KR20250160354A - PANRAS inhibitor antibody-drug conjugates and methods of use thereof - Google Patents

Abstract

An antibody-drug conjugate that binds to a human tumor target is disclosed. The antibody-drug conjugate comprises a panRAS inhibitor drug moiety. The present invention also relates to methods and compositions for treating cancer by administering the antibody-drug conjugate provided herein. Linker-drug conjugates comprising a panRAS inhibitor drug moiety and methods for preparing the same are also disclosed.

Description

PANRAS inhibitor antibody-drug conjugates and methods of use thereof

The present invention relates to antibody-drug conjugates (ADCs) comprising a panRAS inhibitor and an antibody or antigen-binding fragment thereof that binds to an antigen target, e.g., an antigen expressed on a tumor or other cancer cell. The present invention also relates to methods and compositions useful for the treatment and/or diagnosis of cancers that express the target antigen and/or are treatable by modulating the expression and/or activity of panRAS, and to methods for preparing such compositions. Linker-drug conjugates comprising a panRAS inhibitor drug moiety and methods for preparing the same are also disclosed.

Ras proteins (K-Ras, H-Ras, and N-Ras) play essential roles in various human cancers and are therefore attractive targets for anticancer therapies. In fact, mutations in Ras proteins account for approximately 30% of all human cancers in the United States, many of which are lethal. Dysregulation of Ras proteins by activating mutations, overexpression, or upstream activation is common in human tumors, and activating mutations in Ras are frequently found in human cancers. For example, activating mutations at codon 12 of Ras proteins significantly bias the population of mutant Ras proteins to the "on" (GTP-bound) state (Ras(ON)) by inhibiting both the rate of GTPase-activating protein (GAP)-dependent hydrolysis and the rate of intrinsic GTP hydrolysis, resulting in oncogenic MAPK signaling. Notably, Ras exhibits a picomolar affinity for GTP, allowing it to be activated even in the presence of low concentrations of this nucleotide. Mutations in codons 13 (e.g., G13D) and 61 (e.g., Q61K) of Ras are also responsible for oncogenic activity in some cancers.

Despite extensive drug discovery efforts against Ras over the past several decades, further efforts are needed to identify additional drugs for cancers driven by various Ras mutations.

In some embodiments, the present invention provides, in part, novel antibody-drug conjugate (ADC) compounds having biological activity against cancer cells. These compounds may slow, inhibit, and/or reverse tumor growth in mammals and/or may be useful in treating human cancer patients. More specifically, the present invention relates, in some embodiments, to ADC compounds capable of binding to and killing cancer cells. In some embodiments, the ADC compounds disclosed herein comprise a conjugate linker that attaches a panRAS inhibitor to a full-length antibody or antigen-binding fragment. In some embodiments, the ADC compounds may also be internalized into target cells following binding.

In some embodiments, the ADC compound may be represented by Formula 1:

(wherein Ab is an antibody or an antigen-binding fragment thereof;

D is a panRAS inhibitor;

L is a conjugate linker that covalently attaches Ab to D;

p is an integer from 1 to 16). In some embodiments, Ab is an antibody or antigen-binding fragment thereof that targets cancer cells.

In some embodiments, for the ADC compound of formula 1, D comprises a panRAS inhibitor compound of formula Ia covalently attached to a conjugate linker L, or a pharmaceutically acceptable salt thereof:

[Chemical Formula Ia]

[Here,

Dashed lines represent zero, one, two, three, or four non-adjacent double bonds;

A ^D is -N(H or CH ₃ )C(O)-(CH ₂ )- (wherein the amino nitrogen is bonded to a carbon atom of -C(R ^D10a )(R ^D10 )-), an optionally substituted 3 to 6 membered cycloalkylene, an optionally substituted 3 to 6 membered heterocycloalkylene, an optionally substituted 6 membered arylene, or an optionally substituted 5 to 6 membered heteroarylene;

Y ^X is

(Here, represents the point where X connects to ^D3 ; represents the point where W connects to ^X ); or

Y ^X is -N(R ^D11 )-CO-B ^D -L ^D -;

B ^D is -CH(R ^D9 )- or >C=CR ^D9 R ^D9' (wherein the carbon is bonded to the carbonyl carbon of -N(R ^D11 )C(O)-), an optionally substituted 3 to 6 membered cycloalkylene, an optionally substituted 3 to 6 membered heterocycloalkylene, an optionally substituted 6 membered arylene, or a 5 to 6 membered heteroarylene;

L ^D is absent or is a linker;

G ^D is optionally substituted C ₁ -C ₄ alkylene, optionally substituted C ₁ -C ₄ alkenylene, optionally substituted C ₁ -C ₄ heteroalkylene, -C(O)O-CH(R ^D6 )- (wherein -CH(R ^D6 )- is bonded to -C(R ^D7 R ^D8 )-), -C(O)NH-CH(R ^D6 )- (wherein -CH(R ^D6 )- is bonded to -C(R ^D7 R ^D8 )-), optionally substituted C ₁ -C ₄ heteroalkylene, or 3 to 8 membered heteroarylene;

W ^X is hydrogen, cyano, optionally substituted C ₁ -C ₃ heteroalkyl, optionally substituted amino, optionally substituted C ₁ -C ₄ alkoxy, optionally substituted C ₁ -C ₄ hydroxyalkyl, optionally substituted C ₁ -C ₄ aminoalkyl, optionally substituted C ₁ -C ₄ haloalkyl, optionally substituted C ₁ -C ₄ alkyl, optionally substituted C ₁ -C ₄ guanidinoalkyl, C ₀ -C ₄ alkyl, optionally substituted 3 to 11 membered heterocycloalkyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 3 to 8 membered heteroaryl;

X ^D1 is optionally substituted C ₁ -C ₂ alkylene, NR ^D , O, or S(O) _nD ;

X ^D2 is O or NH;

X ^D3 is N or CH;

nD is 0, 1, or 2;

R ^D is hydrogen, cyano, optionally substituted C ₁ -C ₄ alkyl, optionally substituted C ₂ -C ₄ alkenyl, optionally substituted C ₂ -C ₄ alkynyl, C(O)R ^D ', C(O)OR ^D ', C(O)N(R ^D ') ₂ , S(O)R ^D ', S(O) ₂ R ^D ', or S(O) ₂ N(R ^D ') ₂ ; each R ^D' is independently H or optionally substituted C ₁ -C ₄ alkyl;

Y ^D1 is C, CH, or N;

Y ^D2 , Y ^D3 , Y ^D4 , and Y ^D7 are independently C or N;

Y ^D5 is CH, CH ₂ , or N;

Y ^D6 is C(O), CH, CH ₂ , or N;

R ^D1 is cyano, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 6 membered cycloalkenyl, optionally substituted 3 to 6 membered heterocycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 5 to 10 membered heteroaryl, or

R ^D1 and R ^D2 are combined with the atoms to which they are attached to form an optionally substituted 3 to 14 membered heterocycloalkyl;

R ^D2 is absent, hydrogen, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 7 membered heterocycloalkyl, optionally substituted 6 membered aryl, optionally substituted 5 or 6 membered heteroaryl;

R ^D3 is absent or R ^D2 and R ^D3 are combined with the atoms to which they are attached to form an optionally substituted 3 to 8 membered cycloalkyl or an optionally substituted 3 to 14 membered heterocycloalkyl;

R ^D4 is absent, hydrogen, halogen, cyano, or methyl (optionally substituted with 1 to 3 halogens);

R ^D5 is hydrogen, C ₁ -C ₄ alkyl (optionally substituted with halogen, cyano, hydroxy, or C ₁ -C ₄ alkoxy), cyclopropyl, or cyclobutyl;

R ^D6 is hydrogen or methyl;

R ^D7 is hydrogen, halogen, or optionally substituted C ₁ -C ₃ alkyl, or

R ^D6 and R ^D7 are combined with the carbon atom to which they are attached to form an optionally substituted 3 to 6 membered cycloalkyl or an optionally substituted 3 to 7 membered heterocycloalkyl;

R ^D8 is hydrogen, halogen, hydroxy, cyano, optionally substituted C ₁ -C ₃ alkoxy, optionally substituted C ₁ -C ₃ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or

R ^D7 and R ^D8 are combined with the carbon atom to which they are attached to form C=CR ^D7' R ^D8' ; C=N(OH), C=N(OC ₁ -C ₃ alkyl), C=O, C=S, C=NH, an optionally substituted 3 to 6 membered cycloalkyl, or an optionally substituted 3 to 7 membered heterocycloalkyl;

R ^D7a and R ^D8a are, independently, hydrogen, halo, optionally substituted C ₁ -C ₃ alkyl, or combined with the carbon to which they are attached, form carbonyl;

R ^D7' is hydrogen, halogen, or optionally substituted C ₁ -C ₃ alkyl;

R ^D8' is hydrogen, halogen, hydroxyl, cyano, optionally substituted C ₁ -C ₃ alkoxyl, optionally substituted C ₁ -C ₃ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or

R ^D7' and R ^D8' are combined with the carbon atom to which they are attached to form an optionally substituted 3 to 6 membered cycloalkyl or an optionally substituted 3 to 7 membered heterocycloalkyl;

R ^D9 is hydrogen, F, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, or optionally substituted 3 to 7 membered heterocycloalkyl;

R ^D9 and L ^D are combined with the atoms to which they are attached to form an optionally substituted 3 to 14 membered heterocycloalkyl;

R ^D9' is hydrogen or optionally substituted C ₁ -C ₆ alkyl;

R ^D10 is hydrogen, halo, hydroxyl, C ₁ -C ₃ alkoxyl, or C ₁ -C ₃ alkyl;

R ^D10a is hydrogen or halogen;

R ^D11 is hydrogen or C ₁ -C ₃ alkyl;

R ^D16 is hydrogen or C ₁ -C ₃ alkyl].

