spacer (SEQ ID NO: 923)] [PL07-0002 LC (SEQ ID NO: 1147)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGLSGRSGNHGGSDIQMTQSPSSLSASVGDRV TITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1146)

Amino acid sequence PL02-002

GIALCPSHFCQLPQTGGGSSGGSGGSGGLSGRSGNHGGSDIQMTQSPSSLSASVGDRVTITCRASQS ISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDNGYP STFGGGTKVEIKR (SEQ ID NO: 1147)

PL07-1001

[Spacer (SEQ ID NO: 923)] [PL07-1001 LC (SEQ ID NO: 1149)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSSGGSDIQMTQSPSSLSASVGDRV

TITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1148)

Amino acid sequence PL07-1001

- 170 039736

GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSSGGSDIQMTQSPSSLSASVGDRVTITCRASQS

ISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDNGYP

STFGGGTKVEIKR (SEQ ID NO: 1149)

PL07-1002

[Spacer (SEQ ID NO: 923)] [PL07-1002 LC (SEQ ID NO: 1151)]

[spacer (SEQ ID NO: 923)][PL07-1002 LC (SEQ ID NO: 1151)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGQNQALRMAGGSDIQMTQSPSSLSASVGDRV

TITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY

YCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1150)

Amino acid sequence PL07-1002

GIALCPSHFCQLPQTGGGSSGGSGGSGGQNQALRMAGGSDIQMTQSPSSLSASVGDRVTITCRASQS

ISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDNGYP

STFGGGTKVEIKR (SEQ ID NO: 1151)

PL07-1003

[Spacer [SEQ ID NO: 923)] [PL07-1003 LC (SEQ ID NO: 1153)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGVHMPLGFLGPGGSDIQMTQSPSSLSASVGD

RVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA

TYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1152)

Amino acid sequence PL07-1003

GIALCPSHFCQLPQTGGGSSGGSGGSGGVHMPLGFLGPGGSDIQMTQSPSSLSASVGDRVTITCRAS

QSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDNG YPSTFGGGTKVEIKR (SEQ ID NO: 1153)

PL07-1004

[Spacer (SEQ ID NO: 923)] [PL07-1004 LC (SEQ ID NO: 1201)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGAVGLLAPPGGSDIQMTQSPSSLSASVGDRV

TITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY

YCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1200)

Amino acid sequence PL07-1004

GIALCPSHFCQLPQTGGGSSGGSGGSGGAVGLLAPPGGSDIQMTQSPSSLSASVGDRVTITCRASQS

ISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDNGYP

STFGGGTKVEIKR (SEQ ID NO: 1201)

PL07-2002

[Spacer (SEQ ID NO: 923)] [PL07-2002 LC (SEQ ID NO: 1155)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSGNHGGSDIQMTQSPSSLSAS

VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE

DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1154)

- 171 039736

Amino acid sequence PL07-2002

GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSGNHGGSDIQMTQSPSSLSASVGDRVTITC RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKR (SEQ ID NO: 1155)

PL07-2003

[Spacer (SEQ ID NO: 923)] [PL07-2003 LC (SEQ ID NO: 1157)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSANPRGGGSDIQMTQSPSSLS

ASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1156)

Amino acid sequence PL07-2003

GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSANPRGGGSDIQMTQSPSSLSASVGDRVTI TCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQDNGYPSTFGGGTKVEIKR (SEQ ID NO: 1157)

PL07-2004

[Spacer (SEQ ID NO: 923)] [PL07-2004 LC (SEQ ID NO: 1159)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGAVGLLAPPTSGRSANPRGGGSDIQMTQSPS SLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1158)

Amino acid sequence PL07-2004

(SEQ ID NO: 1159)

PL07-2005

[Spacer (SEQ ID NO: 923)] [PL07-2005 LC (SEQ ID NO: 1161)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGAVGLLAPPSGRSANPRGGGSDIQMTQSPSS LSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1160)

Amino acid sequence PL07-2005

GIALCPSHFCQLPQTGGGSSGGSGGSGGAVGLLAPPSGRSANPRGGGSDIQMTQSPSSLSASVGDRV TITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQDNGYPSTFGGGTKVEIKR (SEQ ID NO: 1161)

PL07-2006

[Spacer (SEQ ID NO: 923)] [PL07-2006 LC (SEQ ID NO: 1163)]

- 172 039736

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDDHGGSDIQMTQSPSSLSAS

VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE

DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1162)

Amino acid sequence PL07-2006

GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDDHGGSDIQMTQSPSSLSASVGDRVTITC RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKR (SEQ ID NO: 1163)

PL07-2007 [Spacer (SEQ ID NO: 923)] [PL07-2007 LC (SEQ ID NO: 1165)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDIHGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1164)

Amino acid sequence PL07-2007

GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDIHGGSDIQMTQSPSSLSASVGDRVTITC RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKR (SEQ ID NO: 1165)

PL07-2008 [Spacer (SEQ ID NO: 923)] [PL07-2008 LC (SEQ ID NO: 1167)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDQHGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1166)

Amino acid sequence PL07-2008

GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDQHGGSDIQMTQSPSSLSASVGDRVTITC RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKR (SEQ ID NO: 1167)

PL07-2009 [Spacer (SEQ ID NO: 923)] [PL07-2009 LC (SEQ ID NO: 1169)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDTHGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1168)

Amino acid sequence PL07-2009

GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDTHGGSDIQMTQSPSSLSASVGDRVTITC RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKR (SEQ ID NO: 1169)

PL07-2010 [Spacer (SEQ ID NO: 923)] [PL07-2010 LC (SEQ ID NO: 1171)]

- 173 039736

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDYHGGSDIQMTQSPSSLSAS

VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE

DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1170)

Amino acid sequence PL07-2010

GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDYHGGSDIQMTQSPSSLSASVGDRVTITC RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKR (SEQ ID NO: 1171)

PL07-2011 [Spacer (SEQ ID NO: 923)] [PL07-2011 LC (SEQ ID NO: 1173)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDNPGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1172)

Amino acid sequence PL07-2011

GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDNPGGSDIQMTQSPSSLSASVGDRVTITC RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKR (SEQ ID NO: 1173)

PL07-2012 [Spacer (SEQ ID NO: 923)] [PL07-2012 LC (SEQ ID NO: 1175)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSANPGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1174)

Amino acid sequence PL07-2012

GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSANPGGSDIQMTQSPSSLSASVGDRVTITC RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKR (SEQ ID NO: 1175)

PL07-2013 [Spacer (SEQ ID NO: 923)] [PL07-2013 LC (SEQ ID NO: 1177)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSANIGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1176)

Amino acid sequence PL07-2013

GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSANIGGSDIQMTQSPSSLSASVGDRVTITC RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKR (SEQ ID NO: 1177)

PL07-2014

[Spacer (SEQ ID NO: 923)] [PL07-2017 LC (SEQ ID NO: 1179)]

- 174 039736

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDNIGGSDIQMTQSPSSLSAS

VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1178)

Amino acid sequence PL07-2014

GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDNIGGSDIQMTQSPSSLSASVGDRVTITC RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKR (SEQ ID NO: 1179)

PL07-1004/GG/0003

[Spacer (SEQ ID NO: 923)] [PL07-1004/GG/0003 LC (SEQ ID

NO: 1181)]

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGAVGLLAPPGGTSTSGRSANPRGGGSDIQMT

QSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQDNGYPSTFGGGTKVEIKR] (SEQ ID NO: 1180)

Amino acid sequence PL07-1004/GG/0003

GIALCPSHFCQLPQTGGGSSGGSGGSGGAVGLLAPPGGTSTSGRSANPRGGGSDIQMTQSPSSLSAS

VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKR (SEQ ID NO: 1181)

Example 11 Activated Anti-PDL1 Antibodies of the Invention.

This example shows that activated anti-PDL1 antibodies can be generated with various combinations of MM, CM, VL and VH regions, as well as with various human isotypes.

