JPWO2021226440A5

JPWO2021226440A5 -

Info

Publication number: JPWO2021226440A5
Application number: JP2022566686A
Authority: JP
Publication date: 2024-04-24

Claims

An immunoconjugate comprising a cell-binding agent covalently attached to one or more immunostimulatory moieties by a linker comprising an elastase-substrate peptide linker unit.

The immunoconjugate of claim 1, wherein the cell-binding agent is an antibody.

The immunoconjugate of claim 2 , wherein the antibody is an antibody construct having an antigen-binding domain that binds to a target selected from PD-L1 , HER2, CEA, and Trop2.

4. The immunoconjugate of claim 3, wherein the antibody is selected from the group consisting of atezolizumab, durvalumab , avelumab , trastuzumab, pertuzumab, labetuzumab, and sacituzumab .

The immunoconjugate of claim 1, wherein the one or more immunostimulatory moieties are pattern recognition receptors.

2. The immunoconjugate of claim 1 , wherein the one or more immunostimulatory moieties interact with or modulate a receptor selected from the group consisting of TLR, STING, NOD2, RIG-1, and NLRP3.

Formula I:
Ab-[L-Ims] _p I
or a pharma- ceutically acceptable salt thereof ;
In the formula,
Ab is an antibody;
L is a linker comprising an elastase substrate peptide linker unit;
Ims is an immunostimulatory moiety;
The immunoconjugate of claim 1 , wherein p is an integer from 1 to 8 .

The immunoconjugate of claim 7 , wherein the I ms is a Toll-like receptor (TLR) agonist.

I ms is selected from formulae Ia-f:

In the formula,
R ¹ , R ² , R ³ , R ⁴ , and R ⁵ are independently selected from the group consisting of H, C ₁ -C ₁₂ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₁₂ carbocyclyl, C ₆ -C ₂₀ aryl, C ₂ -C ₉ heterocyclyl, and C ₁ -C ₂₀ heteroaryl, where alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl are:
-(C ₁ -C ₁₂ alkyldiyl)-N(R ⁶ )- ^* ;
-(C ₁ -C ₁₂ alkyldiyl)-N(R ⁶ ) ₂ ;
-(C ₁ -C ₁₂ alkyldiyl)-OR ⁶
-( _C3 - _C12 carbocyclyl);
-( _C3 - _C12 carbocyclyl)- ^* ;
-(C ₃ -C ₁₂ carbocyclyl)-(C ₁ -C ₁₂ alkyldiyl)-NR ⁶ - ^* ;
-( _C3 - _C12 carbocyclyl)-( _C1 - _C12 alkyldiyl)-N( ^R6 ) ₂ ;
-( _C3 - _C12 carbocyclyl) ^-NR5 -C(= ^NR6a ) ^NR6- ^* ;
-( _C6 - _C20 aryl);
-( _C6 - _C20 aryldiyl)- ^* ;
-(C ₆ -C ₂₀ aryldiyl)-N(R ⁶ )- ^* ;
-(C ₆ -C ₂₀ aryldiyl)-(C ₁ -C ₁₂ alkyldiyl)-N(R ⁶ )- ^* ;
-(C ₆ -C ₂₀ aryldiyl)-(C ₁ -C ₁₂ alkyldiyl)-(C ₂ -C ₂₀ heterocyclyldiyl)- ^* ;
-(C ₆ -C ₂₀ aryldiyl)-(C ₁ -C ₁₂ alkyldiyl)-N(R ⁶ ) ₂ ;
-(C ₆ -C ₂₀ aryldiyl)-(C ₁ -C ₁₂ alkyldiyl)-NR ⁶ -C(═NR ^6a )N(R ⁶ )- ^* ;
-( _C2 - _C20 heterocyclyl);
-(C ₂ -C ₂₀ heterocyclyl)- ^* ;
-(C ₂ -C ₉ heterocyclyl)-(C ₁ -C ₁₂ alkyldiyl)-NR ⁶ - ^* ;
-(C ₂ -C ₉ heterocyclyl)-(C ₁ -C ₁₂ alkyldiyl)-N(R ⁶ ) ₂ ;
-(C ₂ -C ₉ heterocyclyl)-C(═O)-(C ₁ -C ₁₂ alkyldiyl)-N(R ⁶ )- ^* ;
-( _C2 - _C9 heterocyclyl) ^-NR5 -C(= ^NR6a ) ^NR6- ^* ;
-(C ₂ -C ₉ heterocyclyl)-NR ⁶ -(C ₆ -C ₂₀ aryldiyl)-(C ₁ -C ₁₂ alkyldiyl)-N(R ⁶ )- ^* ;
-(C ₂ -C ₉ heterocyclyl)-(C ₆ -C ₂₀ aryldiyl)- ^* ;
-( _C1 - _C20 heteroaryl);
-(C ₁ -C ₂₀ heteroaryldiyl)- ^* ;
-(C ₁ -C ₂₀ heteroaryldiyl)-(C ₁ -C ₁₂ alkyldiyl)-N(R ⁶ )- ^* ;
-(C ₁ -C ₂₀ heteroaryldiyl)-(C ₁ -C ₁₂ alkyldiyl)-N(R ⁶ ) ₂ ;
-(C ₁ -C ₂₀ heteroaryldiyl)-NR ⁶ -C(═NR ^6a )N(R ⁶ )- ^* ;
-(C ₁ -C ₂₀ heteroaryldiyl)-N(R ⁶ )C(═O)-(C ₁ -C ₁₂ alkyldiyl)-N(R ⁶ )- ^* ;
-C(=O)- ^* ;
-C(=O)-( _C1 - _C12 alkyldiyl)-N( ^R6 )- ^* ;
-C(=O)-( _C2 - _C20 heterocyclyldiyl)- ^* ;
-C(=O)N( ^R6 ) ₂ ;
-C(=O)N( ^R6 )- ^* ;
-C(=O)N( ^R6 )-( _C1 - _C12 alkyldiyl)-N( ^R6 )C(=O) ^R5 ;
-C(=O)N( ^R6 )-( _C1 - _C12 alkyldiyl)-N( ^R6 )C(=O)N( ^R6 ) ₂ ;
-C(=O) ^NR6- ( _C1 - _C12 alkyldiyl)-N( ^R6 ) _CO2R6 ^;
-C(=O) ^NR6- ( _C1 - _C12 alkyldiyl)-N( ^R6 )C(= ^NR6a )N( ^R6 ) ₂ ;
-C(=O)NR ⁶ -(C ₁ -C ₁₂ alkyldiyl)-NR ⁶ C(=NR ^6a )R ⁶ ;
-C(=O) ^NR6- ( _C1 - _C8 alkyldiyl) ^-NR6 ( _C2 - _C5 heteroaryl);
-C(=O) ^NR6- ( _C1 - _C20 heteroaryldiyl)-N( ^R6 )- ^* ;
-C(=O) ^NR6- ( _C1 - _C20 heteroaryldiyl)- ^* ;
-C(=O) ^NR6- ( _C1 - _C20 heteroaryldiyl)-( _C1 - _C12 alkyldiyl)N( ^R6 ) ₂ ;
-C(=O) ^NR6- ( _C1 - _C20 heteroaryldiyl)-( _C2 - _C20 heterocyclyldiyl)-C(=O) ^NR6- ( _C1 - _C12 alkyldiyl) ^-NR6- ^* ;
-N( ^R6 ) ₂ ;
-N( ^R6 )- ^* ;
-N( ^R6 )C(=O) ^R6 ;
-N( ^R6 )C(=O)- ^* ;
-N( ^R6 )C(=O)N( ^R6 ) ₂ ;
-N( ^R6 )C(=O)N( ^R6 )- ^* ;
-N( ^R6 ) _CO2R6 ^;
-N( ^R6 ) _CO2 ( ^R6 )- ^* ;
^-NR6C (= ^NR6a )N( ^R6 ) ₂ ;
^-NR6C (= ^NR6a )N( ^R6 )- ^* ;
^-NR6C (= ^NR6a ) ^R6 ;
-N(R ⁶ )C(═O)-(C ₁ -C ₁₂ alkyldiyl)-N(R ⁶ )- ^* ;
-N( ^R6 )-( _C2 - _C5 heteroaryl);
-N( ^R6 )-S(=O) _2- ( _C1 - _C12 alkyl);
-O-( _C1 - _C12 alkyl);
-O-(C ₁ -C ₁₂ alkyldiyl)-N(R ⁶ ) ₂ ;
-O-(C ₁ -C ₁₂ alkyldiyl)-N(R ⁶ )- ^* ;
-OC(=O)N( ^R6 ) ₂ ;
-OC(=O)N( ^R6 )- ^* ;
-S(=O) _2- ( _C2 - _C20 heterocyclyldiyl)- ^* ;
-S(=O) _2- ( _C2 - _C20 heterocyclyldiyl)-( _C1 - _C12 alkyldiyl)-N( ^R6 ) ₂ ;
-S(=O) _2- ( _C2 - _C20 heterocyclyldiyl)-( _C1 - _C12 alkyldiyl) ^-NR6- ^* ; and
-S(=O) _2- ( _C2 - _C20 heterocyclyldiyl)-( _C1 - _C12 alkyldiyl)-OH;
or R ² and R ³ of formulae Ic-If taken together form a 5- or 6-membered heterocyclyl ring;
X ¹ , X ² , X ³ , X ⁴ , and X ⁵ are independently selected from the group consisting of a bond, C(═O), C(═O)N(R ⁶ ), O, N(R ⁶ ), S, S(O) ₂ , and S(O) ₂ N(R ⁶ );
R ⁶ is selected from the group consisting of H, C ₆ -C ₂₀ aryl, C ₆ -C ₂₀ aryldiyl, C ₁ -C ₁₂ alkyl, and C ₁ -C ₁₂ alkyldiyl, or two R ⁶ groups taken together form a 5- or 6-membered heterocyclyl ring;
R ^6a is selected from the group consisting of C ₆ -C ₂₀ aryl and C ₁ -C ₂₀ heteroaryl;
where the asterisk ^* indicates the attachment site of L, where one of R ¹ , R ² , R ³ , R ⁴ and R ⁵ is attached to L;
Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are F, Cl, Br, I, -CN _, _-CH3 , _-CH2CH3 , -CH= _CH2 , _-C≡CH , -C≡CCH3, _-CH2CH2CH3 _, -CH ₍ _CH3 ) ₂ , _-CH2CH ₍ CH3) ₂ , -CH2OH, _-CH2OCH3 _, _-CH2CH2OH _, -C ₍ _CH3 ) _2OH , _-CH ₍ OH)CH( _CH3 ₎ ₂ , -C(CH3) _2CH2OH , _-CH2CH2SO _2CH3 _, _-CH2OP (O)(OH) ₂ , -CH2F _, _-CHF2 _, -CF3 _, -CH2CF3 _, _-CH2CHF2 _, -CH( _CH3 )CN, -C( _CH3 )2CN _, _-CH2CN, _-CH2NH2 _, _-CH2NHSO2CH3 _, _-CH2NHCH3 _{, -CH2N(CH3)2} _, _-CO2H , _-COCH3 , _-CO2CH3 _, _-CO2C ₍ CH3 ₎ ₃ , -COCH(OH) _CH3 , _-CONH2 , _-CONHCH3 , -CON( _CH3 ) ₂ , _-C ( _CH3 ₎ _2CONH ₂ , _-NH2 , _-NHCH3 , _-N ( _CH3 ) ₂ , _-NHCOCH3 , -N( _CH3 )COCH3, -NHS(O ₎ _2CH3 _, _-N ( _CH3 ) _C ( _CH3 )2CONH2 _, -N(CH3) _CH2CH2S (O) _2CH3 , -NHC(=NH)H, -NHC(=NH) _CH3 _, -NHC(=NH)NH2, -NHC(=O ₎ _{NH2, -NO2} _, ₌ _O _, -OH _, _-OCH3 _, -OCH2CH3 _, _-OCH2CH2OCH3 , -OCH2CH2OH _, _-OCH2CH2N ( _CH3 ) ₂ , -O(CH ₂ CH ₂ O) _n -(CH ₂ ) _m CO ₂ H, -O(CH ₂ CH ₂ O) _n H, -OP(O)( OH ) ₂ , -S(O) ₂ N(CH ₃ ) ₂ , -SCH ₃ , -S(O) ₂ CH ₃ , and -S( O ) ₃ H.