In some embodiments, p is an integer from 1 to 8. In some embodiments, p is an integer from 1 to 6. In some embodiments, p is an integer from 1 to 5. In some embodiments, p is an integer from 2 to 4. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 4. In some embodiments, p is determined by liquid chromatography-mass spectrometry (LC-MS).

In some embodiments, the conjugate linker (L) comprises an attachment group, at least one spacer group, and at least one cleavable group. In some cases, the cleavable group comprises a pyrophosphate group and/or a self-immolative group. In certain embodiments, L comprises an attachment group; at least one bridging spacer group; and at least one cleavable group comprising a pyrophosphate group and/or a self-immolative group.

In some embodiments, the antibody-drug conjugate comprises a linker-drug (or "linker-payload") moiety -(L-D) of formula A:

[Chemical Formula A]

(wherein R ¹ is an attachment group, L ₁ is a crosslinking spacer group, and E is a cleavable group).

In some embodiments, the cleavable group comprises a pyrophosphate group. In some embodiments, the cleavable group comprises:

.

In some embodiments, the crosslinking spacer group comprises a polyoxyethylene (PEG) group. In some cases, the PEG group can be selected from PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, and PEG15. In some embodiments, the crosslinking spacer group can comprise: -CO-CH ₂ -CH ₂ -PEG12-. In other embodiments, the crosslinking spacer group comprises a butanoyl, pentanoyl, hexanoyl, heptanoyl, or octanoyl group. In some embodiments, the crosslinking spacer group comprises a hexanoyl group.

In some embodiments, the attachment group is formed by at least one reactive group selected from a maleimide group, a thiol group, a cyclooctyne group, and an azido group. For example, the maleimide group may have the following structure:

.

Ajidogi can have the following structure: -N=N ⁺ =N ^- .

The cyclooctene group has the following structure:

can have, where, is binding to an antibody or an antigen-binding fragment thereof.

In some cases, the cyclooctene group has the following structure:

, where, is binding to an antibody or an antigen-binding fragment thereof.

In some embodiments, the attachment device is has a chemical formula including, wherein, is binding to an antibody or an antigen-binding fragment thereof.

In some embodiments, the antibody or antigen-binding fragment thereof is is bonded to the conjugate linker (L) by an attachment group selected from

Here, is binding to an antibody or an antigen-binding fragment thereof, is a bond to a cross-linking spacer group. As used herein, the term "bonded" refers to covalently attached or covalently linked.

In some embodiments, the crosslinking spacer group is bonded or covalently linked to the cleavable group.

In some embodiments, the bridging spacer group is -CH ₂ CH ₂ -O-CH ₂ CH ₂ -CO-.

In some embodiments, the cleavable group is -pyrophosphate-CH ₂ -CH ₂ -NH ₂ -.

In some embodiments, the cleavable group is bound to or covalently linked to a panRAS inhibitor (D).

In some embodiments, the conjugate linker comprises: an attachment group, at least one crosslinking spacer group, a peptide group, and at least one cleavable group.

In some embodiments, the antibody-drug conjugate comprises a linker-drug moiety, -(L-D), which is of formula B:

[Chemical Formula B]

(wherein R ¹ is an attachment group, L ₁ is a cross-linking spacer, Lp is a peptide group containing 1 to 6 amino acid residues, E is a cleavable group, L ₂ is a cross-linking spacer, m is 0 or 1; and D is a panRAS inhibitor). In some cases, m is 1 and the cross-linking spacer comprises:

.

In some embodiments, the at least one crosslinking spacer comprises a PEG group. In some cases, the PEG group is selected from PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, and PEG15. In some cases, the at least one crosslinking spacer is selected from *-C(O)-CH ₂ -CH ₂ -PEG1-**, *-C(O)-CH ₂ -PEG3-**, *-C(O)-CH ₂ -CH ₂ -PEG12**, *-NH-CH ₂ -CH ₂ -PEG1-**, a polyhydroxyalkyl group, *-C(O)-N(CH ₃ )-CH ₂ -CH ₂ -N(CH ₃ )-C(O)-**, *-C(O)-CH ₂ -CH ₂ -PEG12-NH-C(O)CH ₂ -CH ₂ -**, wherein ** represents a direct or indirect attachment point of the at least one crosslinking spacer to an attachment group, and * represents a direct or indirect attachment point of the at least one crosslinking spacer to a peptide group.

In some embodiments, L ₁ is selected from *-C(O)-CH ₂ -CH ₂ -PEG1-**, *-C(O)-CH ₂ -PEG3-**, *-C(O)-CH ₂ -CH ₂ -PEG12**, *-NH-CH ₂ -CH ₂ -PEG1-**, and polyhydroxyalkyl groups, wherein ** represents a direct or indirect point of attachment of L ₁ to R ¹ , and * represents a direct or indirect point of attachment of L ₁ to Lp.

In some embodiments, m is 1 and L ₂ is -C(O)-N(CH ₃ )-CH ₂ -CH ₂ -N(CH ₃ )-C(O)-.

In some embodiments, the peptide group comprises 1 to 12 amino acid residues. In some embodiments, the peptide group (Lp) comprises 1 to 10 amino acid residues. In some embodiments, the peptide group (Lp) comprises 1 to 8 amino acid residues. In some embodiments, the peptide group (Lp) comprises 1 to 6 amino acid residues. In some embodiments, the peptide group comprises 1 to 4 amino acid residues. In some embodiments, the peptide group comprises 1 to 3 amino acid residues. In some embodiments, the peptide group comprises 1 to 2 amino acid residues. In some cases, the amino acid residues are selected from L-glycine (Gly), L-valine (Val), L-citrulline (Cit), L-cysteic acid (sulfo-Ala), L-lysine (Lys), L-isoleucine (Ile), L-phenylalanine (Phe), L-methionine (Met), L-asparagine (Asn), L-proline (Pro), L-alanine (Ala), L-leucine (Leu), L-tryptophan (Trp), and L-tyrosine (Tyr). For example, the peptide group may comprise Val-Cit, Val-Ala, Val-Lys, and/or sulfo-Ala-Val-Ala. In some embodiments, the peptide group (Lp) comprises one amino acid residue. In some embodiments, the peptide group (Lp) comprises: .

In some cases, the peptide group comprises a group selected from:

.

In some embodiments, the self-immolative group comprises para-aminobenzyl-carbamate, para-aminobenzyl-ammonium, para-amino-(sulfo)benzyl-ammonium, para-amino-(sulfo)benzyl-carbamate, para-amino-(alkoxy-PEG-alkyl)benzyl-carbamate, para-amino-(polyhydroxycarboxytetrahydropyranyl)alkyl-benzyl-carbamate, or para-amino-(polyhydroxycarboxytetrahydropyranyl)alkyl-benzyl-ammonium.

In some embodiments, m is 1 and the crosslinking spacer is Includes.

In some embodiments, the linker-drug moiety, -(L-D), is formed of a compound selected from:

.

In some embodiments, the antibody-drug conjugate comprises a linker-drug group, -(L-D), which

Contains a chemical formula selected from,

Here, is binding to an antibody or an antigen-binding fragment thereof.

In some embodiments, the antibody-drug conjugate comprises a linker-drug group, -(L-D), which is of formula C:

[Chemical Formula C]

[Wherein, R ¹ is an attachment group, L ₁ is a crosslinking spacer; L _p is a peptide group containing 1 to 6 amino acids; D is a panRAS inhibitor; G ₁ -L ₂ -A is a self-immolative spacer; L ₂ is a bond, methylene, neopentylene or C ₂ -C ₃ alkenylene; A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl and the * of A represents the point of attachment to D; L ₃ is a spacer moiety; R ² is a hydrophilic moiety.

In some embodiments, the antibody-drug conjugate comprises a linker-drug group, -(L-D), which is of formula D:

[Chemical Formula D]

[Wherein, R ¹ is an attachment group; L ₁ is a bridging group; Lp is a peptide group containing 1 to 6 amino acids; A is a bond, -OC(=O)-*, , - OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl and the * of A represents the point of attachment to D; L ₃ is a spacer moiety; R ² is a hydrophilic moiety.

In some embodiments, L ₁ is , or *-CH(OH)CH(OH)CH(OH)CH(OH)-**, wherein each n is an integer from 1 to 12, the * of L ₁ represents a direct or indirect attachment point to Lp, and the ** of L ₁ represents a direct or indirect attachment point to R ¹ .

In some embodiments, L ₁ is , and n is an integer from 1 to 12, where * of L ₁ represents a direct or indirect attachment point to Lp, and ** of L ₁ represents a direct or indirect attachment point to R ¹ .

In some embodiments, L ₁ is , n is 1, where * of L ₁ represents a direct or indirect attachment point to Lp, and ** of L ₁ represents a direct or indirect attachment point to R ¹ .

In some embodiments, L ₁ is , n is 12, where * of L ₁ indicates a direct or indirect attachment point to Lp, and ** of L ₁ indicates a direct or indirect attachment point to R ¹ .

In some embodiments, L ₁ is , and n is an integer from 1 to 12, where * of L ₁ represents a direct or indirect attachment point to Lp, and ** of L ₁ represents a direct or indirect attachment point to R ¹ .

In some embodiments, L ₁ is , wherein * of L ₁ represents a direct or indirect attachment point to Lp, and ** of L ₁ represents a direct or indirect attachment point to R ¹ .