Table 18 Components of Activated Anti-PDL1 Antibodies

Masking sequence (MM) Substrate sequence (CM) VL vh YCEVSELFVLPWCMG (SEQ ID NO: 208) LSGRSDNH (SEQ ID NO: 341) SEQ ID NO: 12 SEQ ID NO: fourteen SCLMHPHYAHDYCYV (SEQ ID NO: 426) TGRGPSWV (SEQ ID NO: 338) SEQ ID NO: 58 SEQ ID NO: 16

- 175 039736

LCEVLMLLQHPWCMG (SEQ ID NO: 59) PLTGRSGG (SEQ ID NO: 344) SEQ ID NO:18 IACRHFMEQLPFCHH (SEQ ID NO: 60) TARGPSFK (SEQ ID NO: 340) SEQ ID NO: 20 FGPRCGEASTCVPYE (SEQ ID NO: 61) NTLSGRSENHSG (SEQ ID NO: 435) SEQ ID NO: 22 ILYCDSWGAGCLTRP (SEQ ID NO: 62) NTLSGRSGNHGS (SEQ ID NO: 436) SEQ ID NO: 2 4 GIALCPSHFCQLPQT (SEQ ID NO: 63) TSTSGRSANPRG (SEQ ID NO: 437) SEQ ID NO:26 DGPRCFVSGECSPIG (SEQ ID NO: 64) TSGRSANP (SEQ ID NO: 438) SEQ ID NO:28

- 176 039736

LCYKLDYDDRSYCHI (SEQ ID NO: 65) VHMPLGFLGP (SEQ ID NO: 352) SEQ ID NO: 3 0 PCHPHPYDARPYCNV (SEQ ID NO: 66) AVGLAPP (SEQ ID NO: 372) SEQ ID NO:32 PCYWHPFFAYRYCNT (SEQ ID NO: 67) AQNLLGMV (SEQ ID NO: 360) SEQ ID NO:34 VCYYMDWLGRNWCSS (SEQ ID NO: 68) QNQALRMA (SEQ ID NO: 359) SEQ ID NO: 3 6 LCDLFKLREFPYCMG (SEQ ID NO: 69) LAAPLGLL (SEQ ID NO: 371) SEQ ID NO: 3 8 YLPCHFVPIGACNNK (SEQ ID NO: 70) STPFGMF (SEQ ID NO: 361) SEQ ID NO:40

- 177 039736

IFCHMGVWPQCANY (SEQ ID NO: 71) ISSGLLSS (SEQ ID NO: 364) SEQ ID NO:42 ACHPHPYDARPYCNV (SEQ ID NO: 72) PAGLWLDP (SEQ ID NO: 374) SEQ ID NO: 4 4 PCHPAPYDARPYCNV (SEQ ID NO: 73) VAGRSMRP (SEQ ID NO: 439) SEQ ID NO: 4 6 PCHPHAYDARPYCNV (SEQ ID NO: 74) WPEGRRS (SEQ ID NO: 440) SEQ ID NO: 4 8 PCHPHPADARPYCNV (SEQ ID NO: 75) ILPRSPAF (SEQ ID NO: 441) SEQ ID NO: 50 PCHPHPYAARPYCNV (SEQ ID NO: 76) MVLGRSLL (SEQ ID NO: 442) SEQ ID NO: 52 PCHPHPYDAAPYCNV (SEQ ID NO: 77) QGRAITFI (SEQ ID NO: 443) SEQ ID NO: 54 PCHPHPYDARPACNV (SEQ ID NO: 78) SPRSIMLA (SEQ ID NO: 444) SEQ ID NO: 56 PCHPHPYDARPYCAV (SEQ ID NO: 79) SMLRSMPL (SEQ ID NO: 445)

- 178 039736

PCHAHPYDARPYCNV (SEQ ID NO: 80) ISSGLLSGRSDNH (SEQ ID NO: 377) PCHPHPYDARAYCNV (SEQ ID NO: 81) AVGLLAPPGGLSGRSDNH (SEQ ID NO: 383) ISSGLLSSGGSGGSLSGRDN H (SEQ ID NO: 378) LSGRSGNH (SEQ ID NO: 883) SGRSANPRG (SEQ ID NO: 884) LSGRSDDH (SEQ ID NO: 885) LSGRSDIH (SEQ ID NO: 886) LSGRSDQH (SEQ ID NO: 887) LSGRSDTH (SEQ ID NO: 888) LSGRSDYH (SEQ ID NO: 889) LSGRSDNP (SEQ ID NO: 890) LSGRSANP (SEQ ID NO: 891) LSGRSANI (SEQ ID NO: 892)

- 179 039736

LSGRSDNI (SEQ ID NO: 893) MIAPVAYR (SEQ ID NO: 894) RPSPMWAY (SEQ ID NO: 895) WATPRPMR (SEQ ID NO: 896) FRLLDWQW (SEQ ID NO: 897) ISSGL (SEQ ID NO: 898) ISSGLLS (SEQ ID NO: 899) ISSGLL (SEQ ID NO: 900) ISSGLLSGRSANPRG (SEQ ID NO: 901) AVGLLAPPTSGRSANPRG (SEQ ID NO: 902) AVGLLAPPSGRSANPRG (SEQ ID NO: 903) ISSGLLSGRSDDH (SEQ ID NO: 904) ISSGLLSGRSDDIH (SEQ ID NO: 905)

- 180 039736

ISSGLLSGRSDQH (SEQ ID NO: 906) ISSGLLSGRSDTH (SEQ ID NO: 907) ISSGLLSGRSDYH (SEQ ID NO: 908) ISSGLLSGRSDNP (SEQ ID NO: 909) ISSGLLSGRSANP (SEQ ID NO: 910) ISSGLLSGRSANI (SEQ ID NO: 911) AVGLLAPPGGLSGRSDDDH (SEQ ID NO: 912) AVGLLAPPGGLSGRSDIH (SEQ ID NO: 913) AVGLLAPPGGLSGRSDQH (SEQ ID NO: 914) AVGLLAPPGGLSGRSDTH (SEQ ID NO: 915) AVGLLAPPGGLSGRSDYH (SEQ ID NO: 916) AVGLLAPPGGLSGRSDNP (SEQ ID NO: 917) AVGLLAPPGGLSGRSANP (SEQ ID NO: 918)

- 181 039736

AVGLLAPPGGLSGRSANI (SEQ ID NO: 919) ISSGLLSGRSDNI (SEQ ID NO: 920) AVGLLAPPGGLSGRSDNI (SEQ ID NO: 921) GLSGRSDNHGGAVGLLAPP (SEQ ID NO: 1009) GLSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 1010)

Any of the combinations described in Table. 18 can be combined with human immunoglobulin constant regions to produce fully human IgGs, including IgG1, IgG2, IgG4, or mutated constant regions to produce human IgGs with altered functions, such as IgG1 N297A, IgG1 N297Q, or IgG4 S22 8P. The combinations shown in Table 18 are not limited to the specific combinations shown in any given row, and include any masking sequence from column 1, corresponding to any substrate sequence from column 2, corresponding to any VL sequence from column 3, corresponding to any VH sequence from column 4. In addition to the substrate sequences listed in column 2, any CM disclosed herein may be used.

As an example, a spacer sequence (SEQ ID NO: 923) and a masking sequence SEQ ID NO: 63 can be combined with the substrate ISSGLLSGRSDNH (SEQ ID NO: 377), VL SEQ ID NO: 58, and combined with the human kappa constant region to give SEQ ID NO: 428; or a masking sequence of SEQ ID NO: 63 can be combined with the substrate ISSGLLSGRSDNH (SEQ ID NO: 377), VL SEQ ID NO: 58, and combined with a human kappa constant region to give SEQ ID NO: 1008. In addition, VH SEQ ID NO : 46 can be combined with human immunoglobulin heavy chain constant regions to produce human IgG1 (SEQ ID NO: 430), mutant human IgG4 S228P (SEQ ID NO: 432), mutant human IgG1 N297A (SEQ ID NO: 434), or mutant human IgG1 N297Q (SEQ ID NO: 1202). Co-expression of SEQ ID NO: 427 with SEQ ID NO: 429 produces a fully human IgG1-activated anti-PDL1 antibody. Co-expression of SEQ ID NO: 427 with SEQ ID NO: 431 yields fully human IgG4 activated anti-PDL1 antibody S228P. Co-expression of SEQ ID NO: 427 with SEQ ID NO: 433 yields fully human activated IgG1 anti-PDL1 antibody N297A. Co-expression of SEQ ID NO: 427 with the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1202 produces the fully human IgG1 activated anti-PDL1 antibody N297Q.