One of R ¹ , R ² , R ³ , R ⁴ and R ⁵ is a group represented by the formula:

The immunoconjugate of claim 9 , selected from:

The immunoconjugate of claim 7 , wherein the I ms is a STING agonist.

I ms has the formula Ig:

wherein ^Xa and ^Xb are independently selected from 5-membered heteroaryl;
R ¹ is selected from the group consisting of F, Cl, Br, I, -CN, -OH, and -O-(C ₁ -C ₆ alkyldiyl);
R ^2a and R ^2b are independently selected from -C(=O)N(R ⁵ ) ₂ ;
R ³ is selected from C 1 -C 6 alkyldiyl, -(C 1 -C 3 alkyldiyl)-O- ₍ _C 1 -C ₃ alkyldiyl ₎ - , _C ₂ -C ₆ _alkenyldiyl _, and C 2 -C 6 alkynyldiyl, _optionally substituted with one or more groups selected from _F , Cl , -OH , _-OCH ₃ , -OCH ₂ CH _{3 , -OCH 2 CH 2} _OCH ₃ , _-OCH 2 _CH ₂ OH, -OCH ₂ CH ₂ N(CH 3 ) 2 ;
^R4 is:
-(C ₁ -C ₁₂ alkyldiyl)-N(R ⁵ )- ^* ;
-(C ₁ -C ₁₂ alkyldiyl)-(C ₂ -C ₂₀ heterocyclyldiyl)- ^* ;
-O-(C ₁ -C ₁₂ alkyldiyl)-N(R ⁵ )- ^* ;
-O-(C ₁ -C ₁₂ alkyldiyl)-(C ₂ -C ₂₀ heterocyclyldiyl)- ^* ;
-O-(C ₁ -C ₁₂ alkyldiyl)-(C ₂ -C ₂₀ heterocyclyldiyl)-N(R ⁵ )- ^* ;
-OC(=O)N( ^R5 )- ^* ;
-N(R ⁵ )-(C ₁ -C ₁₂ alkyldiyl)-N(R ⁵ )- ^* ;
-N(R ⁵ )-(C ₁ -C ₁₂ alkyldiyl)-(C ₂ -C ₂₀ heterocyclyldiyl)- ^* ;
-C(=O)N( ^R5 )- ^* ;
-(C ₂ -C ₂₀ heterocyclyldiyl)- ^* ;
-S(=O) _2- ( _C2 - _C20 heterocyclyldiyl)- ^* ;
-S(=O) _2- ( _C2 - _C20 heterocyclyldiyl)-( _C1 - _C12 alkyldiyl) ^-NR5- ^*
selected from the group consisting of:
where the asterisk ^* indicates the binding site of L;
R ⁵ is independently H or C ₁ -C ₆ alkyl, or two R ⁵ groups taken together form a 5- or 6-membered heterocyclyl ring;
Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are F, Cl, Br, I, -CN _, _-CH3 _, _-CH2CH3 , -CH= _CH2 , _-C≡CH , -C≡CCH3, _-CH2CH2CH3 _, -CH ₍ _CH3 ) ₂ , -CH2CH ₍ CH3) ₂ , -CH2OH, _-CH2OCH3 _, _-CH2CH2OH _, -C ₍ _CH3 ) _2OH , _-CH ₍ OH)CH( _CH3 ₎ ₂ , -C(CH3) _2CH2OH , _-CH2CH2SO _2CH3 _, _-CH2OP (O)(OH) ₂ , -CH2F _, _-CHF2 _, -CF3 _, -CH2CF3 _, _-CH2CHF2 _, -CH( _CH3 )CN, -C( _CH3 )2CN _, _-CH2CN, _-CH2NH2 _, _-CH2NHSO2CH3 _, _-CH2NHCH3 _{, -CH2N(CH3)2} _, _-CO2H , _-COCH3 , _-CO2CH3 _, _-CO2C ₍ CH3 ₎ ₃ , -COCH(OH) _CH3 , _-CONH2 , _-CONHCH3 , -CON( _CH3 ) ₂ , _-C ( _CH3 ₎ _2CONH ₂ , _-NH2 , _-NHCH3 , _-N ( _CH3 ) ₂ , _-NHCOCH3 , -N( _CH3 )COCH3, -NHS(O ₎ _2CH3 _, _-N ( _CH3 ) _C ( _CH3 )2CONH2 _, -N(CH3) _CH2CH2S (O) _2CH3 , -NHC(=NH)H, -NHC(=NH) _CH3 _, -NHC(=NH)NH2, -NHC(=O ₎ _{NH2, -NO2} _, ₌ _O _, -OH _, _-OCH3 _, -OCH2CH3 _, _-OCH2CH2OCH3 , -OCH2CH2OH _, _-OCH2CH2N ( _CH3 ) ₂ , -O(CH ₂ CH ₂ O) _n -(CH ₂ ) _m CO ₂ H, -O(CH ₂ CH ₂ O) _n H, -OCH ₂ F, -OCHF ₂ , -OCF ₃ , -OP(O)(OH) ₂ , -S( O ) ₂ N ( CH ₃ ) ₂ , -SCH ₃ , -S (O) ₂ CH ₃ , and -S(O) ₃ H.