In some embodiments, L ₁ is

*-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**;

*-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)O(CH ₂ ) _m SSC(R ³ ) ₂ (CH ₂ ) _m C(=O)NR ³ (CH ₂ ) _m NR ³ C(=O)(CH ₂ ) _m -**;

*-C(=O)O(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**;

*-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**; _{* -C} ( ₌ O)(CH ₂ ) _m

_* -C(=O)(( _{CH 2 ) m} O) _t (CH ₂ ) _n *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n -**; * _-C (=O) _{( CH 2} ) _m NHC(=O)(CH ₂ ) _n

*-C(=O)(( _{CH 2} ) _m O _{) t (} CH ₂ ) _n NHC(=O)(CH ₂ ) _n

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m C(R ³ ) ₂ -** or

*-C(=O)(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -** as a bridge spacer, wherein * of L ₁ represents a direct or indirect attachment point to Lp, ** of L ₁ represents a direct or indirect attachment point to R ¹ , and X ₁ is and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

In some embodiments, R ² is selected from the group consisting of polyethylene glycol, polyalkylene glycol, polyol, polysarcosine, sugar, oligosaccharide, polypeptide, 1 to 3 C ₂ -C ₆ alkyl substituted with a group, or C 2 -C 6 alkyl substituted with _{one to two} substituents independently selected from -OC(=O)NHS(O) ₂ NHCH ₂ CH ₂ OCH ₃ , -NHC(=O)C _1-4 alkylene-P(O)(OCH ₂ CH _{3 )} 2 and -COOH groups. In some embodiments, _R ² is

(where n is an integer from 1 to 6),

am.

In some embodiments, the hydrophilic moiety has the chemical formula , wherein R is H, -CH ₃ CH ₂ CH ₂ NHC(=O)OR _a , -CH ₂ CH ₂ NHC(=O)R _a , or -CH ₂ CH ₂ C(=O)OR _a , R' is OH, -OCH ₃ , CH ₂ CH ₂ NHC(=O)OR _a , -CH ₂ CH ₂ NHC(=O)R _a , or -OCH ₂ CH ₂ C(=O)OR _a , and each of m and n is an integer from 2 to 25 (e.g., from 3 to 25).

In some embodiments, the hydrophilic moiety is Includes.

In some embodiments, the hydrophilic moiety is, for example, a moiety

A polysarcosine having, wherein, n is an integer from 3 to 25; and R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

In some embodiments, L ₃ is structured is a spacer moiety having,

Here,

W is -CH ₂ -, -CH ₂ O-, -CH ₂ N(R ^b )C(=O)O-, -NHC(=O)C(R ^b ) ₂ NHC(=O)O-, -NHC(=O)C(R ^b ) ₂ NH-, -NHC(=O)C(R ^b ) ₂ NHC(=O)-, -CH ₂ N(XR ² )C(=O)O-, -C(=O)N(XR ² )-, -CH ₂ N(XR ² )C(=O)-, -C(=O)NR ^b -, -C(=O)NH-, -CH ₂ NR ^b C(=O)-, -CH ₂ NR ^b C(=O)NH-, -CH ₂ NR ^b C(=O)NR ^b -, -NHC(=O)-, -NHC(=O)O-, -NHC(=O)NH-, -OC(=O)NH-, -S(O) ₂ NH-, -NHS(O) ₂ -, -C(=O)-, -C(=O)O- or -NH- (wherein each R ^b is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl);

X is a bond, triazolyl or -CH ₂ -triazolyl- (wherein X is connected to R ² ).

In some embodiments, L ₃ is structured is a spacer moiety having,

Here,

W is -CH ₂ -, -CH ₂ O-, -CH ₂ N(R ^b )C(=O)O-, -NHC(=O)C(R ^b ) ₂ NHC(=O)O-, -NHC(=O)C(R ^b ) ₂ NH-, -NHC(=O)C(R ^b ) ₂ NHC(=O)-, -CH ₂ N(XR ² )C(=O)O-, -C(=O)N(XR ² )-, -CH ₂ N(XR ² )C(=O)-, -C(=O)NR ^b -, -C(=O)NH-, -CH ₂ NR ^b C(=O)-, -CH ₂ NR ^b C(=O)NH-, -CH ₂ NR ^b C(=O)NR ^b -, -NHC(=O)-, -NHC(=O)O-, -NHC(=O)NH-, -OC(=O)NH-, -S(O) ₂ NH-, -NHS(O) ₂ -, -C(=O)-, -C(=O)O- or -NH- (wherein each R ^b is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl);

X is -CH ₂ -triazolyl-C _1-4 alkylene-OC(O)NHS(O) ₂ NH-, -C _4-6 cycloalkylene-OC(O)NHS(O) ₂ NH-, -(CH ₂ CH ₂ O) _n -C(O)NHS(O) ₂ NH-, -(CH ₂ CH ₂ O) _n -C(O)NHS(O) ₂ NH-(CH ₂ CH ₂ O) _n -, -CH ₂ -triazolyl-C _1-4 alkylene-OC(O)NHS(O) ₂ NH-(CH ₂ CH ₂ O) _n -, or -C _4-6 cycloalkylene-OC(O)NHS(O) ₂ NH-(CH ₂ CH ₂ O) _n - (wherein each n is independently 1, 2, or 3 and X is connected to R ² ).

In some embodiments, the attachment group is formed by a reaction involving at least one reactive group. In some cases, the attachment group is formed by the reaction of: a first reactive group attached to the conjugate linker, and a second reactive group attached to the antibody or antigen-binding fragment thereof or being an amino acid residue of the antibody or antigen-binding fragment thereof.

In some embodiments, at least one of the reactors

thiol,

maleimide,

Haloacetamide,

Azid,

Alkyne,

cyclooctene,

Triaryl phosphine,

Oxanobornadiene,

cyclooctyne,

Diaryl tetrazine,

Monoaryl tetrazine,

Norbornene,

aldehyde,

Hydroxylamine,

Hydrazine,

NH ₂ -NH-C(=O)-,

ketones,

vinyl sulfone,

Aziridine,

amino acid residue,

Includes;

Here,

Each R ³ is independently selected from H and C ₁ -C ₆ alkyl;

Each R ⁴ is 2-pyridyl or 4-pyridyl;

Each R ⁵ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl, and -OH;

Each R ⁶ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl, -NH ₂ , -OCH ₃ , -OCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -CN, -NO ₂ and -OH;

Each R ⁷ is independently selected from H, C _1-6 alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C _1-4 alkoxy substituted with -C(=O)OH, and C _1-4 alkyl substituted with -C(=O)OH.

In some embodiments, the first reactor and the second reactor comprise:

thiols and maleimides,

thiols and haloacetamides,

thiols and vinyl sulfones,

thiols and aziridines,

Azides and alkynes,

Azide and cyclooctyne,

Azide and cyclooctene,

Azide and triaryl phosphines,

Azides and oxanobornadienes,

Diaryl tetrazine and cyclooctene,

Monoaryl tetrazine and norbornene,

Aldehydes and hydroxylamine,

Aldehydes and hydrazines,

Aldehyde and NH ₂ -NH-C(=O)-,

Ketones and hydroxylamines,

Ketones and hydrazines,

Ketones and NH2-NH-C(=O)-,

Hydroxylamine and

Amine and

, or

CoA or CoA analogue and serine residue.

In some embodiments, the attachment device is

Disulfide

Includes a flag selected from,

Here,

R ³² is H, C _1-4 alkyl, phenyl, pyrimidine or pyridine;

R ³⁵ is H, C _1-6 alkyl, phenyl or C _1-4 alkyl substituted with 1 to 3 -OH groups;

Each R ⁷ is independently selected from H, C _1-6 alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C _1-4 alkoxy substituted with -C(=O)OH, and C _1-4 alkyl substituted with -C(=O)OH;

R ³⁷ is independently selected from H, phenyl and pyridine;

q is 0, 1, 2, or 3;

R ⁸ is H or methyl;

R ⁹ is H, -CH ₃ or phenyl.

In some embodiments, the peptide group (Lp) comprises 1 to 6 amino acid residues. In some embodiments, the peptide group (Lp) comprises 1 to 4 amino acid residues. In some embodiments, the peptide group comprises 1 to 3 amino acid residues. In some embodiments, the peptide group comprises 1 to 2 amino acid residues. In some embodiments, the amino acid residues are selected from L-glycine (Gly), L-valine (Val), L-citrulline (Cit), L-cysteic acid (sulfo-Ala), L-lysine (Lys), L-isoleucine (Ile), L-phenylalanine (Phe), L-methionine (Met), L-asparagine (Asn), L-proline (Pro), L-alanine (Ala), L-leucine (Leu), L-tryptophan (Trp), and L-tyrosine (Tyr). In some embodiments, the peptide group comprises Val-Cit, Phe-Lys, Val-Ala, Val-Lys, Leu-Cit, sulfo-Ala-Val, and/or sulfo-Ala-Val-Ala. In some embodiments, Lp is selected from:

.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a bond to an antibody or an antigen-binding fragment thereof; A, D and R are as defined above). In some embodiments, A is a bond or -OC(=O)-*; and R is -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a bond to an antibody or an antigen-binding fragment thereof; A, D and R are as defined above). In some embodiments, A is a bond or -OC(=O)-*; and R is -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a bond to an antibody or an antigen-binding fragment thereof; A, D and R are as defined above). In some embodiments, A is a bond or -OC(=O)-*; and R is -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

Each R is independently selected from H, -CH ₃ , and -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a bond to an antibody or an antigen-binding fragment thereof; A, D and R are as defined above). In some embodiments, A is a bond or -OC(=O)-*; and R is -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