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Light chain sequence (SEQ ID NO: 1007) with spacer (SEQ ID NO: 1006)] (SEQ ID NO: 427)

[CAGGGCCAGTCCGGCTCA][TATCTGCCCTGCCACTTCGTGTCCAATCGGGGCCTGTAACAATAAGG

GCGGTGGATCTAGTGGTGGCTCAGGCGGGTCTGGCGGCATTTCCAGTGGACTCTTGTCAGGACGATC CGATAATCATGGCGGGTCCGACATCCAGATGACACAGAGCCCTTCTTCCCTCTCCGCAAGCGTTGGC GACAGGGTCACCATTACCTGTAGGGCTTCTCAGAGCATCTCAAGCTATCTGAACTGGTACCAGCAGA AACCTGGAAAGGCTCCAAAACTGCTGATTTACGCTGCCTCCAGTCTTCAGTCAGGCGTCCCCTCCAG ATTTAGCGGATCAGGTAGTGGAACTGATTTTACCCTTACAATATCTTCTCTGCAGCCAGAGGACTTC GCCACATACTATTGTCAGCAAGACAATGGTTACCCCAGTACATTTGGCGGAGGGACAAAGGTCGAGA TCAAAAGGACCGTAGCAGCACCAAGCGTCTTTATTTTCCCCCCCAGTGACGAACAGCTGAAGAGCGG AACAGCTTCAGTGGTGTGTCTCCTGAATAACTTCTATCCACGCGAGGCAAAGGTGCAGTGGAAGGTG GACAATGCACTGCAGTCTGGTAATTCCCAAGAAAGTGTTACTGAGCAGGATTCCAAGGATTCAACTT ACTCTCTGTCTAGCACCCTGACTCTTTCTAAAGCAGATTATGAGAAGCATAAGGTCTACGCTTGCGA GGTGACCCACCAGGGGCTTTCCTCTCCAGTTACCAAGTCATTCAACCGGGGTGAGTGTTGATGAGAA TTC] (SEQ ID NO: 427)

Light chain sequence without spacer

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TATCTGCCCTGCCACTTCGTGCCAATCGGGGCCTGTAACAATAAGGGCGGTGGATCTAGTGGTGGCT CAGGCGGGTCTGGCGGCATTTCCAGTGGACTCTTGTCAGGACGATCCGATAATCATGGCGGGTCCGA CATCCAGATGACACAGAGCCCTTCTTCCCTCTCCGCAAGCGTTGGCGACAGGGTCACCATTACCTGT AGGGCTTCTCAGAGCATCTCAAGCTATCTGAACTGGTACCAGCAGAAACCTGGAAAGGCTCCAAAAC TGCTGATTTACGCTGCCTCCAGTCTTCAGTCAGGCGTCCCCTCCAGATTTAGCGGATCAGGTAGTGG AACTGATTTTACCCTTACAATATCTTCTCTGCAGCCAGAGGACTTCGCCACATACTATTGTCAGCAA GACAATGGTTACCCCAGTACATTTGGCGGAGGGACAAAGGTCGAGATCAAAAGGACCGTAGCAGCAC CAAGCGTCTTTATTTTCCCCCCCAGTGACGAACAGCTGAAGAGCGGAACAGCTTCAGTGGTGTGTCT CCTGAATAACTTCTATCCACGCGAGGCAAAGGTGCAGTGGAAGGTGGACAATGCACTGCAGTCTGGT AATTCCCAAGAAAGTGTTACTGAGCAGGATTCCAAGGATTCAACTTACTCTCTGTCTAGCACCCTGA CTCTTTCTAAAGCAGATTATGAGAAGCATAAGGTCTACGCTTGCGAGGTGACCCACCAGGGGCTTTC CTCTCCAGTTACCAAGTCATTCAACCGGGGTGAGTGTTGATGAGAATTC (SEQ ID NO: 1007)

[Light chain sequence (SEQ ID NO: 1008) with spacer (SEQ ID NO: 923)] (SEQ ID NO: 428)

[QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQDNGYPSTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 428)

Light chain sequence without spacer

GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSASVGDRVTITC

RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ DNGYPSTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ID08 (SE00)

SEQ ID NO: 429

GAAGTGCAGCTGCTCGAAAGCGGCGGAGGCTTGGTGCAGCCAGGAGGGAGCCTGCGACTGTCTTGCG CAGCCAGCGGATTCACTTTCTCTTCCTATGCCATGAGCTGGGTTCGACAGGCACCCGGCAAAGGTCT CGAGTGGGTGTCTAGCATCTGGCGAAACGGAATAGTTACAGTGTATGCCGATAGCGTGAAGGGTCGC TTTACTATTTCACGGGATAATTCTAAGAACACCCTCTACCTGCAAATGAATAGCCTTAGGGCAGAAG ATACCGCCGTGTACTACTGTGCCAAATGGTCCGCAGCCTTTGACTACTGGGGCCAGGGGACACTGGT GACCGTGTCCTCTGCATCAACCAAGGGGCCATCAGTGTTCCCACTCGCCCCATCTTCCAAGAGTACT TCCGGCGGAACCGCAGCCCTTGGCTGCCTTGTTAAGGACTATTTCCCAGAACCCGTGACCGTAAGTT

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GGAACTCTGGCGCCCTTACTTCTGGGGTGCACACCTTCCCAGCAGTGTTGCAGTCCAGTGGCCTTTA CTCTCTGTCTAGTGTAGTGACTGTGCCTTCCTCTAGTCTCGGTACCCAGACCTATATTTGTAATGTT AACCATAAGCCCAGCAATACAAAGGTTGATAAGAAAGTGGAACCCAAGAGCTGCGATAAGACACATA CCTGCCCACCTTGTCCAGCTCCCGAGCTGCTGGGCGGACCCTCAGTCTTTCTCTTCCCACCTAAACC CAAGGATACCCTTATGATCTCCAGGACTCCTGAGGTGACCTGCGTTGTGGTCGACGTGTCACATGAG GACCCTGAGGTAAAGTTTAACTGGTACGTGGACGGTGTGGAGGTACATAACGCTAAGACTAAGCCAC GAGAGGAGCAATACGCTTCCACTTACAGGGTGGTCAGCGTCCTGACCGTTCTCCATCAGGACTGGCT GAACGGGAAGGAATATAAGTGTAAGGTTAGCAACAAAGCTCTCCCTGCACCAATCGAGAAGACAATC AGCAAGGCAAAAGGGCAGCCTCGGGAACCTCAGGTCTACACCCTCCCTCCTAGCAGGGAAGAGATGA CAAAGAACCAGGTCTCTCTCACCTGCCTGGTGAAAGGCTTCTATCCATCTGACATTGCTGTGGAGTG GGAATCCAACGGCCAGCCTGAAAATAATTATAAGACCACACCCCCCGTCCTTGATTCCGATGGATCT TTCTTCCTGTACAGTAAACTCACCGTCGACAAATCACGGTGGCAGCAAGGTAACGTGTTCAGCTGTT CTGTCATGCATGAGGCTCTGCATAACCATTACACACAAAAGTCTTTGTCATTGTCTCCAGGATGATG AGAATTCATTGATCATAATCAGCCATACCAC

SEQ ID NO: 430

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE dpevkfnwyvdgvevhnaktkpreeqy|n|styrwsvltvlhqdwlngkeykckvsnkalpapiekti SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO: 431

GAAGTGCAGCTGCTCGAAAGCGGCGGAGGCTTGGTGCAGCCAGGAGGGAGCCTGCGACTGTCTTGCG CAGCCAGCGGATTCACTTTCTCTTCCTATGCCATGAGCTGGGTTCGACAGGCACCCGGCAAAGGTCT CGAGTGGGTGTCTAGCATCTGGCGAAACGGAATAGTTACAGTGTATGCCGATAGCGTGAAGGGTCGC TTTACTATTTCACGGGATAATTCTAAGAACACCCTCTACCTGCAAATGAATAGCCTTAGGGCAGAAG ATACCGCCGTGTACTACTGTGCCAAATGGTCCGCAGCCTTTGACTACTGGGGCCAGGGGACACTGGT GACCGTGTCCTCTGCATCAACCAAGGGGCCATCAGTGTTCCCACTCGCCCCATGTAGCAGATCAACA TCTGAATCCACCGCAGCCCTTGGCTGCCTTGTTAAGGACTATTTCCCAGAACCCGTGACCGTAAGTT GGAACTCTGGCGCCCTTACTTCTGGGGTGCACACCTTCCCAGCAGTGTTGCAGTCCAGTGGCCTTTA CTCTCTGTCTAGTGTAGTGACTGTGCCTTCCTCTAGTCTCGGTACCAAGACCTATACCTGCAACGTA GATCATAAGCCCAGCAATACAAAGGTTGATAAGAGAGTAGAGTCAAAGTACGGCCCACCCTGCCCAC CTTGTCCAGCTCCCGAGTTCCTGGGCGGACCCTCAGTCTTTCTCTTCCCACCTAAACCCAAGGATAC CCTTATGATCTCCAGGACTCCTGAGGTGACCTGCGTTGTGGTCGACGTGTCACAAGAGGACCCTGAG