13. The immunoconjugate of claim 12 , ^wherein Xa and ^Xb are independently selected from the group consisting of imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, oxadiazolyl, and thiadiazolyl.

14. The immunoconjugate of claim 13 , ^wherein Xa and ^Xb are each pyrazolyl substituted with one or more C ₁ -C ₁₂ alkyl groups.

13. The immunoconjugate of claim 12 , wherein R ¹ is selected from the group consisting of -OCH ₃ , -OCH ₂ CH ₃ , -OCH ₂ CH ₂ OCH ₃ , -OCH ₂ CH ₂ OH, and -OCH ₂ CH ₂ N(CH ₃ ) ₂ .

^16. The immunoconjugate of claim 15 , wherein R1 is _-OCH3 .

The immunoconjugate of claim 12 , wherein R ¹ is F.

13. The immunoconjugate of claim 12 , wherein R ^2a and R ^2b are each -C(=O) _NH2 .

13. The immunoconjugate of claim 12 , wherein R3 is selected from -CH ₂ CH ₂ -, -CH=CH-, and -C≡C- ^.

^13. The immunoconjugate of claim 12 , wherein R3 is a _C2 - _C4 alkenyldiyl substituted with one or more groups selected from F, -OH, and _-OCH3.

The immunoconjugate of claim 12 , wherein R ⁴ is -O-(C ₁ -C ₁₂ alkyldiyl)-(C ₂ -C ₂₀ heterocyclyldiyl)- ^*.

The immunoconjugate of claim 21 , wherein the C ₁ -C ₁₂ alkyldiyl is propyldiyl and the C ₂ -C ₂₀ heterocyclyldiyl is piperidiyl.

Lbut:
-C(=O)-(PEG)-C(=O)-(EsPEP)-;
-C(=O)-(PEG)-C(=O)-(EsPEP)-N(R⁶) -;
-C(=O)-(PEG)-C(=O)-(EsPEP)-N(R⁶)-(C₁-C₁₂Alkyldiyl)-;
-C(=O)-(PEG)-C(=O)-(EsPEP)-N(R⁶)-(C₁-C₁₂alkyldiyl)-N(R⁶) C(=O)-(C₂-C₅Monoheterocyclyldiyl)-;
-C(=O)-(PEG)-N(R⁶)-(PEG)-C(=O)-(EsPEP)-;
-C(=O)-(PEG)-N⁺(R⁶)₂-(PEG)-C(=O)-(EsPEP)-;
-C(=O)-(PEG)-C(=O)-N(R⁶) CH(AA₁)C(=O)-(PEG)-C(=O)-(EsPEP)-;
-C(=O)-(C₁-C₁₂alkyldiyl)-C(=O)-(EsPEP)-;
-C(=O)-(C₁-C₁₂alkyldiyl)-C(=O)-(EsPEP)-N(R⁶)-(C₁-C₁₂Alkyldiyl)-;
-C(=O)-(C₁-C₁₂alkyldiyl)-C(=O)-(EsPEP)-N(R⁶)-(C₁-C₁₂alkyldiyl)-N(R⁶) -C(=O);
-C(=O)-(C₁-C₁₂alkyldiyl)-C(=O)-(EsPEP)-N(R⁶)-(C₁-C₁₂alkyldiyl)-N(R⁶) C(=O)-(C₂-C₅Monoheterocyclyldiyl)-;
-Succinimidyl-(CH₂)_m-C(=O)N(R⁶)-PEG-C(═O)-(EsPEP)-;
-Succinimidyl-(CH₂)_m-C(=O)-(EsPEP)-N(R⁶)-(C₁-C₁₂Alkyldiyl)-;
-Succinimidyl-(CH₂)_m-C(=O)-(EsPEP)-N(R⁶)-(C₁-C₁₂Alkyldiyl)N(R⁶)C(═O)—; and
-(Succinimidyl)-(CH₂)_m-C(=O)-(EsPEP)-N(R⁶)-(C₁-C₁₂alkyldiyl)-N(R⁶) C(=O)-(C₂-C₅Monoheterocyclyldiyl)-
selected from the group consisting of;
PEG is a group represented by the formula: -(CH₂CH₂O)_n- (CH₂)_m- m is an integer from 1 to 5 and n is an integer from 2 to 50;
EsPEP is an elastase substrate peptide linker unit comprising 2-12 amino acid residues;
R⁶But, H, C₆-C₂₀Aryl, C₆-C₂₀Aryldiyl, C₁-C₁₂Alkyl, and C₁-C₁₂alkyldiyl, or two R⁶the groups taken together form a 5- or 6-membered heterocyclyl ring;
Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are each independently selected from the group consisting of F, Cl, Br, I, -CN, -CH₃, -CH₂CH₃, -CH=CH₂, -C≡CH, -C≡CCH₃, -CH₂CH₂CH₃, -CH(CH₃)₂, -CH₂CH (CH₃)₂, -CH₂OH, -CH₂OCH₃, -CH₂CH₂OH, -C(CH₃)₂OH, -CH(OH)CH(CH₃)₂, -C(CH₃)₂CH₂OH, -CH₂CH₂SO₂CH₃, -CH₂OP(O)(OH)₂, -CH₂F, -CHF₂, -CF₃, -CH₂CF₃, -CH₂CHF₂, -CH(CH₃)CN, -C(CH₃)₂CN, -CH₂CN, -CH₂N.H.₂, -CH₂N.H.S.O.₂CH₃, -CH₂N.H.C.H.₃, -CH₂N (CH₃)₂, -CO₂H, -COCH₃, -CO₂CH₃, -CO₂C (CH₃)₃, -COCH(OH)CH₃, -CONH₂, -CONHCH₃, -CON(CH₃)₂, -C(CH₃)₂CONH₂, -NH₂, -NHCH₃, -N(CH₃)₂, -NHCOCH₃, -N(CH₃) COCH₃, -NHS(O)₂CH₃, -N(CH₃) C(CH₃)₂CONH₂, -N(CH₃) CH₂CH₂S (O)₂CH₃, -NHC(=NH)H, -NHC(=NH)CH₃, -NHC(=NH)NH₂, -NHC(=O)NH₂, -NO₂, =O, -OH, -OCH₃, -OCH₂CH₃, -OCH₂CH₂OCH₃, -OCH₂CH₂OH, -OCH₂CH₂N (CH₃)₂, -O(CH₂CH₂O)_n- (CH₂)_mCO₂H, -O(CH₂CH₂O)_nH, -OP(O)(OH)₂, -S(O)₂N (CH₃)₂, -SCH₃, -S(O)₂CH₃, and -S(O)₃ H orand independently one or more groups selected fromandOptionally replacedattitudeClaims7The immunoconjugate described in