Each R is independently selected from H, -CH ₃ , and -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a bond to an antibody or an antigen-binding fragment thereof; A, D and R are as defined above). In some embodiments, A is a bond or -OC(=O)-*; and R is -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

Xa is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ -, and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a bond to an antibody or an antigen-binding fragment thereof; Xa, A, D and R are as defined above). In some embodiments, Xa is -CH ₂ - or -NHCH ₂ -; A is a bond or -OC(=O)-*; and R is -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a bond to an antibody or an antigen-binding fragment thereof; A, D and R are as defined above). In some embodiments, A is a bond or -OC(=O)-*; and R is -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

Xb is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ -, and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a bond to an antibody or an antigen-binding fragment thereof; Xb, A, D and R are as defined above). In some embodiments, A is a bond or -OC(=O)-*; and R is -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a binding to an antibody or an antigen-binding fragment thereof; A and are as defined above). In some embodiments, A is a bond or -OC(=O)-*.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a binding to an antibody or an antigen-binding fragment thereof; A and D are as defined above). In some embodiments, A is a bond or -OC(=O)-*.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a binding to an antibody or an antigen-binding fragment thereof; A and D are as defined above). In some embodiments, A is a bond or -OC(=O)-*.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a binding to an antibody or an antigen-binding fragment thereof; A and D are as defined above). In some embodiments, A is a bond or -OC(=O)-*.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a binding to an antibody or an antigen-binding fragment thereof; A and D are as defined above). In some embodiments, A is a bond or -OC(=O)-*.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a binding to an antibody or an antigen-binding fragment thereof; A and D are as defined above). In some embodiments, A is a bond or -OC(=O)-*.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a binding to an antibody or an antigen-binding fragment thereof; A and D are as defined above). In some embodiments, A is a bond or -OC(=O)-*.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

Each R is independently H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,

Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a bond to an antibody or an antigen-binding fragment thereof; A, D and R are as defined above). In some embodiments, A is a bond or -OC(=O)-*; and R is -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

Each R is independently H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,

Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;

D is a panRAS inhibitor). In some embodiments, the linker-drug group -(L-D) comprises the following formula:

(Here, is a bond to an antibody or an antigen-binding fragment thereof; A, D and R are as defined above). In some embodiments, A is a bond or -OC(=O)-*; and R is -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of the following formula:

(Here,

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,

Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;

D is a panRAS inhibitor).

In some embodiments, A is a bond.

In some embodiments, A is -OC(=O)-*.

In some embodiments, R is -CH ₃ .

In some embodiments, R is —CH ₂ CH ₂ COOH.

In some embodiments, the antibody-drug conjugate comprises a linker-drug group, -(L-D), which is formed from a compound selected from:

In some embodiments, the antibody-drug conjugate comprises a linker-drug group, -(L-D), which

Contains a chemical formula selected from, wherein, is binding to an antibody or an antigen-binding fragment thereof.

In some embodiments, the panRAS inhibitor (D) comprises a compound of formula Ia: or a pharmaceutically acceptable salt thereof.

[Chemical Formula Ia]

(wherein the variables are as described above for chemical formula Ia).

In some embodiments, the panRAS inhibitor (D) comprises a compound of formula I or a pharmaceutically acceptable salt thereof:

[Chemical Formula I]

[Here,

Dashed lines represent zero, one, two, three, or four non-adjacent double bonds;

A ^D is -N(H or CH ₃ )C(O)-(CH ₂ )- (wherein the amino nitrogen is bonded to a carbon atom of -C(R ^D10a )(R ^D10 )-), an optionally substituted 3 to 6 membered cycloalkylene, an optionally substituted 3 to 6 membered heterocycloalkylene, an optionally substituted 6 membered arylene, or an optionally substituted 5 to 6 membered heteroarylene;

B ^D is -CH(R ^D9 )- or >C=CR ^D9 R ^D9' (wherein the carbon is bonded to the carbonyl carbon of -N(R ^D11 )C(O)-), an optionally substituted 3 to 6 membered cycloalkylene, an optionally substituted 3 to 6 membered heterocycloalkylene, an optionally substituted 6 membered arylene, or a 5 to 6 membered heteroarylene;

G ^D is optionally substituted C ₁ -C ₄ alkylene, optionally substituted C ₁ -C ₄ alkenylene, optionally substituted C ₁ -C ₄ heteroalkylene, -C(O)O-CH(R ^D6 )- (wherein -CH(R ^D6 )- is bonded to -C(R ^D7 R ^D8 )-), -C(O)NH-CH(R ^D6 )- (wherein -CH(R ^D6 )- is bonded to -C(R ^D7 R ^D8 )-), optionally substituted C ₁ -C ₄ heteroalkylene, or 3 to 8 membered heteroarylene;

L ^D is absent or is a drug linker;

W ^D is hydrogen, cyano, optionally substituted amino, optionally substituted C ₁ -C ₄ alkoxy, optionally substituted C ₁ -C ₄ hydroxyalkyl, optionally substituted C ₁ -C ₄ aminoalkyl, optionally substituted C _{1 -C 4 haloalkyl, optionally substituted C 1 -C 4 alkyl ,} optionally substituted C ₁ -C ₄ guanidinoalkyl, C ₀ -C ₄ alkyl optionally substituted 3 to 11 membered heterocycloalkyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 3 to 8 membered heteroaryl;

X ^D1 is optionally substituted C ₁ -C ₂ alkylene, NR ^D , O, or S(O) _nD ;

X ^D2 is O or NH;

X ^D3 is N or CH;

nD is 0, 1, or 2;

R ^D is hydrogen, cyano, optionally substituted C ₁ -C ₄ alkyl, optionally substituted C ₂ -C ₄ alkenyl, optionally substituted C ₂ -C ₄ alkynyl, C(O)R ^D ', C(O)OR ^D ', C(O)N(R ^D ') ₂ , S(O)R ^D ', S(O) ₂ R ^D ', or S(O) ₂ N(R ^D ') ₂ ; each R ^D' is independently H or optionally substituted C ₁ -C ₄ alkyl;

Y ^D1 is C, CH, or N;

Y ^D2 , Y ^D3 , Y ^D4 , and Y ^D7 are independently C or N;

Y ^D5 is CH, CH ₂ , or N;

Y ^D6 is C(O), CH, CH ₂ , or N;

R ^D1 is cyano, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 6 membered cycloalkenyl, optionally substituted 3 to 6 membered heterocycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 5 to 10 membered heteroaryl, or

R ^D1 and R ^D2 are combined with the atoms to which they are attached to form an optionally substituted 3 to 14 membered heterocycloalkyl;

R ^D2 is absent, hydrogen, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 7 membered heterocycloalkyl, optionally substituted 6 membered aryl, optionally substituted 5 or 6 membered heteroaryl;

R ^D3 is absent or R ^D2 and R ^D3 are combined with the atoms to which they are attached to form an optionally substituted 3 to 8 membered cycloalkyl or an optionally substituted 3 to 14 membered heterocycloalkyl;

R ^D4 is absent, hydrogen, halogen, cyano, or methyl (optionally substituted with 1 to 3 halogens);

R ^D5 is hydrogen, C ₁ -C ₄ alkyl (optionally substituted with halogen, cyano, hydroxy, or C ₁ -C ₄ alkoxy), cyclopropyl, or cyclobutyl;

R ^D6 is hydrogen or methyl;

R ^D7 is hydrogen, halogen, or optionally substituted C ₁ -C ₃ alkyl, or

R ^D6 and R ^D7 are combined with the carbon atom to which they are attached to form an optionally substituted 3 to 6 membered cycloalkyl or an optionally substituted 3 to 7 membered heterocycloalkyl;

R ^D8 is hydrogen, halogen, hydroxy, cyano, optionally substituted C ₁ -C ₃ alkoxy, optionally substituted C ₁ -C ₃ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or

R ^D7 and R ^D8 are combined with the carbon atom to which they are attached to form C=CR ^D7' R ^D8' ; C=N(OH), C=N(OC ₁ -C ₃ alkyl), C=O, C=S, C=NH, an optionally substituted 3 to 6 membered cycloalkyl, or an optionally substituted 3 to 7 membered heterocycloalkyl;

R ^D7a and R ^D8a are, independently, hydrogen, halo, optionally substituted C ₁ -C ₃ alkyl, or combined with the carbon to which they are attached, form carbonyl;

R ^D7' is hydrogen, halogen, or optionally substituted C ₁ -C ₃ alkyl;

R ^D8' is hydrogen, halogen, hydroxyl, cyano, optionally substituted C ₁ -C ₃ alkoxyl, optionally substituted C ₁ -C ₃ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or

R ^D7' and R ^D8' are combined with the carbon atom to which they are attached to form an optionally substituted 3 to 6 membered cycloalkyl or an optionally substituted 3 to 7 membered heterocycloalkyl;

R ^D9 is hydrogen, F, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, or optionally substituted 3 to 7 membered heterocycloalkyl;

R ^D9 and L ^D are combined with the atoms to which they are attached to form an optionally substituted 3 to 14 membered heterocycloalkyl;

R ^D9' is hydrogen or optionally substituted C ₁ -C ₆ alkyl;

R ^D10 is hydrogen, halo, hydroxyl, C ₁ -C ₃ alkoxyl, or C ₁ -C ₃ alkyl;

R ^D10a is hydrogen or halogen;

R ^D11 is hydrogen or C ₁ -C ₃ alkyl;

R ^D16 is hydrogen or C ₁ -C ₃ alkyl].

In some embodiments, the panRAS inhibitor (D) comprises a compound of formula Ic: or a pharmaceutically acceptable salt thereof.