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GTACAGTTTAACTGGTACGTGGACGGTGTGGAGGTACATAACGCTAAGACTAAGCCACGAGAGGAGC AATTTAACTCCACTTACAGGGTGGTCAGCGTCCTGACCGTTCTCCATCAGGACTGGCTGAACGGGAA GGAATATAAGTGTAAGGTTAGCAACAAAGGTCTGCCCAGTTCTATCGAGAAGACAATCAGCAAGGCA AAAGGGCAGCCTCGGGAACCTCAGGTCTACACCCTCCCTCCTAGCCAGGAAGAGATGACAAAGAACC AGGTCTCTCTCACCTGCCTGGTGAAAGGCTTCTATCCATCTGACATTGCTGTGGAGTGGGAATCCAA CGGCCAGCCTGAAAATAATTATAAGACCACACCCCCCGTCCTTGATTCCGATGGATCTTTCTTCCTG TACAGTCGCCTCACCGTCGACAAATCACGGTGGCAGGAAGGTAACGTGTTCAGCTGTTCTGTCATGC ATGAGGCTCTGCATAACCATTACACACAAAAGTCTTTGTCATTGTCTCTCGGATGATGAGAATTCAT T GAT CATAAT CAGC CATAC CAC

SEQ ID NO: 432

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDYWGQGTLVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNV DHKPSNTKVDKRVESKYGPPCP|P|CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

SEQ ID NO:433

GAAGTGCAGCTGCTCGAAAGCGGCGGAGGCTTGGTGCAGCCAGGAGGGAGCCTGCGACTGTCTTGCG CAGCCAGCGGATTCACTTTCTCTTCCTATGCCATGAGCTGGGTTCGACAGGCACCCGGCAAAGGTCT CGAGTGGGTGTCTAGCATCTGGCGAAACGGAATAGTTACAGTGTATGCCGATAGCGTGAAGGGTCGC TTTACTATTTCACGGGATAATTCTAAGAACACCCTCTACCTGCAAATGAATAGCCTTAGGGCAGAAG ATACCGCCGTGTACTACTGTGCCAAATGGTCCGCAGCCTTTGACTACTGGGGCCAGGGGACACTGGT GACCGTGTCCTCTGCATCAACCAAGGGGCCATCAGTGTTCCCACTCGCCCCATCTTCCAAGAGTACT TCCGGCGGAACCGCAGCCCTTGGCTGCCTTGTTAAGGACTATTTCCCAGAACCCGTGACCGTAAGTT GGAACTCTGGCGCCCTTACTTCTGGGGTGCACACCTTCCCAGCAGTGTTGCAGTCCAGTGGCCTTTA CTCTCTGTCTAGTGTAGTGACTGTGCCTTCCTCTAGTCTCGGTACCCAGACCTATATTTGTAATGTT AACCATAAGCCCAGCAATACAAAGGTTGATAAGAAAGTGGAACCCAAGAGCTGCGATAAGACACATA CCTGCCCACCTTGTCCAGCTCCCGAGCTGCTGGGCGGACCCTCAGTCTTTCTCTTCCCACCTAAACC CAAGGATACCCTTATGATCTCCAGGACTCCTGAGGTGACCTGCGTTGTGGTCGACGTGTCACATGAG GACCCTGAGGTAAAGTTTAACTGGTACGTGGACGGTGTGGAGGTACATAACGCTAAGACTAAGCCAC GAGAGGAGCAATACGCTTCCACTTACAGGGTGGTCAGCGTCCTGACCGTTCTCCATCAGGACTGGCT GAACGGGAAGGAATATAAGTGTAAGGTTAGCAACAAAGCTCTCCCTGC ACCAATCGAGAAGACAATC AGCAAGGCAAAAGGGCAGCCTCGGGAACCTCAGGTCTACACCCTCCCTCCTAGCAGGGAAGAGATGA CAAAGAACCAGGTCTCTCTCACCTGCCTGGTGAAAGGCTTCTATCCATCTGACATTGCTGTGGAGTG

GGAATCCAACGGCCAGCCTGAAAATAATTATAAGACCACACCCCCCGTCCTTGATTCCGATGGATCT TTCTTCCTGTACAGTAAACTCACCGTCGACAAATCACGGTGGCAGCAAGGTAACGTGTTCAGCTGTT CTGTCATGCATGAGGCCTCTGCATAACCATTACACACAAAAGTCTTTGTCATTGTCTCCAGGATGATG AGAATTCATTGATCATAATCAGCCATACCAC

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SEQ ID NO: 434

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYADSVKGR

FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKST

SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNV

NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE

DPEVKFNWYVDGVEVHNAKTKPREEQY^STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI

SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO: 1202

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHE dpevkfnwyvdgvevhnaktkpreeqy|q|styrwsvltvlhqdwlngkeykckvsnkalpapiekti SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Additional examples of heavy chain constant regions include, but are not limited to, a heavy chain constant region having a mutation or deletion of one or more of the following amino acids: Ser 228, Leu234, Leu235, Gly236, Gly237, Asp265, Asp270, Asn297, Lys 320, Lys 322 , Glu328, Pro329, Pro331, according to the EC index set forth by Kabat, so that the Fc region has a lower affinity for the Fc gamma receptor as a result.

Example 12 Evaluation of the effectiveness of masking fragments.

This assay assessed the ability of the masking peptide to block the binding of the activated antibody to the antigen compared to the binding of the unmodified antibody to the antigen.

The general scheme of this assay is as follows: Nunc, Maxisorp plates are coated overnight at 4°C at 100 μl/well with a 1 ppm/ml solution of human PDL1 (R&D, Systems) in PBS, pH 7.4. The plates are washed 3 times with PBST (PBS, pH 7.4, 0.05% Tween-20) and the wells are blocked with 200 ml/well of 10 mg/ml BSA in PBST for 2 hours at room temperature. The plates are washed 3 times with PBST (PBS, pH 7.4, 0.05% Tween-20). Dilution curves were prepared in 10 mg/ml BSA in PBST as shown in Table 10 below.

Table 10. Layout of samples in the masking efficiency assay plate, one plate per time point

[antibody]=nM Rows 1-3 [activated antibody 1]=nM Rows 4-6 [activated antibody 2] = nM Rows 7-9 [activated antibody 3] = nM Rows 10-12 BUT 37 1000 1000 1000 AT 12.3 333 333 333 FROM 4.1 111 111 111 D 1.34 37 37 37 E 0.45 12.3 12.3 12.3 F 0.15 4.1 4.1 4.1 G 0.03 1.34 1.34 1.34 H 0.01 0.45 0.45 Control

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Binding solutions are added to plates which are then incubated for 1 hour at room temperature (RT) and then washed 3 times with PBST (PBS, pH 7.4, 0.05% Tween-20). 100 μl/well 1:4000 dilution of goat anti-human IgG (Fab specific, Sigma cat # A0293) in 10 mg/ml BSA in PBST is added and the plate is incubated for 1 hour at room temperature. The tablet develop with TMB and 1N HCl. In FIG. 15-17 are plots of binding isotherms for activated anti-PDL1 antibodies, which include the C5H9v2 anti-PDLl antibody described herein. Plots were obtained using the Prizm program (Sigma Plot) and the data was fitted to a single site saturation model and Kd determined. In FIG. 17 PL03+ is an effector activated antibody, wild-type IgG1, which is used as a positive control. PL03- is an effector activated antibody, mutated IgG1 N297Q, which is used as a negative control.

The masking efficiency is calculated by dividing the Kd for binding of activated antibodies by the Kd of the parent antibody. The effectiveness of the masking is shown in Fig. 15-17.

Example 13 Activated Anti-PDL1 Antibodies of the Invention.

Slow the induction of diabetes in NOD mice.

In this example, the activated anti-PDL1 antibodies PL15-0003-C5H9v2 and PL18-0003-C5H9v2 were tested for their ability to induce diabetes in NOD mice. NOD mice, subspecies NOD/ShiLtJ, were obtained from the Jackson Laboratory nursery at 6 weeks of age and acclimatized in situ. After 9.5 weeks, mice were tested for the presence of diabetes prior to inclusion in the experiment, division into groups and dosing, as indicated in table. 19.