24. The immunoconjugate of claim 23 , wherein EsPEP is composed of one or more unnatural amino acid residues.

EsPEP:

24. The immunoconjugate of claim 23 , consisting of amino acid residues of an amino acid selected from the group consisting of:

24. The immunoconjugate of claim 23, wherein EsPEP is selected from the group consisting of Ala-Pro-Val, Asn-Pro-Val, Ala-Ala-Val, Ala-Ala-Pro-Ala (SEQ ID NO:639), Ala-Ala-Pro-Val (SEQ ID NO : 640), and Ala-Ala-Pro-Nva (SEQ ID NO:641).

EsPEP has the formula:

having
wherein AA is independently selected from a natural or unnatural amino acid side chain or one or more AAs, and adjacent nitrogen atoms form a 5-membered ring of a proline amino acid, with the wavy line indicating the point of attachment;
Cyc is selected from C ₆ -C ₂₀ aryldiyl and C ₁ -C ₂₀ heteroaryldiyl, optionally including F, Cl, NO ₂ , —OH, —OCH ₃ , and the structure:

and glucuronic acid having the formula :
R ⁷ is selected from the group consisting of -CH(R ⁸ )O-, -CH ₂ -, -CH ₂ N(R ⁸ )-, and -CH(R ⁸ )O-C(=O)-, R ⁸ is selected from H, C ₁ -C ₆ alkyl, C(=O)-C ₁ -C ₆ alkyl, and -C(=O)N(R ⁹ ) ₂ , R ⁹ is independently selected from the group consisting of H, C ₁ -C ₁₂ alkyl, and -(CH ₂ CH ₂ O) _n -(CH ₂ ) _m -OH, m is an integer from 1 to 5, n is an integer from 2 to 50, or two R ⁹ groups taken together form a 5- or 6-membered heterocyclyl ring;
y is an integer from 2 to 12;
24. The immunoconjugate of claim 23 , wherein z is 0 or 1.

28. The immunoconjugate of claim 27 , wherein y is selected from 2, 3, and 4.

EsPEP has the formula:

is a tripeptide having the formula
wherein AA ₁ , AA ₂ , and AA ₃ are independently selected from natural or unnatural amino acids and the wavy line indicates the point of attachment;
Cyc is selected from C ₆ -C ₂₀ aryldiyl and C ₁ -C ₂₀ heteroaryldiyl, optionally including F, Cl, NO ₂ , —OH, —OCH ₃ , and the structure:

and glucuronic acid having the formula :
R ⁷ is selected from the group consisting of -CH(R ⁸ )O-, -CH ₂ -, -CH ₂ N(R ⁸ )-, and -CH(R ⁸ )O-C(=O)-, R ⁸ is selected from H, C ₁ -C ₆ alkyl, C(=O)-C ₁ -C ₆ alkyl, and -C(=O)N(R ⁹ ) ₂ , R ⁹ is independently selected from the group consisting of H, C ₁ -C ₁₂ alkyl, and -(CH ₂ CH ₂ O) _n -(CH ₂ ) _m -OH, m is an integer from 1 to 5, n is an integer from 2 to 50, or two R ⁹ groups taken together form a 5- or 6-membered heterocyclyl ring;
24. The immunoconjugate of claim 23 , wherein z is 0 or 1.

30. The immunoconjugate of claim 29 , wherein AA ₁ is methyl, AA ₂ forms a proline, and AA ₃ is isopropyl.

EsPEP has the formula:

30. The immunoconjugate of claim 29 , having the formula:

EsPEP has the formula:

The immunoconjugate of claim 31 , selected from:

L is:
24. The immunoconjugate of claim 23 , which is -C(=O)-(PEG)-C(=O)-(EsPEP)-.