[Chemical formula Ic]

[Here,

Dashed lines represent zero, one, two, three, or four non-adjacent double bonds;

A ^D is -N(H or CH ₃ )C(O)-(CH ₂ )- (wherein the amino nitrogen is bonded to a carbon atom of -CH(R ^D10 )-), an optionally substituted 3 to 6 membered cycloalkylene, an optionally substituted 3 to 6 membered heterocycloalkylene, an optionally substituted 6 membered arylene, or an optionally substituted 5 to 6 membered heteroarylene;

B ^D is -CH(R ^D9 )- (wherein the carbon is bonded to the carbonyl carbon of -N(R ^D11 )C(O)-), an optionally substituted 3 to 6 membered cycloalkylene, an optionally substituted 3 to 6 membered heterocycloalkylene, an optionally substituted 6 membered arylene, or a 5 to 6 membered heteroarylene;

L ^D is absent or is a drug linker;

W ^D is hydrogen, optionally substituted amino, optionally substituted C ₁ -C ₄ alkoxy, optionally substituted C ₁ -C ₄ hydroxyalkyl, optionally substituted C ₁ -C ₄ aminoalkyl, optionally substituted C ₁ -C ₄ haloalkyl, optionally substituted C ₁ -C ₄ alkyl, optionally substituted C ₁ -C ₄ guanidinoalkyl, C ₀ -C ₄ alkyl, optionally substituted 3 to 11 membered heterocycloalkyl, optionally substituted 3 to 8 membered cycloalkyl, or optionally substituted 3 to 8 membered heteroaryl;

X ^D2 is O or NH;

X ^D3 is N or CH;

R ^D is hydrogen, cyano, optionally substituted C ₁ -C ₄ alkyl, optionally substituted C ₂ -C ₄ alkenyl, optionally substituted C ₂ -C ₄ alkynyl, C(O)R ^D ', C(O)OR ^D ', C(O)N(R ^D ') ₂ , S(O)R ^D ', S(O) ₂ R ^D ', or S(O) ₂ N(R ^D ') ₂ ;

Each R ^D' is independently H or optionally substituted C ₁ -C ₄ alkyl;

Y ^D1 is C, CH, or N;

Y ^D2 , Y ^D3 , Y ^D4 , and Y ^D7 are independently C or N;

Y ^D5 and Y ^D6 are independently CH or N;

R ^D1 is cyano, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 6 membered cycloalkenyl, optionally substituted 3 to 6 membered heterocycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 5 to 10 membered heteroaryl;

R ^D2 is hydrogen, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 7 membered heterocycloalkyl, optionally substituted 6 membered aryl, optionally substituted 5 or 6 membered heteroaryl;

R ^D3 is absent; or

R ^D2 and R ^D3 are combined with the atoms to which they are attached to form an optionally substituted 3 to 8 membered cycloalkyl or an optionally substituted 3 to 14 membered heterocycloalkyl;

R ^D4 is absent, hydrogen, halogen, cyano, or methyl (optionally substituted with 1 to 3 halogens);

R ^D5 is hydrogen, C ₁ -C ₄ alkyl (optionally substituted with halogen, cyano, hydroxy, or C ₁ -C ₄ alkoxy), cyclopropyl, or cyclobutyl;

R ^D6 is hydrogen or methyl;

R ^D7 is hydrogen, halogen, or optionally substituted C ₁ -C ₃ alkyl, or

R ^D6 and R ^D7 are combined with the carbon atom to which they are attached to form an optionally substituted 3 to 6 membered cycloalkyl or an optionally substituted 3 to 7 membered heterocycloalkyl;

R ^D8 is hydrogen, halogen, hydroxy, cyano, optionally substituted C ₁ -C ₃ alkoxy, optionally substituted C ₁ -C ₃ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or

R ^D7 and R ^D8 are combined with the carbon atom to which they are attached to form C=CR ^7' R ^8' ; C=N(OH), C=N(OC ₁ -C ₃ alkyl), C=O, C=S, C=NH, an optionally substituted 3 to 6 membered cycloalkyl, or an optionally substituted 3 to 7 membered heterocycloalkyl;

R ^D7' is hydrogen, halogen, or optionally substituted C ₁ -C ₃ alkyl;

R ^D8' is hydrogen, halogen, hydroxy, cyano, optionally substituted C ₁ -C ₃ alkoxy, optionally substituted C ₁ -C ₃ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or

R ^D7' and R ^D8' are combined with the carbon atom to which they are attached to form an optionally substituted 3 to 6 membered cycloalkyl or an optionally substituted 3 to 7 membered heterocycloalkyl;

R ^D9 is optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, or optionally substituted 3 to 7 membered heterocycloalkyl;

R ^D10 is hydrogen, hydroxy, C ₁ -C ₃ alkoxy or C ₁ -C ₃ alkyl;

R ^D11 is hydrogen or C ₁ -C ₃ alkyl].

In some embodiments, the panRAS inhibitor (D) comprises a compound of formula If or a pharmaceutically acceptable salt thereof:

[Chemical formula If]

[Here,

A ^D is -N(H or CH ₃ )C(O)-(CH ₂ )- (wherein the amino nitrogen is bonded to the carbon atom of -CH ₂ -), optionally substituted 3 to 6 membered cycloalkylene, optionally substituted 3 to 6 membered heterocycloalkylene, optionally substituted 6 membered arylene, or optionally substituted 5 to 6 membered heteroarylene;

B ^D is -CH(R ^D9 )- (wherein the carbon is bonded to the carbonyl carbon of -NHC(O)-), an optionally substituted 3-6 membered cycloalkylene, an optionally substituted 3-6 membered heterocycloalkylene, an optionally substituted 6-membered arylene, or a 5-6 membered heteroarylene;

L ^D is absent or is a drug linker;

W ^D is hydrogen, optionally substituted amino, optionally substituted C ₁ -C ₄ alkoxy, optionally substituted C ₁ -C ₄ hydroxyalkyl, optionally substituted C ₁ -C ₄ aminoalkyl, optionally substituted C ₁ -C ₄ haloalkyl, optionally substituted C ₁ -C ₄ alkyl, optionally substituted C ₁ -C ₄ guanidinoalkyl, C ₀ -C ₄ alkyl, optionally substituted 3 to 11 membered heterocycloalkyl, optionally substituted 3 to 8 membered cycloalkyl, or optionally substituted 3 to 8 membered heteroaryl;

R ^D1 is cyano, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 6 membered cycloalkenyl, optionally substituted 3 to 6 membered heterocycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 5 to 10 membered heteroaryl;

R ^D2 is C ₁ -C ₆ alkyl or 3 to 6 membered cycloalkyl;

R ^D7 is C ₁ -C ₃ alkyl;

R ^D8 is C ₁ -C ₃ alkyl;

R ^D9 is optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, or optionally substituted 3 to 7 membered heterocycloalkyl.

In some embodiments, in any one of the embodiments described above, R ^D1 is a 5-10 membered heteroaryl.

In some embodiments, R ^D1 is an optionally substituted 6-membered aryl or an optionally substituted 6-membered heteroaryl.

In some embodiments, the panRAS inhibitor D is attached to a conjugate linker represented by L at the A ^D or R ^D1 position.

In some embodiments, the panRAS inhibitor D comprises a compound of formula Ig: or a pharmaceutically acceptable salt thereof.

[Chemical formula Ig]

[Here,

A ^D is an optionally substituted 3 to 6 membered cycloalkylene, an optionally substituted 3 to 6 membered heterocycloalkylene, an optionally substituted 6 membered arylene, or an optionally substituted 5 to 6 membered heteroarylene;

B ^D is -CH(R ^D9 )- (wherein the carbon is bonded to the carbonyl carbon of -NHC(O)-), an optionally substituted 3-6 membered cycloalkylene, an optionally substituted 3-6 membered heterocycloalkylene, an optionally substituted 6-membered arylene, or a 5-6 membered heteroarylene;

L ^D is absent or is a drug linker;

W ^D is hydrogen, optionally substituted amino, optionally substituted C ₁ -C ₄ alkoxy, optionally substituted C ₁ -C ₄ hydroxyalkyl, optionally substituted C ₁ -C ₄ aminoalkyl, optionally substituted C ₁ -C ₄ haloalkyl, optionally substituted C ₁ -C ₄ alkyl, optionally substituted C ₁ -C ₄ guanidinoalkyl, C ₀ -C ₄ alkyl, optionally substituted 3 to 11 membered heterocycloalkyl, optionally substituted 3 to 8 membered cycloalkyl, or optionally substituted 3 to 8 membered heteroaryl;

R ^D2 is C ₁ -C ₆ alkyl or 3 to 6 membered cycloalkyl;

R ^D7 is C ₁ -C ₃ alkyl;

R ^D8 is C ₁ -C ₃ alkyl;

R ^D9 is optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, or optionally substituted 3 to 7 membered heterocycloalkyl;

X ^De is N, CH, or CR ^D17 ;

X ^Df is N or CH;

R ^D12 is optionally substituted C ₁ -C ₆ alkyl or optionally substituted C ₁ -C ₆ heteroalkyl;

R ^D17 is optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 6 membered cycloalkenyl, optionally substituted 3 to 6 membered heterocycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 5 to 10 membered heteroaryl.

In some embodiments, A ^D is an optionally substituted 6-membered arylene.

In some embodiments, A ^D is an optionally substituted 5-6 membered heteroarylene.

In some embodiments, B ^D is -CHR ^D9 -.

In some embodiments, R ^D9 is optionally substituted C ₁ -C ₆ alkyl or optionally substituted 3 to 6 membered cycloalkyl.