Table 19. Groups and doses in the diabetes trial with activated anti-PDL1 antibodies

Group Number of mice Treatment Dose (mg/kg) Volumetric dose (ml/kg) Scheme Route of administration one eight mlgG2a (Cl.18.4) 3 ten Day 0 i/b 2 eight anti-PDL1 (C5H9v2) one ten Day 0 i/b 3 eight anti-PDL1 (C5H9v2) 0.3 ten Day 0 i/b four eight activated antibody PL15-0003C5H9v2 one ten Day 0 i/b 5 eight activated antibody PL18-0003C5H9v2 one ten Day 0 i/b

In FIG. 18A, which plots % of diabetic mice versus number of days after initial dose, showing that anti-PDL1 antibody C5H9v2 induced diabetes in NOD mice at 1 mg/kg, while activated anti-PDL1- the PL15-0003-C5H9v2 antibody (here referred to as Gr4-PF15) did not induce diabetes, and the upregulated anti-PDL1 antibody PL180003-C5H9v2 (here referred to as Gr 5-PL18) caused a delay in the development of diabetes when administered at a dose of 1 mg /kg. Control mIgG2a and anti-PDL1 antibody C5H9v2 at 0.3 mg/kg did not induce diabetes in NOD mice. In an additional group, a single dose of 3mg/kg of PL15-0003-C5H9v2, an upregulated anti-PDL1 antibody administered i.p. at a volume dose of 10 ml/kg, induced diabetes in 2 of 8 mice (75% of mice without diabetes), providing more than threefold safety margin compared to anti-PDL1 antibody C5H9v2 (group 2). The results are shown in FIG. 18V.

Example 14 Activated anti-PDL1 antibodies of the invention suppress the growth of MC38 tumors in mice.

In this example, the activated anti-PDL1 antibodies PL15-0003-C5H9v2 and PL18-0003-C5H9v2 were analyzed for their ability to inhibit the growth of syngeneic MC38 tumors.

Mouse colon carcinoma MC38 cells were obtained from ATCC. MC38 cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37° C. in an atmosphere with 5% CO ₂ in air. Cells were harvested during logarithmic growth phase, resuspended in PBS with appropriate cell titer, and left on ice to induce tumors.

Each mouse was inoculated subcutaneously in the right flank with 0.5x10 ⁶ MC38 cells in PBS for tumor growth. Treatments were started when the average size of the tumors reached about 100-200 mm ³ (no more than 200 mm ³ ). The sizes of the tumors were determined twice a week in two dimensions using a caliper and the volume was expressed in mm ³ using the formula: V=0.5 axb ² where a and b are the long and short diameters of the tumor, respectively.

Mice were divided into groups and dosed as indicated in Table 20.

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Table 20. Groups and doses in the experiment with syngeneic MC38 tumors with activated anti-PDL1 antibodies

Group Number of mice Treatment Dose (mg/kg) Volumetric dose (ml/kg) Scheme Entry way one 9 mlgG2a (Cl.18.4) ten ten t.i.w. within 2 weeks i/b 2 9 anti-PDL1 (C5H9v2) ten ten t.i.w. within 2 weeks i/b four 9 PL18-0003C5H9v2 ten ten t.i.w. within 2 weeks i/b 6 9 PL15-0003C5H9v2 ten ten t.i.w. within 2 weeks i/b

In FIG. 19, which plots tumor volume versus number of days after initial dose, showing that upregulated anti-PDL1 antibodies PL15-0003-C5H9v2 and PL18-0003-C5H9v2 inhibited the growth of syngeneic MC38 tumors, similarly to anti-PDL1 antibody C5H9v2, which was used as a positive control.

Example 15 An activated anti-PDL1 antibody of the invention demonstrates reduced PDL1 occupancy in the blood and spleen of C57B1/6 mice.

Unlike an antibody, which will arbitrarily bind to a target antigen, an activated antibody will be a molecule that is inert unless activated in the tumor microenvironment. Once activated, the cleaved activated antibody binds to the target antigen only within the tumor, bypassing antigens in peripheral regions. PDL1 is expressed in tumor cells as well as T cells (CD4+ and CD8+) in the bloodstream and spleen. This example shows that animals treated with an activated anti-PDL1 antibody lacked detectable activated antibody on T cells in the periphery, while animals treated with an anti-PDL1 antibody showed a detectable dose-dependent presence of antibody on T cells at the periphery. periphery.

To determine how activated anti-PDL1 antibody PL15-0003-C5H9v2 is protected from T-cell binding in the circulation and spleen compared to anti-PDL1 antibody C5H9v2, 14-week-old C57B1/6 mice (with tumors of size ranging from 38-671 mm ³ ) were processed according to the scheme shown in Table 21. The size of the tumors was determined in two dimensions using a caliper and the volume was expressed in mm ³ using the formula: V=0.5 aho, where a and b are the long and short diameters of the tumor, respectively. Approximately 20 hours later, the animals were euthanized; and blood, spleen and plasma were collected from each animal.

Table 21 Groups and doses in the PDL1 occupancy study

Group Number of mice Treatment Dose (mg/kg) Volumetric dose (ml/kg) Scheme Route of administration one 2 mlgG2a (Cl.18.4) one ten Day 0 i/b 2 2 Anti- PDLl (C5H9v2) one ten Day 0 i/b 3 2 AntiPDLl (C5H9v2) 0.3 ten Day 0 i/b four 2 Anti- PDLl (C5H9v2) ο,ι ten Day 0 i/b 5 2 PL15-0003C5H9v2 one ten Day 0 i/b 6 2 PL15-0003C5H9v2 0.3 ten Day 0 i/b

Whole blood was processed as follows: one volume of blood was added to 10 volumes of RBC lysis solution (130-094-183 Miltenyi Biotec) and processed following the manufacturer's protocol. Lymphocytes were resuspended in staining buffer (HBSS 2% FBS). The spleen was processed as follows: the spleen was placed on a 70 μM mesh filter (352350 Coming/BD Falcon) with 1 ml of RPMI-1640 serum-free medium. The posterior end of a 3 ml syringe was used to crush the splenic tissue in a circular motion. The filter was washed with medium. Splenocytes were pelleted using a centrifuge and blood was lysed using 10 volumes of RBC lysis solution (130-094183 Miltenyi Biotec) following the manufacturer's protocol. Splenocytes were refiltered after resuspension in staining buffer (HBSS 2% FBS). The number of lymphocytes was counted from

- 189 039736 whole blood and lymphocytes from the spleen and aliquot portions were poured into the wells of 96-well round bottom plates (5e5 cells/well). Cells were centrifuged and mouse FcR reagent 1:10 was added following the manufacturer's protocol (130-092-575 Miltenyi Biotec). One row of cells was stained with a saturation amount, 100 nM, of anti-PDL1 antibody C5H9v2 for 60 minutes to obtain the maximum saturation value. Cells were washed 3 times and stained with biotinylated a allotype antibody, anti-mIgG2a antibody (a) clone 8.3 (553502 BD Biosciences) at 300 ng/mL for 60 min. Cells were washed 3 times and then stained with 1 μg/ml (also referred to here as μg/ml) anti-CD4-Pacific Blue (558107 BD Bioscience), 1 μg/ml anti-CD8-APC (553035 BD Bioscience) and 1:500 dilution of SAPE (S-866 Life Technologies) within 30 min. Cells were washed 2 times and stained with 7-AAD following the manufacturer's protocol (559925 BD Bioscience). A MACSQuant flow cytometer was used to measure the amount of anti-PDL1 antibody C5H9v2 and activated anti-PDL1 antibody PL150003-C5H9v2 bound to T cells from whole blood and spleen. Briefly, a maximum of 30,000 events per lymphocyte gate were collected. Live cells were gated from the 7AAD negative gate. CD4+ and CD8+ cells were isolated separately, and anti-PDL1 mAb and anti-PDL1 activated antibody MFI were detected for CD4 and CD8. Percentage occupancy was determined by sampling the MFI sample compared to the mean MFI for maximum MFI binding. In FIG. 20A-D show the percentage of anti-PDL1 antibody C5H9v2 and activated anti-PDL1 antibody PL15-0003-C5H9v2 bound to CD4+ and CD8+ T cells from peripheral blood (FIGS. 20A, 20B) or spleen (FIGS. 20C, 20D). In all plots, circles represent isotype antibody, squares represent C5H9v2 anti-PDL1 antibody, and triangles represent PL15-0003-C5H9v2 activated anti-PDL1 antibody. The percentage occupancy of anti-PDL1 antibody C5H9v2 was a reflection of dose titration. The percentage occupancy of the activated anti-PDL1 antibody PL15-0003-C5H9v2 at 1 mg/kg was similar to that of the mIgG2a isotype control at 1 mg/kg.