PEG :
The immunoconjugate of claim 33 , which is -(CH ₂ CH ₂ O) ₂₅ -(CH ₂ ) ₂ - or -(CH ₂ CH ₂ O) ₁₀ -(CH ₂ ) ₂ -.

formula:

The immunoconjugate of claim 34 , selected from:

I ms has the structure:

36. The immunoconjugate of claim 35 , having the formula:

EsPEP has the formula:

is a tetrapeptide having the formula
wherein AA ₁ , AA ₂ , AA ₃ , and AA ₄ are independently selected from natural or unnatural amino acids, and the wavy line indicates the point of attachment;
Cyc is selected from C ₆ -C ₂₀ aryldiyl and C ₁ -C ₂₀ heteroaryldiyl, optionally including F, Cl, NO ₂ , —OH, —OCH ₃ , and the structure:

A A ₁ is selected from the group consisting of Abu, Ala, and Val;
_AA2 is selected from the group consisting of Nle(O-Bzl), Oic, and Pro;
_AA3 is selected from the group consisting of Ala and Met(O) ₂ ;
38. The immunoconjugate of claim 37 , wherein AA ₄ is selected from the group consisting of Oic, Arg(NO ₂ ), Bpa, and Nle(O-Bzl).

EsPEP has the formula:

The immunoconjugate of claim 38 , having the formula:

EsPEP has the formula:

40. The immunoconjugate of claim 39 , having the formula:

IIe-h:

and a wavy line indicates attachment to the antibody via L.

IIi to IIl:

and a wavy line indicates attachment to the antibody via L.

The immunoconjugate of claim 42 , wherein R ² and R ³ are each C ₁ -C ₈ alkyl.

The immunoconjugate of claim 43 , wherein R ² and R ³ are each -CH ₂ CH ₂ CH ₃ .

43. The immunoconjugate of claim 42 , wherein X2 and ^X3 are each a bond and ^R2 or ^R3 is ^-O- ( _C1 - _C12 alkyl).

_46. The immunoconjugate of claim 45 ^, wherein R2 or ^R3 is _-OCH2CH3 .

The immunoconjugate of claim 1 , wherein the elastase substrate peptide linker is cleaved by elastase.

Formula IIa~f:

are selected from
In the formula, R¹, R², R³, R⁴, and R⁵But, H, C₁-C₁₂Alkyl, C₂-C₆Alkenyl, C₂-C₆Alkynyl, C₃-C₁₂Carbocyclyl, C₆-C₂₀Aryl, C₂-C₉Heterocyclyl, and C₁-C₂₀heteroaryl, where alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl are independently selected from the group consisting of:
- (C₁-C₁₂alkyldiyl)-N(R⁶) -^*;
- (C₁-C₁₂alkyldiyl)-N(R⁶)₂;
- (C₁-C₁₂Alkyldiyl)-OR⁶ ;
- (C₃-C₁₂carbocyclyl);
- (C₃-C₁₂Carbocyclyl)-^*;
- (C₃-C₁₂carbocyclyl)-(C₁-C₁₂Alkyldiyl)-NR⁶-^*;
- (C₃-C₁₂carbocyclyl)-(C₁-C₁₂alkyldiyl)-N(R⁶)₂;
- (C₃-C₁₂carbocyclyl)-NR⁵-C(=NR^6a) N.R.⁶-^*;
- (C₆-C₂₀aryl);
- (C₆-C₂₀Aryldiyl)-^*;
- (C₆-C₂₀Aryldiyl)-N(R⁶) -^*;
- (C₆-C₂₀Aryldiyl)-(C₁-C₁₂alkyldiyl)-N(R⁶) -^*;
- (C₆-C₂₀Aryldiyl)-(C₁-C₁₂Alkyldiyl)-(C₂-C₂₀Heterocyclyldiyl)-^*;
- (C₆-C₂₀Aryldiyl)-(C₁-C₁₂alkyldiyl)-N(R⁶)₂;
- (C₆-C₂₀Aryldiyl)-(C₁-C₁₂Alkyldiyl)-NR⁶-C(=NR^6a) N (R⁶) -^*;
- (C₂-C₂₀Heterocyclyl);
- (C₂-C₂₀Heterocyclyl)-^*;
- (C₂-C₉Heterocyclyl)-(C₁-C₁₂Alkyldiyl)-NR⁶-^*;
- (C₂-C₉Heterocyclyl)-(C₁-C₁₂alkyldiyl)-N(R⁶)₂;
- (C₂-C₉Heterocyclyl)-C(=O)-(C₁-C₁₂alkyldiyl)-N(R⁶) -^*;
- (C₂-C₉Heterocyclyl)-NR⁵-C(=NR^6a) N.R.⁶-^*;
- (C₂-C₉Heterocyclyl)-NR⁶- (C₆-C₂₀Aryldiyl)-(C₁-C₁₂alkyldiyl)-N(R⁶) -^*;
- (C₂-C₉Heterocyclyl)-(C₆-C₂₀Aryldiyl)-^*;
- (C₁-C₂₀Heteroaryl);
- (C₁-C₂₀Heteroaryldiyl)-^*;
- (C₁-C₂₀Heteroaryldiyl)-(C₁-C₁₂alkyldiyl)-N(R⁶) -^*;
- (C₁-C₂₀Heteroaryldiyl)-(C₁-C₁₂alkyldiyl)-N(R⁶)₂;
- (C₁-C₂₀Heteroaryldiyl)-NR⁶-C(=NR^6a) N (R⁶) -^*;
- (C₁-C₂₀Heteroaryldiyl)-N(R⁶) C(=O)-(C₁-C₁₂alkyldiyl)-N(R⁶) -^*;
-C(=O)-^*;
-C(=O)-(C₁-C₁₂alkyldiyl)-N(R⁶) -^*;
-C(=O)-(C₂-C₂₀Heterocyclyldiyl)-^*;
-C(=O)N(R⁶)₂;
-C(=O)N(R⁶) -^*;
-C(=O)N(R⁶)-(C₁-C₁₂alkyldiyl)-N(R⁶) C(=O)R⁵;
-C(=O)N(R⁶)-(C₁-C₁₂alkyldiyl)-N(R⁶)C(=O)N(R⁶)₂;
-C(=O)NR⁶- (C₁-C₁₂alkyldiyl)-N(R⁶) CO₂R⁶;
-C(=O)NR⁶- (C₁-C₁₂alkyldiyl)-N(R⁶) C(=NR^6a) N (R⁶)₂;
-C(=O)NR⁶- (C₁-C₁₂Alkyldiyl)-NR⁶C (=NR^6a) R⁶;
-C(=O)NR⁶- (C₁-C₈Alkyldiyl)-NR⁶(C₂-C₅Heteroaryl);
-C(=O)NR⁶- (C₁-C₂₀Heteroaryldiyl)-N(R⁶) -^*;
-C(=O)NR⁶- (C₁-C₂₀Heteroaryldiyl)-^*;
-C(=O)NR⁶- (C₁-C₂₀Heteroaryldiyl)-(C₁-C₁₂Alkyldiyl)N(R⁶)₂;
-C(=O)NR⁶- (C₁-C₂₀Heteroaryldiyl)-(C₂-C₂₀Heterocyclyldiyl)-C(=O)NR⁶- (C₁-C₁₂Alkyldiyl)-NR⁶-^*;
-N(R⁶)₂;
-N(R⁶) -^*;
-N(R⁶) C(=O)R⁶;
-N(R⁶) C(=O)-^*;
-N(R⁶)C(=O)N(R⁶)₂;
-N(R⁶)C(=O)N(R⁶) -^*;
-N(R⁶) CO₂R⁶;
-N(R⁶) CO₂(R⁶) -^*;
-NR⁶C (=NR^6a) N (R⁶)₂;
-NR⁶C (=NR^6a) N (R⁶) -^*;
-NR⁶C (=NR^6a) R⁶;
-N(R⁶) C(=O)-(C₁-C₁₂alkyldiyl)-N(R⁶) -^*;
-N(R⁶)-(C₂-C₅Heteroaryl);
-N(R⁶) -S(=O)₂- (C₁-C₁₂alkyl);
-O-(C₁-C₁₂alkyl);
-O-(C₁-C₁₂alkyldiyl)-N(R⁶)₂;
-O-(C₁-C₁₂alkyldiyl)-N(R⁶) -^*;
-OC(=O)N(R⁶)₂;
-OC(=O)N(R⁶) -^*;
-S(=O)₂- (C₂-C₂₀Heterocyclyldiyl)-^*;
-S(=O)₂- (C₂-C₂₀Heterocyclyldiyl)-(C₁-C₁₂alkyldiyl)-N(R⁶)₂;
-S(=O)₂- (C₂-C₂₀Heterocyclyldiyl)-(C₁-C₁₂Alkyldiyl)-NR⁶-^*;as well as
-S(=O)₂- (C₂-C₂₀Heterocyclyldiyl)-(C₁-C₁₂Alkyldiyl)-OH orand optionally substituted independently with one or more groups selected from:
or R of formula Ic or Id²and R³taken together form a 5- or 6-membered heterocyclyl ring;
X¹, X², X³, X⁴, and X⁵is a bond, C(=O), C(=O)N(R⁶), O, N (R⁶), S, S(O)₂, and S(O)₂N (R⁶) independently selected from the group consisting of:
R⁶But, H, C₆-C₂₀Aryl, C₆-C₂₀Aryldiyl, C₁-C₁₂Alkyl, and C₁-C₁₂Alkyl diRu oror two R⁶the groups taken together form a 5- or 6-membered heterocyclyl ring;
R^6aBut, C₆-C₂₀Aryl and C₁-C₂₀heteroaryl;
Here, an asterisk^*indicates the binding site of L, where R¹, R², R³, R⁴and R⁵is attached to L;
L is:
Q-C(=O)-(PEG)-C(=O)-(EsPEP)-;
Q-C(=O)-(PEG)-C(=O)-(EsPEP)-N(R⁶)-(C₁-C₁₂Alkyldiyl)-;
Q-C(=O)-(PEG)-C(=O)-(EsPEP)-N(R⁶)-(C₁-C₁₂alkyldiyl)-N(R⁶) C(=O)-(C₂-C₅Monoheterocyclyldiyl)-;
Q-C(=O)-(PEG)-N(R⁶)-(PEG)-C(=O)-(EsPEP)-;
Q-C(=O)-(PEG)-N⁺(R⁶)₂-(PEG)-C(=O)-(EsPEP)-;
Q-C(=O)-(PEG)-C(=O)-N(R⁶) CH(AA₁)C(=O)-(PEG)-C(=O)-(EsPEP)-;
Q-C(=O)-(C₁-C₁₂alkyldiyl)-C(=O)-(EsPEP)-;
Q-C(=O)-(C₁-C₁₂alkyldiyl)-C(=O)-(EsPEP)-N(R⁶)-(C₁-C₁₂Alkyldiyl)-;
Q-C(=O)-(C₁-C₁₂alkyldiyl)-C(=O)-(EsPEP)-N(R⁶)-(C₁-C₁₂alkyldiyl)-N(R⁶) -C(=O);
Q-C(=O)-(C₁-C₁₂alkyldiyl)-C(=O)-(EsPEP)-N(R⁶)-(C₁-C₁₂alkyldiyl)-N(R⁶) C(=O)-(C₂-C₅Monoheterocyclyldiyl)-;
Q-(CH₂)_m-C(=O)N(R⁶)-PEG-C(═O)-(EsPEP)-;
Q-(CH₂)_m-C(=O)-(EsPEP)-N(R⁶)-(C₁-C₁₂Alkyldiyl)-;
Q-(CH₂)_m-C(=O)-(EsPEP)-N(R⁶)-(C₁-C₁₂Alkyldiyl)N(R⁶)C(═O)—; and
Q-(CH₂)_m-C(=O)-(EsPEP)-N(R⁶)-(C₁-C₁₂alkyldiyl)-N(R⁶) C(=O)-(C₂-C₅Monoheterocyclyldiyl-mosquitoselected from the group consisting of:
PEG is a group represented by the formula: -(CH₂CH₂O)_n- (CH₂)_m- m is an integer from 1 to 5 and n is an integer from 2 to 50;
EsPEP is an elastase-substrate peptide linker unit comprising 2-12 amino acid residues;
Q is F, Cl, NO₂and S.O.₃ ^-N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, maleimide, and phenoxy, each substituted with one or more groups independently selected from
Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are each independently selected from the group consisting of F, Cl, Br, I, -CN, -CH₃, -CH₂CH₃, -CH=CH₂, -C≡CH, -C≡CCH₃, -CH₂CH₂CH₃, -CH(CH₃)₂, -CH₂CH (CH₃)₂, -CH₂OH, -CH₂OCH₃, -CH₂CH₂OH, -C(CH₃)₂OH, -CH(OH)CH(CH₃)₂, -C(CH₃)₂CH₂OH, -CH₂CH₂SO₂CH₃, -CH₂OP(O)(OH)₂, -CH₂F, -CHF₂, -CF₃, -CH₂CF₃, -CH₂CHF₂, -CH(CH₃)CN, -C(CH₃)₂CN, -CH₂CN, -CH₂N.H.₂, -CH₂N.H.S.O.₂CH₃, -CH₂N.H.C.H.₃, -CH₂N (CH₃)₂, -CO₂H, -COCH₃, -CO₂CH₃, -CO₂C (CH₃)₃, -COCH(OH)CH₃, -CONH₂, -CONHCH₃, -CON(CH₃)₂, -C(CH₃)₂CONH₂, -NH₂, -NHCH₃, -N(CH₃)₂, -NHCOCH₃, -N(CH₃) COCH₃, -NHS(O)₂CH₃, -N(CH₃) C(CH₃)₂CONH₂, -N(CH₃) CH₂CH₂S (O)₂CH₃, -NHC(=NH)H, -NHC(=NH)CH₃, -NHC(=NH)NH₂, -NHC(=O)NH₂, -NO₂, =O, -OH, -OCH₃, -OCH₂CH₃, -OCH₂CH₂OCH₃, -OCH₂CH₂OH, -OCH₂CH₂N (CH₃)₂, -O(CH₂CH₂O)_n- (CH₂)_mCO₂H, -O(CH₂CH₂O)_nH, -OP(O)(OH)₂, -S(O)₂N (CH₃)₂, -SCH₃, -S(O)₂CH₃, and -S(O)₃ H orone or more groups independently selected fromandOptionally replacedattitudeAn immunostimulant-elastase substrate peptide linker compound.