In some embodiments, the drug linker in the panRAS inhibitor described in any one of the above embodiments has the structure of Formula II:

[Chemical Formula II]

(Here,

A ^D1 is a bond between the drug linker and B; A ^D2 is a bond between W and the drug linker;

B ^D1 , B ^D2 , B ^D3 , and B ^D4 are each independently selected from optionally substituted C ₁ -C ₂ alkylene, optionally substituted C ₁ -C ₃ heteroalkylene, O, S, and NR ^DN ; R ^DN is hydrogen, optionally substituted C ₁ -C ₄ alkyl, optionally substituted C ₁ -C ₃ cycloalkyl, optionally substituted C ₂ -C ₄ alkenyl, optionally substituted C ₂ -C ₄ alkynyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted C ₁ -C ₇ heteroalkyl;

C ^D1 and C ^D2 are each independently selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl;

fD, gD, hD, iD, jD, and kD are each independently 0 or 1;

D ^D1 is an optionally substituted C ₁ -C ₁₀ alkylene, an optionally substituted C ₂ -C ₁₀ alkenylene, an optionally substituted C ₂ -C ₁₀ alkynylene, an optionally substituted 3 to 14 membered heterocycloalkylene, an optionally substituted 5 to 10 membered heteroarylene, an optionally substituted 3 to 8 membered cycloalkylene, an optionally substituted 6 to 10 membered arylene, an optionally substituted C ₂ -C ₁₀ polyethylene glycolene, or an optionally substituted C ₁ -C ₁₀ heteroalkylene, or a chemical bond connecting A ^D1 -(B ^D1 ) _fD -(C ^D1 ) _gD -(B ^D2 ) _hD - to -(B ^D3 ) _iD -(C ^D2 ) _Dj -(B ^D4 ) _Dk -A ^D2 .

In some embodiments, the drug linker has the structure of Formula IIa:

[Chemical Formula IIa]

(Here,

X ^Da is absent or N;

R ^D14 is absent, hydrogen, optionally substituted C ₁ -C ₆ alkyl, or optionally substituted C ₁ -C ₃ cycloalkyl;

L ^D2 is absent, -C(O)-, -SO ₂ -, optionally substituted C ₁ -C ₄ alkylene or optionally substituted C ₁ -C ₄ heteroalkylene, wherein at least one of X ^Da , R ^D14 , or L ^D2 is present.

In some embodiments, in the panRAS inhibitor described in any one of the above embodiments, W ^D is hydrogen.

In some embodiments, in the panRAS inhibitor described in any one of the above embodiments, W ^D is a C ₀ -C ₄ alkyl optionally substituted 3 to 11 membered heterocycloalkyl.

In some embodiments, the panRAS inhibitor D is attached to a conjugate linker represented by L at position A ^D or R ^D17 .

In some embodiments, the panRAS inhibitor D comprises a compound of formula Ih: or a pharmaceutically acceptable salt thereof.

[chemical formula Ih]

(Here,

R ^D2 is C ₁ -C ₃ alkyl;

R ^D7 is C ₁ -C ₃ alkyl;

R ^D8 is C ₁ -C ₃ alkyl;

R ^D9 is C ₁ -C ₆ alkyl;

R ^D14 is hydrogen, or C ₁ -C ₆ alkyl,

R ^D17 is an optionally substituted 3 to 6 membered cycloalkyl or an optionally substituted 3 to 6 membered heterocycloalkyl;

W ^D is an optionally substituted 3 to 11 membered heterocycloalkyl).

In some embodiments, R ^D9 is C ₁ -C ₃ alkyl; R ^D14 is C ₁ -C ₃ alkyl; R ^D17 is an optionally substituted 3 to 6 membered heterocycloalkyl; and W ^D is an optionally substituted 5 to 6 membered heterocycloalkyl.

In some embodiments, the panRAS inhibitor D is

A compound represented by or a pharmaceutically acceptable salt thereof.

In some embodiments, the panRAS inhibitor D comprises a compound of formula Ij: or a pharmaceutically acceptable salt thereof.

[Chemical formula Ij]

(where aD is 0 or 1). Definitions of other variables are provided in any of the embodiments described above.

In some embodiments, the panRAS inhibitor D comprises a compound of formula Ik: or a pharmaceutically acceptable salt thereof.

[chemical formula Ik]

(where aD is 0 or 1). Definitions of other variables are provided in any of the embodiments described above.

In some embodiments, the panRAS inhibitor D comprises a compound of formula Im or a pharmaceutically acceptable salt thereof:

[Chemical formula Im]

(where aD is 0 or 1). Definitions of other variables are provided in any of the embodiments described above.

In some embodiments, the panRAS inhibitor D comprises a compound of formula In or a pharmaceutically acceptable salt thereof:

[Chemical formula In]

(where aD is 0 or 1). Definitions of other variables are provided in any of the embodiments described above.

In some embodiments, D represents a panRAS inhibitor attached to a conjugate linker L by a covalent bond, wherein the panRAS inhibitor is selected from a compound of Table A1:

Table A1

or a salt acceptable to the patient.

In some embodiments, the panRAS inhibitor D comprises a formula selected from any one of the formulas in Table A2, or a pharmaceutically acceptable salt thereof.

Table A2

(Here, ) indicates binding to the conjugate linker.

In some embodiments, -(L-D) is formed from a compound selected from Table B or an enantiomer, diastereomer and/or pharmaceutically acceptable salt thereof.

In some embodiments, the maleimide group in the compound of Table B forms a covalent bond with an antibody or an antigen-binding fragment thereof (Ab), ADC compounds of formula 1 comprising a moiety, wherein * represents a linkage point to Ab. In the compounds of Table A1, Table A2, Table B and Table 1, depending on their electronic charge, these compounds may contain one pharmaceutically acceptable monovalent anionic counterion M ₁ ^- . In some embodiments, the monovalent anionic counterion M ₁ - may be selected from bromide, chloride, iodide, acetate ^, trifluoroacetate, benzoate, mesylate, tosylate, triflate, formate and the like. In some embodiments, the monovalent anionic counterion M ₁ ^- is trifluoroacetate or formate.

Table B. Exemplary linker-drug groups

In some embodiments, the antibody-drug conjugate has a chemical formula according to any one of the structures exemplified in Table 1.

Table 1. ADC structure

or Ab: any antibody or antigen-binding fragment thereof described herein, e.g., an anti-EphA2 antibody, an anti-B7-H3 antibody or an antigen-binding fragment thereof.

The ADC shown above can also be represented by the following chemical formula 1:

[Chemical Formula 1]

(Here, Ab or represents an antibody or antigen-binding fragment thereof covalently linked to the linker-payload (LD) as described above; and p is an integer from 1 to 16). In some embodiments, p is an integer from 1 to 8. In some embodiments, p is an integer from 1 to 5. In some embodiments, p is an integer from 2 to 4. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 4. In some embodiments, p is determined by liquid chromatography-mass spectrometry (LC-MS).

As used herein, “L-D” refers to a linker-payload, linker-drug, or linker-compound disclosed herein, and the term “L#-D#” is used to refer to a particular linker-drug disclosed herein, while the code “D#” is used to refer to a particular compound, unless otherwise specified, including enantiomers, diastereomers, atropisomers, deuterated derivatives and/or pharmaceutically acceptable salts of any of the aforementioned compounds.

In some embodiments, in the ADCs shown in Table 1, Ab is an antibody or antigen-binding fragment thereof described herein. In some embodiments, in the ADCs shown in Table 1, Ab is an anti-EphA2 antibody or antigen-binding fragment thereof. In some embodiments, Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof.

In some embodiments, the antibody or antigen-binding fragment binds to a target antigen on a cancer cell. In some embodiments, the target antigen is EphA2 or B7-H3 (CD276).

In some embodiments, the target antigen is EphA2. In some embodiments, the target antigen is B7-H3 (CD276).

In some embodiments, the antibody or antigen-binding fragment is an anti-EphA2 antibody or antigen-binding fragment. In some embodiments, the anti-EphA2 antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) selected from the group consisting of:

1) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 17, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 18, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 19; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 26, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 27, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 28;

2) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 20, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 21, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 19; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 29, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 30, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 31;

3) a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 22, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 23, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 24; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 32, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 27, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 31; and

4) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 25, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 21, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 19; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 29, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 30, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 31.

In some embodiments, the anti-EphA2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-EphA2 antibody or antigen-binding fragment comprises an IgG1 heavy chain constant domain or a modified IgG1 heavy chain constant domain. In some embodiments, the IgG1 heavy chain constant domain comprises cysteine residues (C) at positions 152 and 375. In some embodiments, the antibody or antigen-binding fragment comprises an Ig kappa light chain constant domain.

In some embodiments, the anti-EphA2 antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the antibody or antigen-binding fragment is an anti-B7-H3 (CD276) antibody or antigen-binding fragment. In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) selected from the group consisting of:

1) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 33, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 34, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 35; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 42, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 43, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 44;

2) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 36, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 37, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 35; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 45, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 46, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 47;

3) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 38, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 39, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 40; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 48, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 43, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 47;

4) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 41, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 37, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 35; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 45, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 46, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 47;

5) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 49, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 50, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 51; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 58, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 59, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 60;

6) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 52, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 53, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 51; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 61, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 62, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 63;

7) a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 54, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 55, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 56; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 58, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 59, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 63; and

8) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 57, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 53, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 51; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 61, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 62, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 63.

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody or antigen-binding fragment comprises an IgG1 heavy chain constant domain or a modified IgG1 heavy chain constant domain. In some embodiments, the IgG1 heavy chain constant domain comprises cysteine residues (C) at positions 152 and 375. In some embodiments, the antibody or antigen-binding fragment comprises an Ig kappa light chain constant domain.