Example 16 An activated anti-PDL1 antibody of the invention demonstrates reduced PDL1 occupancy in the blood and spleen of C57B1/6 mice.

This example shows that animals treated with an activated anti-PDL1 antibody of the present invention have reduced binding to blood T cells from tumor-bearing mice compared to mice that are treated with an anti-PDL1 antibody of the invention in various doses.

The study was carried out in the same manner as described in Example 15. Briefly, mice bearing MC38 tumors ranging in size from 100-200 mm ³ were treated with a single dose of C5H9v2 anti-PDL1 antibody or PL15-0003-C5H9v2 activated anti-PDL1 antibody, as shown in FIG. 21A and B and 4 days after administration, blood was analyzed for surface-bound antibody by flow cytometry. Plasma antibody and activated antibody concentrations were determined by ELISA. In FIG. 21A and B show that the presence of tumor-derived proteases capable of cleaving the activated antibody did not result in high blood levels of activated activated antibody (FIG. 21A); those. activated antibody (shown as squares) showed a decrease in peripheral PDL1 binding in tumor-bearing mice compared to antibody (shown as circles) or isotype antibody (shown as triangles), despite the fact that plasma concentrations of activated antibody were higher than antibody concentrations ( Fig. 21B).

Example 17 Activity of Anti-PDL1 Antibody and Activated Anti-PDL1 Antibody Embodiments in the Human T Cell Restimulation Assay.

In this example, peripheral blood mononuclear cells from a CMV positive donor were incubated in the presence of a CMV lysate and an anti-PDL1 antibody or an anti-PDL1 activated antibody of the invention to evaluate the effect of such an anti-PDL1 antibody or an activated anti-PDL1 antibody on secretion of the cytokine interferon-gamma (IFN-gamma).

PBMCs from a CMV-positive donor (Hemacare Donor C) were plated at 3.5x105 cells per well in the presence of 4 μg/ml CMV virus lysate (Astarte) and anti-PDL1 antibody C5H9v2, an activated anti-PDL1 antibody PL07-2001-C5H9v2 or hIgG4 isotype control antibodies. Four days later, the supernatant was removed from each well and IFN-gamma levels were analyzed using an IFN-gamma ELISA kit (Life Technologies, Carlsbad, CA). In FIG. 22 shows that the activated anti-PDL1 antibody PL07-2001-C5H9v2 showed increased CMV-stimulated IFN-gamma secretion compared to the hIgG4 control, but decreased activity compared to the parental anti-PDL1 antibody C5H9v2 due to the fact that the activated antibody was masked. .

Example 18 In situ imaging of an activated anti-PDL1 antibody of the invention.

This example demonstrates the ability of the activated anti-PDL1 antibody PL15-0003-C5H9v2 to activate and bind to frozen tissues of MC38 mouse tumors using an in situ imaging technique.

Fluorescently labeled PL15-0003-C5H9v2 activated anti-PDL1 antibody or C5H9v2 anti-PDL1 antibody were incubated on frozen PDL1+ MC38 tumor sections for 1 h in protease-compatible buffer, in the presence or absence of a mixture of broad-spectrum protease inhibitors.

- 190 039736 as described in PCT International Application Publication WO 2014/107599 published July 10, 2014.

The results are shown in FIG. 23. The tissue image in FIG. 23A shows the binding of anti-PDL1 antibody C5H9v2 to a tumor section, indicating the presence of PDL1 antigen in the tumor section. The fabric image in Fig. 23B demonstrates that the anti-PDL1-activated antibody PL15-0003C5H9v2 was activated by proteolytic cleavage of the anti-PDL1-activated antibody by the tumor to form an anti-PDL1 antibody that binds to the PDL1 target in the tumor section. The fabric image in Fig. 23D shows that the fluorescent signal shown in FIG. 23B was inhibited by pretreatment of the tumor section with a mixture of broad spectrum protease inhibitors III and 50 mM EDTA at a dilution of 1:100, while the effect of a mixture of broad spectrum protease inhibitors was not detected when anti-PDL1 antibody C5H9v2 was bound to the tumor section, as shown. in the fabric image in Fig. 23C. These results indicate that the tumor sections have sufficient protease activity to activate the activated antibodies of the invention.

Example 19 The ability of an activated antibody of the invention to exhibit protease dependent binding and blocking activity in vitro.

This example shows the ability of the activated anti-PDL1 antibody PL15-0003-C5H9v2 to exhibit protease-dependent binding and blocking activity in vitro.

Binding and blocking experiments were performed with anti-PDL1 antibody C5H9v2 and anti-PDL1 activated antibody PL15-0003-C5H9v2 in a manner similar to that described in other examples, except that the plates were coated with 0.5 μg/ml PDP1.

In FIG. 24 shows that the activated anti-PDL1 antibody PL15-0003-C5H9v2 had a higher EC ₅₀ for PDL1 binding and blocking PD1 than the anti-PDL1 antibody as determined by ELISA. The figure also shows that activation of the activated anti-PDL1 antibody PL15-0003-C5H9v2 by matriptase completely restored PDL1 binding and PD1 blocking to levels comparable to those for the anti-PDL1 antibody C5H9v2.

Example 20 Ability of an Activated Anti-PDL1 Antibody of the Invention to Reduce PDL1 Binding and Show Protease-Dependent Blocking Activity in Vitro.

In this example, the ability of the masking peptide to reduce the binding of an activated anti-PDL1 antibody of the invention to PDL1 was evaluated compared to the ability of the parent antibody to bind to PDL1 in vitro. This example also demonstrates the ability of an activated anti-PDL1 antibody of the invention to exhibit protease-dependent blocking activity in vitro.

The ability of activated anti-PDL1 antibodies PL07-2001-C5H9v2 and PF07-3001C5H9v2 and anti-PDL1 antibody C5H9 to bind to human or mouse PDL1 was tested in a manner similar to those described in other examples. The results are shown in FIG. 25.

The ability of activated anti-PDL1 antibody PL07-2001-C5H9v2, uPA activated activated anti-PDL1 antibody PL07-2001-C5H9v2, MMP14 activated activated anti-PDL1 antibody PL07-2001-C5H9v2 and anti-PBL1 antibody C5H9v2 were tested to block binding PD1 (PD-1) or B7-1 with PDL1 using a method similar to the methods described in other examples.

Briefly, for the PD1 blocking assay, human PDL1, cynomolgus monkey PDL1, or rat PDL1 were added to the wells of an ELISA plate. Biotinylated human PD1, cynomolgus monkey PD1, or rat PD1 was added to the wells in the absence or presence of increasing concentrations of PL07-2001-C5H9v2 activated anti-PDL1 antibody, uPA-activated PL07-2001-C5H9v2 activated anti-PDL1 antibody, MMP14-activated activated anti-PDL1 antibody PL07-2001-C5H9v2 or anti-PDL1 antibody C5H9v2. The results shown in FIG. 26 indicate that the activated antibody PF07-2001-C5H9v2 had a higher EC ₅₀ value for blocking PB1 than the anti-PDL1 antibody, as determined by ELISA. However, activation of the activated antibody by uPA or MMP14 restored the blocking activity for PB1 to levels comparable to those for the anti-PDL1 antibody C5H9v2.

Briefly, for the B7-1 blocking assay, human PDL1 or Cynomolgus monkey PDL1 was adsorbed into the wells of an ELISA plate. Biotinylated human B7-1 or Cynomolgus monkey B7-1 was added to the wells, in the absence or presence of an increasing concentration of PL07-2001-C5H9v2 activated anti-PDL1 antibody, uPA-activated PL07-2001-C5H9v2 activated anti-PDL1 antibody, MMP14-activated activated anti-PDL1 antibody PL072001-C5H9v2; and anti-PDL1 antibody C5H9v2. The results shown in FIG. 27 show that the activated antibody PF07-2001-C5H9v2 had a higher EC ₅₀ value for blocking B7-1 than the anti-PDL1 antibody as determined by ELISA. However, activation of the activated antibody by uPA or MMP14 restored blocking activity for B7-1 to levels comparable to those for anti-PBL1 antibody C5H9v2.

Example 21 Activated anti-PDL1 antibodies of the invention suppress the growth of MC38 tumors in mice.

In this example, the ability of activated anti-PDL1 antibodies PL15-0003-C5H9v2,

- 191 039736

PL15-2001-C5H9v2 and PL15-3001-C5H9v2 inhibit the growth of syngeneic MC38 tumors.