49. The immunostimulant-elastase substrate peptide linker compound of claim 48 , wherein PEG has the formula: -(CH ₂ CH ₂ O) ₂₅ -(CH ₂ ) ₂ - or -(CH ₂ CH ₂ O) ₁₀ -(CH ₂ ) ₂ -.

^49. The immunostimulant-elastase substrate peptide linker compound of claim 48 , wherein R2 and ^R3 are each _C1 - _C8 alkyl.

^51. The immunostimulant _- _elastase substrate peptide linker compound of claim 50 , wherein R2 and ^R3 are each _--CH2CH2CH3 .

49. The immunostimulant-elastase substrate peptide linker compound of claim 48 , wherein X2 and ^X3 ^are each a bond, and ^R2 or ^R3 is -O-( _C1 - _C12 alkyl).

^53. The immunostimulant-elastase substrate peptide linker compound of claim 52 , wherein R2 or ^R3 _is _-OCH2CH3 .

formula:

49. The immunostimulant-elastase substrate peptide linker compound of claim 48 , selected from the group consisting of: wherein TFP is 2,3,5,6-tetrafluorophenoxy.

IIe-h:

and
49. The immunostimulatory agent-elastase substrate peptide linker compound of claim 48 , wherein the wavy line indicates attachment to the antibody via L.

IIi-l:

wherein the wavy line indicates attachment to the antibody via L.

49. An immunoconjugate prepared by conjugation of a cell binding agent with the immunostimulatory agent-elastase substrate peptide linker compound of claim 48 .

A cell binding agent having the structure:

The immunoconjugate prepared by conjugation of an immunostimulant-elastase substrate peptide linker compound having the formula:

The immunoconjugate of claim 58 , wherein the cell binding agent is an antibody.

A pharmaceutical composition comprising a therapeutically effective amount of the immunoconjugate of claim 1 and one or more pharma- ceutically acceptable diluents, vehicles, carriers, or excipients .

A medicament for treating a cancer selected from bladder cancer , urinary tract cancer , urothelial cancer , lung cancer , non-small cell lung cancer , Merkel cell carcinoma , colon cancer , colorectal cancer , gastric cancer , and breast cancer , comprising the immunoconjugate of claim 1.