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody or antigen-binding fragment comprises an IgG1 heavy chain constant domain or a modified IgG1 heavy chain constant domain. In some embodiments, the IgG1 heavy chain constant domain comprises cysteine residues (C) at positions 152 and 375. In some embodiments, the antibody or antigen-binding fragment comprises an Ig kappa light chain constant domain.

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the anti-B7-H3(CD276) antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10.

In some embodiments, compositions comprising multiple copies of an antibody-drug conjugate (e.g., any of the exemplary antibody-drug conjugates described herein) are also provided herein. In some embodiments, the average p of the antibody-drug conjugates in the composition is from about 2 to about 4.

In some embodiments, also provided herein is a pharmaceutical composition comprising an antibody-drug conjugate (e.g., any of the exemplary antibody-drug conjugates described herein) or composition (e.g., any of the exemplary compositions described herein), and a pharmaceutically acceptable carrier.

Also provided herein, in some embodiments, are therapeutic uses of the described ADC compounds and compositions, for example, for treating cancer. In some embodiments, the invention provides a method of treating cancer (e.g., a cancer expressing an antigen targeted by an antibody or antigen-binding fragment of an ADC, such as EphA2 or B7-H3 (CD276)). In some embodiments, the invention provides a method of reducing or slowing the expansion of a cancer cell population in a subject. In some embodiments, the invention provides a method of determining whether a subject having or suspected of having cancer is responsive to treatment with an ADC compound or composition disclosed herein.

An exemplary embodiment is a method of treating a subject having or suspected of having cancer, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein). In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, B7-H4, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2(NaPi2b), Nectin4, TROP2, LIV1, CD46, MSLN, CD142 (F3), MUC1, MUC16, SLC39A6, TFRC, TACSTD2, GPNMB, EphA2, CD56, SEZ6, CD25, CCR8,CEACAM5, CEACAM6, 4-1BB, 5AC, 5T4, Alpha-fetoprotein, angiopoietin 2, ASLG659, TCLI, BMPRIB, Brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (imitation), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR) I), CD22 (B-cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B-cell differentiation antigen CD72), CD79a, CD80, CD166 (ALCAM), CDH17, CA9, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1, CFC1B), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), Episialin, ERBB3, ETBR (endothelin type B receptor), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extradomain-B, Frizzled receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, human scatter factor receptor kinase, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily receptor translocation-associated 2), Lewis-Y antigen, LY64 (RP105), LY6E, STEAP1, ADAM9, PTK7, MMP14, TM4SF1, ITGB6, FXYD5, MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen (PSMA), PSCA (prostate stem cell antigen precursor), PRLR (prolactin receptor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-related gene I, TAG-72, TEMI, Tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-Rl, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel subfamily M, member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or Vimentin.

In some embodiments, the target antigen is EphA2 or B7-H3 (CD276).

In some embodiments, the target antigen is EphA2.

In some embodiments, the target antigen is B7-H3 (CD276).

In some embodiments, the cancer is a tumor or a blood cancer. In some embodiments, the cancer is breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, stomach cancer or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer.

Another exemplary embodiment is a method of reducing or inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein). In some embodiments, the tumor expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, B7-H4, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2(NaPi2b), nectin4, TROP2, LIV1, CD46, MSLN, CD142 (F3), MUC1, MUC16, SLC39A6, TFRC, TACSTD2, GPNMB, EphA2, CD56, SEZ6, CD25, CCR8, CEACAM5, CEACAM6, 4-1BB, 5AC, 5T4, alpha-fetoprotein, Angiopoietin 2, ASLG659, TCLI, BMPRIB, brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (imitation), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR) I), CD22 (B-cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B-cell differentiation antigen CD72), CD79a, CD80, CD166 (ALCAM), CDH17, CA9, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1, CFC1B), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), episialin, ERBB3, ETBR (endothelin type B receptor), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extradomain-B, frizzled receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, human scatter factor receptor kinase, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily receptor translocation-associated 2), Lewis-Y antigen, LY64 (RP105), LY6E, STEAP1, ADAM9, PTK7, MMP14, TM4SF1, ITGB6, FXYD5, MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen (PSMA), PSCA (prostate stem cell antigen precursor), PRLR (prolactin receptor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-related gene I, TAG-72, TEMI, tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel subfamily M, member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or vimentin. In some embodiments, the target antigen is EphA2 or B7-H3 (CD276). In some embodiments, the target antigen is EphA2. In some embodiments, the target antigen is B7-H3 (CD276). In some embodiments, the tumor is breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, stomach cancer or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer. In some embodiments, administration of the antibody-drug conjugate, composition, or pharmaceutical composition reduces or inhibits tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%.

Another exemplary embodiment is a method of reducing or slowing the expansion of a cancer cell population in a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein). In some embodiments, the cancer cell population expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, B7-H4, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2(NaPi2b), nectin4, TROP2, LIV1, CD46, MSLN, CD142 (F3), MUC1, MUC16, SLC39A6, TFRC, TACSTD2, GPNMB, EphA2, CD56, SEZ6, CD25, CCR8, CEACAM5, CEACAM6, 4-1BB, 5AC, 5T4, alpha-fetoprotein, Angiopoietin 2, ASLG659, TCLI, BMPRIB, brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (imitation), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR) I), CD22 (B-cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B-cell differentiation antigen CD72), CD79a, CD80, CD166 (ALCAM), CDH17, CA9, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1, CFC1B), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), episialin, ERBB3, ETBR (endothelin type B receptor), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extradomain-B, frizzled receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, human scatter factor receptor kinase, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily receptor translocation-associated 2), Lewis-Y antigen, LY64 (RP105), LY6E, STEAP1, ADAM9, PTK7, MMP14, TM4SF1, ITGB6, FXYD5, MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen (PSMA), PSCA (prostate stem cell antigen precursor), PRLR (prolactin receptor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-related gene I, TAG-72, TEMI, tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel subfamily M, member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or vimentin. In some embodiments, the target antigen is EphA2 or B7-H3 (CD276). In some embodiments, the target antigen is EphA2. In some embodiments, the target antigen is B7-H3 (CD276). In some embodiments, the cancer cell population is derived from a tumor or a hematological malignancy. In some embodiments, the cancer cell population is breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, gastric or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer.

In some embodiments, administration of the antibody-drug conjugate, composition, or pharmaceutical composition reduces the cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, administration of the antibody-drug conjugate, composition, or pharmaceutical composition slows the expansion of the cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%.

Another exemplary embodiment is an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein) for use in treating a subject having or suspected of having cancer. In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, B7-H4, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2(NaPi2b), nectin4, TROP2, LIV1, CD46, MSLN, CD142 (F3), MUC1, MUC16, SLC39A6, TFRC, TACSTD2, GPNMB, EphA2, CD56, SEZ6, CD25, CCR8, CEACAM5, CEACAM6, 4-1BB, 5AC, 5T4, alpha-fetoprotein, Angiopoietin 2, ASLG659, TCLI, BMPRIB, brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (imitation), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR) I), CD22 (B-cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B-cell differentiation antigen CD72), CD79a, CD80, CD166 (ALCAM), CDH17, CA9, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1, CFC1B), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), episialin, ERBB3, ETBR (endothelin type B receptor), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extradomain-B, frizzled receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, human scatter factor receptor kinase, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily receptor translocation-associated 2), Lewis-Y antigen, LY64 (RP105), LY6E, STEAP1, ADAM9, PTK7, MMP14, TM4SF1, ITGB6, FXYD5, MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen (PSMA), PSCA (prostate stem cell antigen precursor), PRLR (prolactin receptor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-related gene I, TAG-72, TEMI, tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel subfamily M, member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or vimentin. In some embodiments, the target antigen is EphA2 or B7-H3 (CD276). In some embodiments, the target antigen is EphA2. In some embodiments, the target antigen is B7-H3 (CD276). In some embodiments, the cancer is a tumor or a hematological malignancy. In some embodiments, the cancer is breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, stomach cancer or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer.

Another exemplary embodiment is the use of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein) in treating a subject having or suspected of having cancer. In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, B7-H4, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2(NaPi2b), nectin4, TROP2, LIV1, CD46, MSLN, CD142 (F3), MUC1, MUC16, SLC39A6, TFRC, TACSTD2, GPNMB, EphA2, CD56, SEZ6, CD25, CCR8, CEACAM5, CEACAM6, 4-1BB, 5AC, 5T4, alpha-fetoprotein, Angiopoietin 2, ASLG659, TCLI, BMPRIB, brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (imitation), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR) I), CD22 (B-cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B-cell differentiation antigen CD72), CD79a, CD80, CD166 (ALCAM), CDH17, CA9, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1, CFC1B), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), episialin, ERBB3, ETBR (endothelin type B receptor), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extradomain-B, frizzled receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, human scatter factor receptor kinase, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily receptor translocation-associated 2), Lewis-Y antigen, LY64 (RP105), LY6E, STEAP1, ADAM9, PTK7, MMP14, TM4SF1, ITGB6, FXYD5, MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen (PSMA), PSCA (prostate stem cell antigen precursor), PRLR (prolactin receptor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-related gene I, TAG-72, TEMI, tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel subfamily M, member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or vimentin. In some embodiments, the target antigen is EphA2 or B7-H3 (CD276). In some embodiments, the target antigen is EphA2. In some embodiments, the target antigen is B7-H3 (CD276). In some embodiments, the cancer is a tumor or a hematological malignancy. In some embodiments, the cancer is breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, stomach cancer or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer.