Mouse colon carcinoma MC38 cells were obtained from ATCC. MC38 cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere with 5%

CO ₂ in the air. Cells were harvested during logarithmic growth phase, resuspended in PBS with appropriate cell titer, and left on ice to induce tumors.

Each mouse was inoculated subcutaneously in the right flank with 0.5 x ^{10 6} MC38 cells in PBS for tumor growth. Treatments were started when the average size of the tumors reached about 100-200 mm ³ (no more than 200 mm ³ ). The sizes of the tumors were determined twice a week in two dimensions using a caliper and the volume was expressed in mm ³ using the formula V=0.5 aχb ² where a and b are the long and short diameters of the tumor, respectively.

Mice were divided into groups and dosed as indicated in Table 24.

Table 24. Groups and doses in the experiment with syngeneic MC38 tumors with activated anti-PDL1 antibodies

Group Number of mice Treatment Dose (mg/kg) Volumetric dose (ml/kg) Scheme Entry way one 9 mlgG2a (Cl.18.4) 5 ten b.i.w.; 4 doses i/b 2 9 anti-PDLl (C5H9v2) 5 ten b.i.w.; 4 doses i/b 3 9 PL15-0003-C5H9v2 5 ten b.i.w.; 4 doses i/b four 9 PL15-2001-C5H9v2 5 ten b.i.w.; 4 doses i/b 5 9 PL15-3001-C5H9v2 5 ten b.i.w.; 4 doses i/b

In FIG. 28, which plots tumor volume versus number of days after initial dose, showing that activated anti-PDL1 antibodies PL15-0003-C5H9v2, PL152001-C5H9v2, and PL15-3001-C5H9v2 inhibited the growth of syngeneic MC38 tumors similarly to anti-PDL1 antibody C5H9v2. which was used as a positive control.

Example 22 An activated anti-PDL1 antibody of the invention demonstrates reduced PDL1 occupancy in the blood of C57B1/6 mice.

This example shows that animals treated with the activated anti-PDL1 antibodies of the present invention have reduced binding to blood T cells from tumor-bearing mice compared to animals treated with an anti-PDL1 antibody of the invention.

The study was performed in the same manner as described in Example 15. Briefly, mice bearing MC38 tumors ranging in size from 100-200 mm ³ were treated with a single dose of anti-PDL1 antibody C5H9v2 or activated anti-PDL1 antibody PL15-0003-C5H9v2, PL15 -2001-C5H9v2 or PL15-3001-C5H9v2 as indicated in Table 25 and blood was analyzed for surface-bound antibody by flow cytometry four and eight days after administration. Percentage occupancy was calculated as described in example 15.

- 192 039736

Table 25 Groups and doses in the PDL1 occupancy study

Group Number of mice Treatment Dose (mg/kg) Volumetric dose (ml/kg) Scheme Route of administration one 9 mlgG2a (Cl.18.4) 5 ten b.i.w.; 4 doses i/b 2 9 anti-PD-Ll (C5H9v2) 5 ten b.i.w.; 4 doses i/b 3 9 anti-PD-Ll (C5H9v2) 3 ten b.i.w.; 4 doses i/b four 9 PL15-0003-C5H9v2 5 ten b.i.w.; 4 doses i/b 5 9 PL15-0003-C5H9v2 3 ten b.i.w.; 4 doses i/b 6 9 PL15-2001-C5H9v2 5 ten b.i.w.; 4 doses i/b 7 9 PL15-2001-C5H9v2 3 ten b.i.w.; 4 doses i/b

eight 9 PL15-3001-C5H9v2 5 ten b.i.w.; 4 doses i/b 9 9 PL15-3001-C5H9v2 3 ten b.i.w.; 4 doses i/b

The concentration of antibodies and activated antibodies in plasma was determined using ELISA. In FIG. 29 shows that the presence of tumor-derived proteases capable of degrading activated antibodies did not result in high blood levels of activated activated antibodies.

Example 23 In vivo imaging of an activated anti-PDL1 antibody of the invention.

This example shows the ability of proteases in a tumor implanted in mice to activate an activated anti-PDL1 antibody of the invention.

For these studies, the MDA-MB-231-luc2 orthotopic breast tumor xenograft model was used. Female nu/nu mice were obtained from Charles River Laboratories at 7 weeks of age. After 1 week of acclimation, 3 million MDA-MD-231-luc2-4D3LN cells (Perkin Elmer) in 30 µl (ul also refers here to µl) in RPMI serum-free medium (1:1 matrigel) were injected into the fourth abdominal left mammary fat pad . Tumors grew to a size of 110-159 mm ³ . Three mice per group were injected intravenously with 5 mg/kg A1exa750-conjugated anti-PDL1 antibody C5H9v2 or activated anti-PDL1 antibody PL07-2001-C5H9v2. Four mice were injected intravenously with 5 mg/kg of the control A1exa750-conjugated anti-RSV antibody, palivizumab (Synagis). 96 hours after administration, optical images of each mouse were obtained. The results are shown in FIG. 30. A high-intensity fluorescent signal was only detected in tumors of mice injected with anti-PDL1 antibody or activated anti-PDL1 antibody PL07-2001C5H9v2, suggesting that the activated anti-PDL1 antibody was activated and accumulated in the tumor over account for binding to PDL1.

Example 24 Activation of an Activated Antibody of the Invention in Human Plasma Samples.

In this example, it was determined how activated the activated antibody of the invention is in human plasma samples.

Activated anti-PDL1 antibody PL07-2001-C5H9v2 was conjugated with oregon green (ThermoFisher Cat #06149). The concentration and degree of labeling were determined on a spectrophotometer. 3 μM (also refers here to dCM) of labeled PL07-2001-C5H9v2 activated anti-PDL1 antibody was added to plasma samples obtained from healthy donors, melanoma patients, or lung cancer patients, in a final ratio of activated antibody to plasma of 30:70 . Samples were incubated for 48 hours in a humid chamber at 37°C. Plasma reactions of labeled PL07-2001-C5H9v2 activated anti-PDL1 antibody were stopped after 0 or 48 h by freezing samples at -80° C. or denaturing in Wes buffer (ProteinSimple Cat#PS-MK14). Samples were analyzed using a Wes capillary western blot system (ProteinSimple) using antibodies against oregon green stain (ThermoFisher Cat#A-11095 at 100 µg/mL) followed by an HRP-conjugated secondary antibody (Jackson ImmunoResearch Cat# 705-035-147 at 1/40 dilution). Percent activation analysis of labeled PL07-2001C5H9v2 activated anti-PDL1 antibody was determined using Compass software (ProteinSimple).

There was no detectable activation of the activated antibody in five samples from healthy patients, in five samples from patients with melanoma (three metastases in the lymph nodes, two in the brain and one on the back skin), in five samples from patients with lung cancer (one metastasis in lower lobe of the left lung (stage IV), one in the lower lobe of the right lung (stage IV), one in the upper lobe of the left lung (stage IA), and two in the upper lobe of the right lung (stages IIIA and IB).

Other embodiments

Although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims (23)

1. An activated antibody that, when activated, specifically binds to a mammalian PDL1, wherein said activated antibody comprises an antibody or an antigen-binding fragment (AB) thereof, that specifically binds to a mammalian PDL1, where the AB contains: (a) heavy chain variable domain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 212; (b) heavy chain variable domain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of SEQ ID NO: 246; (c) heavy chain variable domain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of SEQ ID NO: 235; (d) light chain variable domain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 209; (e) a light chain variable domain complementarity determining region 2 (VL CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 215 and 227; and (f) a light chain variable domain complementarity determining region 3 (VL CDR3) comprising the amino acid sequence of SEQ ID NO: 228; a masking fragment (MM) that inhibits AB binding to mammalian PDL1 when the activated antibody is in an uncleaved state, wherein the MM contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 59-81, 208, and 426; and a cleavable fragment (CM) paired with AB, wherein CM is a polypeptide that acts as a substrate for the protease, wherein CM contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 338-394 and 435-445, 883 -921, 1009 and 1010; where MM is linked to SM so that the activated antibody in the uncleaved state contains the following order of structures from the N-terminus to the C-terminus: MM-SM-AB or AB-SM-MM. 2. An activated antibody that, in an activated state, specifically binds to a mammalian PDL1, where said activated antibody contains an antibody or an antigen-binding fragment (AB) thereof, that specifically binds to a mammalian PDL1, where the AB contains: (a) heavy chain variable domain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 212; (b) heavy chain variable domain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of SEQ ID NO: 246; (c) heavy chain variable domain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of SEQ ID NO: 235; (d) light chain variable domain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 209; (e) a light chain variable domain complementarity determining region 2 (VL CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 215 and 227; and (f) a light chain variable domain complementarity determining region 3 (VL CDR3) comprising the amino acid sequence of SEQ ID NO: 228; a masking fragment (MM) that inhibits AB binding to mammalian PDL1 when the activated antibody is in an uncleaved state, wherein the MM contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 59-81, 208, and 426; and - 194 039736 a cleavable fragment (CM) that forms a pair with AB, where CM is a polypeptide that acts as a substrate for the protease, where CM contains an amino acid sequence selected from the group consisting of SEQ ID NO: 338-394 and 435-445 , 883-921, 1009 and 1010; where MM is linked to SM so that the activated antibody in the uncleaved state contains the following order of structures from the N-terminus to the C-terminus: MM-CM-AB or AB-CM-MM, where the activated antibody contains one or more of the following: (i) where the activated antibody contains a linker peptide between MM and SM; (ii) where the activated antibody contains a linker peptide between SM and AB; (iii) where the activated antibody contains a first linker peptide (LP1) and a second linker peptide (LP2) and where the activated antibody in the uncleaved state contains the following order of structures from the N-terminus to the C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM; (iv) where the two linker peptides do not have to be identical to each other; and (v) where each LP1 and LP2 is a peptide of about 1-20 amino acids in length. 3. An activated antibody according to claim 1 or 2, wherein the activated antibody inhibits the induction of type 1 diabetes in the NOD mouse at least 3 times more than AB, and/or where the activated antibody has a safety window that is at least three times the safety window AB. 4. An activated antibody according to claim 1 or 2, wherein the activated antibody in the uncleaved state binds to a lower percentage of the peripheral blood CD4+ CD8+ T lymphocyte population than AB, and/or where the peripheral blood CD4+ CD8+ T lymphocytes are murine; and/or wherein murine peripheral blood CD4+ CD8+ T-lymphocytes are obtained from tumor-bearing mice. 5. The activated antibody of claim 1 or 2, wherein the AB comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, and 56, and the variable a light chain (VL) region comprising SEQ ID NO: 12 and SEQ ID NO: 58; or a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 46 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 58. 6. Activated antibody according to any one of paragraphs.1-5, where the CM is a substrate for a protease that is active in diseased tissue; and/or where the CM contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 377-394, 883-921, 1009 and 1010. 7. An activated antibody according to any one of claims 1 to 6, wherein the antigen binding fragment is selected from the group consisting of Fab fragment, F(ab') ₂ fragment, scFv, scAb, dAb, heavy chain single domain antibody, and single domain antibody light chain based. 8. Activated antibody according to any one of paragraphs.1-7, where the AV is associated with the SM; and/or where AB is directly related to CM; and/or where the AB is linked to the CM via a linker peptide. 9. An activated antibody according to claim 1 or 2, wherein the activated antibody comprises a linker peptide between MM and SM. 10. An activated antibody according to claim 1 or 2, wherein the activated antibody comprises a linker peptide between CM and AB. 11. An activated antibody according to claim 1 or 2, wherein the activated antibody comprises a first linker peptide (LP1) and a second linker peptide (LP2), and wherein the activated antibody in the uncleaved state contains the following order of structures from the N-terminus to the C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. 12. An activated antibody according to claim 11, wherein the two linker peptides need not be identical to each other. 13. An activated antibody according to claim 11, wherein each LP1 and LP2 is a peptide of about 1-20 amino acids in length. 14. An activated antibody conjugate, containing an activated antibody according to any one of claims 1 to 13, conjugated with an agent, where the agent is a toxin or a fragment thereof; and/or where the agent is a microtubule inhibitor; and/or where the agent is a nucleic acid damaging agent; and/or wherein the agent is selected from the group consisting of dolastatin or a derivative thereof, auristatin or a derivative thereof, a maytansinoid or a derivative thereof, duocarmycin or a derivative thereof, and potassium chemamycin or a derivative thereof; and/or where the agent is auristatin E or a derivative thereof; and/or where the agent is monomethylauristatin E (MMAE); and/or where the agent is monomethylauristatin D (MMAD); and/or where the agent is a maytansinoid selected from the group consisting of DM1 and DM4; and/or where the agent is conjugated to the activated antibody via a linker; and/or where the linker is a cleavable linker; and/or where the linker is a non-cleavable linker. 15. The conjugate of claim 14 wherein the agent is a detectable group. 16. Pharmaceutical composition containing an activated antibody according to any one of claims 1-13 or a conjugate according to claim 14 or 15 and a carrier or an activated antibody according to any one of claims 1-13 or a conjugate according to claim 14 or 15 and an additional agent, where the additional agent is a therapeutic agent. 17. An isolated nucleic acid molecule encoding an activated antibody according to any one of claims 1-13. 18. An expression vector containing the isolated nucleic acid molecule according to claim 17. 19. A method for producing an activated antibody by culturing a cell under conditions that result in the expression of an activated antibody, wherein the cell contains the nucleic acid molecule according to claim 17. 20. A method for producing an activated antibody according to any one of claims 1 to 13, which, in an activated state, binds to PDL1, where the method includes: (a) culturing a cell containing a nucleic acid construct that encodes an activated antibody under conditions that result in expression of the activated antibody; and (b) isolating the activated antibody. 21. A method for reducing the activity of PDL1 or blocking the binding of a natural ligand to PDL1 in a PDL1 mediated disorder or disease, comprising administering an effective amount of an activated antibody according to any one of claims 1 to 13, a conjugate according to claim 14 or 15, or a pharmaceutical composition according to claim 16 to a patient ; where mediated PDL1 disorder or disease is a malignant neoplasm; where the malignancy is bladder cancer, bone cancer, breast cancer, carcinoid, cervical cancer, colon cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer , prostate cancer, renal cell carcinoma, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, urogenital cancer and/or urothelial cancer; where the malignancy is selected from the group consisting of melanoma (MEL), renal cell carcinoma (RCC), squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer (CRC), castration-resistant prostate cancer (CRPC), hepatocellular carcinoma ( HCC), head and neck squamous cell carcinoma, carcinoma of the esophagus, ovary, gastrointestinal tract, and breast, or hematologic malignancy such as multiple myeloma, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma/primary mediastinal B-cell lymphoma, and chronic myeloid leukemia; where the method includes the introduction of an additional agent, where the additional agent is a therapeutic agent. 22. A method for producing a conjugate according to any one of claims 14 or 15, which, in an activated state, binds to PDL1, where the method includes: (a) culturing a cell containing a nucleic acid construct that encodes an activated antibody under conditions that result in expression of the activated antibody; (b) isolating the activated antibody; and (c) conjugating the agent to the isolated activated antibody, wherein the agent is a toxin or a fragment thereof; and/or where the agent is a microtubule inhibitor; and/or where the agent is a nucleic acid damaging agent; and/or wherein the agent is selected from the group consisting of dolastatin or a derivative thereof, auristatin or a derivative thereof, maytansinoid or a derivative thereof, duocarmycin or a derivative thereof, and calicheamicin or a derivative thereof; and/or where the agent is auristatin E or a derivative thereof; and/or where the agent is monomethylauristatin E (MMAE); and/or where the agent is monomethylauristatin D (MMAD); and/or where the agent is a maytansinoid selected from the group consisting of DM1 and DM4; and/or where the agent is conjugated to the activated antibody via a linker; and/or - 196 039736 where the linker is a cleavable linker; and/or where the linker is a non-cleavable linker. 23. A method of treating, reducing symptoms, or slowing the progression of a PDL1-mediated disorder or disease, comprising administering an effective amount of an activated antibody according to any one of claims 1-13, a conjugate according to claim 14 or 15, or a pharmaceutical composition according to claim 16 to a patient, where mediated by PDL1 the disorder or disease is a malignancy; where the malignancy is bladder cancer, bone cancer, breast cancer, carcinoid, cervical cancer, colon cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer , prostate cancer, renal cell carcinoma, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, urogenital cancer and/or urothelial cancer; where the malignancy is selected from the group consisting of melanoma (MEL), renal cell carcinoma (RCC), squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer (CRC), castration-resistant prostate cancer (CRPC), hepatocellular carcinoma ( HCC), head and neck squamous cell carcinoma, carcinoma of the esophagus, ovary, gastrointestinal tract, and breast, or hematologic malignancy such as multiple myeloma, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma/primary mediastinal B-cell lymphoma, and chronic myeloid leukemia; where the method includes the introduction of an additional agent, where the additional agent is a therapeutic agent.