Another exemplary embodiment is the use of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein) in a method of preparing a medicament for treating a subject having or suspected of having cancer. In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, B7-H4, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2(NaPi2b), nectin4, TROP2, LIV1, CD46, MSLN, CD142 (F3), MUC1, MUC16, SLC39A6, TFRC, TACSTD2, GPNMB, EphA2, CD56, SEZ6, CD25, CCR8, CEACAM5, CEACAM6, 4-1BB, 5AC, 5T4, alpha-fetoprotein, Angiopoietin 2, ASLG659, TCLI, BMPRIB, brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (imitation), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR) I), CD22 (B-cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B-cell differentiation antigen CD72), CD79a, CD80, CD166 (ALCAM), CDH17, CA9, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1, CFC1B), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), episialin, ERBB3, ETBR (endothelin type B receptor), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extradomain-B, frizzled receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, human scatter factor receptor kinase, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily receptor translocation-associated 2), Lewis-Y antigen, LY64 (RP105), LY6E, STEAP1, ADAM9, PTK7, MMP14, TM4SF1, ITGB6, FXYD5, MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen (PSMA), PSCA (prostate stem cell antigen precursor), PRLR (prolactin receptor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-related gene I, TAG-72, TEMI, tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel subfamily M, member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or vimentin. In some embodiments, the target antigen is EphA2 or B7-H3 (CD276). In some embodiments, the target antigen is EphA2. In some embodiments, the target antigen is B7-H3 (CD276). In some embodiments, the cancer is a tumor or a hematological malignancy. In some embodiments, the cancer is breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, stomach cancer or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer.

Another exemplary embodiment is a method of determining whether a subject having or suspected of having cancer is responsive to treatment with an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein), comprising: providing a biological sample from the subject; contacting the sample with an antibody-drug conjugate; and detecting binding of the antibody-drug conjugate to cancer cells in the sample. In some embodiments, the cancer cells in the sample express a target antigen. In some embodiments, the cancer expresses the target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, B7-H4, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2(NaPi2b), nectin4, TROP2, LIV1, CD46, MSLN, CD142 (F3), MUC1, MUC16, SLC39A6, TFRC, TACSTD2, GPNMB, EphA2, CD56, SEZ6, CD25, CCR8, CEACAM5, CEACAM6, 4-1BB, 5AC, 5T4, alpha-fetoprotein, Angiopoietin 2, ASLG659, TCLI, BMPRIB, brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (imitation), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR) I), CD22 (B-cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B-cell differentiation antigen CD72), CD79a, CD80, CD166 (ALCAM), CDH17, CA9, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1, CFC1B), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), episialin, ERBB3, ETBR (endothelin type B receptor), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extradomain-B, frizzled receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, human scatter factor receptor kinase, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily receptor translocation-associated 2), Lewis-Y antigen, LY64 (RP105), LY6E, STEAP1, ADAM9, PTK7, MMP14, TM4SF1, ITGB6, FXYD5, MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen (PSMA), PSCA (prostate stem cell antigen precursor), PRLR (prolactin receptor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-related gene I, TAG-72, TEMI, tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel subfamily M, member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or vimentin. In some embodiments, the target antigen is EphA2 or B7-H3 (CD276). In some embodiments, the target antigen is EphA2. In some embodiments, the target antigen is B7-H3 (CD276). In some embodiments, the cancer is a tumor or a hematological malignancy. In some embodiments, the cancer is breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, stomach cancer or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer. In some embodiments, the sample is a tissue biopsy sample, a blood sample, or a bone marrow sample.

Methods for preparing the described ADC compounds and compositions are also disclosed. An exemplary embodiment is a method for preparing an antibody-drug conjugate by reacting an antibody or antigen-binding fragment with a cleavable conjugate linker linked or covalently attached to a panRAS inhibitor under conditions permitting conjugation.

Figure 1 shows the in vitro activity of panRAS ADC, isotype ADC, and sotorasib in multiple cancer cell lines (LU65, HPAC, H727, and SW1271).

The disclosed compositions and methods may be more readily understood by reference to the following detailed description taken in conjunction with the accompanying drawings, which form a part hereof.

Throughout this specification, the descriptions describe compositions and methods of using the compositions. Where this specification discloses or claims features or embodiments relating to a composition, such features or embodiments are equally applicable to methods of using the composition. Similarly, where this specification discloses or claims features or embodiments relating to methods of using a composition, such features or embodiments are equally applicable to the composition.

When expressing a range of values, this includes embodiments using any particular value within that range. Furthermore, reference to a value specified in a range includes each and every value within that range. All ranges include their endpoints and are combinable. When expressing values as approximations using the adjective "about," it will be understood that the particular value forms another embodiment. Reference to a specific numerical value includes at least that particular value unless the context clearly indicates otherwise. The use of "or" means "and/or," unless the context clearly indicates otherwise. All references cited herein are incorporated by reference for any purpose. In the event of a conflict between a reference and this specification, this specification shall control.

Unless the context of the description indicates otherwise, for example, when a structure or fragment of a structure is depicted without a symbol indicating a specific point(s) of connection, it may be used by itself or attached to other components of the ADC, and this may be done by using an antibody or antigen-binding fragment thereof attached to any suitable point of attachment to a chemical moiety, such as a linker-drug, in any orientation. However, when indicated, the components of the ADC are attached in the orientation illustrated in the given chemical formula. For example, if Chemical Formula 1 is described as Ab-(LD) _p and the group "-(LD)" is When described as, the elaborate structure of chemical formula 1 is This is. This is This is not it.

It should be understood that certain features of the disclosed compositions and methods, which are, for clarity, described in the context of individual embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed compositions and methods, which are, for brevity, described in the context of a single embodiment, may also be provided individually or in any subcombination.

As used throughout this application, antibody drug conjugates may be identified using the naming convention in the general format of "target antigen/antibody-linker-payload". By way of example only, if an antibody drug conjugate is referred to as "target X-L0-P0", the conjugate comprises an antibody that binds target X, a conjugate linker designated L0, and a payload designated P0. Alternatively, if an antibody drug conjugate is referred to as "anti-target X-L0-P0", the conjugate comprises an antibody that binds target X, a conjugate linker designated L0, and a payload designated P0. In yet another alternative, if an antibody drug conjugate is referred to as "AbX-L0-P0", the conjugate comprises an antibody designated AbX, a conjugate linker designated L0, and a payload designated P0. A control antibody drug conjugate comprising a non-specific isotype control antibody may be referred to as an “isotype control IgG1-L0-P0” or “IgG1-L0-P0”.

Any of the chemical formulas provided herein are also intended to represent unlabeled and isotopically labeled forms of the compounds. Isotopically labeled compounds have a structure represented by the chemical formulas provided herein, except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Isotopes that may be included in the compounds of the present invention include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, such as ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ F, and ³⁶ Cl. It should therefore be understood that the ^present invention includes compounds comprising one or more of any of the aforementioned isotopes, including, for example, radioactive isotopes such as 3 H and ¹⁴ C, or those in which non-radioactive isotopes such as ² H and ¹³ C are present. Such isotopically labeled compounds are useful for metabolic studies (using ¹⁴ C), reaction kinetic studies (e.g., using ² H or ³ H), detection or imaging techniques such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) (including drug or substrate tissue distribution analysis), or radiotherapy of patients. In particular, ¹⁸ F or labeled compounds may be particularly desirable for PET or SPECT studies. Isotopically labeled compounds can generally be prepared by conventional techniques known to those skilled in the art, for example, by substituting an appropriate isotopically labeled reagent for a previously used unlabeled reagent.

definition

Various terms relating to aspects of the description are used throughout this specification and claims. Unless otherwise indicated, these terms should be given their customary meanings in the art. Other specifically defined terms should be interpreted in a manner consistent with the definitions provided herein.

As used herein, the singular forms include plural forms unless the context clearly dictates otherwise. The terms "comprising," "having," "having a chemical formula," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise indicated. Additionally, whenever "comprising" or another open-ended term is used in an embodiment, it should be understood that the same embodiment may be more narrowly claimed using the intermediate term "consisting essentially of" or the closed term "consisting of."

The terms "about" or "approximately" as used in connection with values and ranges indicate a value or range that is close or near the recited value or range such that the embodiment may be performed as intended, as would be apparent to one of ordinary skill in the art from the teachings contained herein. In some embodiments, "about" means ±20%, 15%, 10%, 5%, 1%, 0.5%, or 0.1% of the numerical amount. In one embodiment, the term "about" refers to a range of values that are 10% more or less than a stated value. In another embodiment, the term "about" refers to a range of values that are 5% more or less than a stated value. In yet another embodiment, the term "about" refers to a range of values that are 1% more or less than a stated value.

The terms "antibody-drug conjugate," "antibody conjugate," "conjugate," "immunoconjugate," and "ADC" are used interchangeably and refer to one or more therapeutic compounds (e.g., panRAS inhibitors) linked to one or more antibodies or antigen-binding fragments. In some embodiments, the ADC is defined by the general formula 1:

[Chemical Formula 1]

Ab-(LD) _p [wherein Ab = antibody or antigen-binding fragment (e.g., anti-EphA2 antibody or anti-B7-H3 antibody, or antigen-binding fragment thereof), L = conjugate linker moiety, D = drug moiety (e.g., panRAS inhibitor drug moiety), and p = number of drug moieties per antibody or antigen-binding fragment]. In an ADC comprising a panRAS inhibitor drug moiety, “ p ” represents the number of panRAS inhibitor compounds linked to the antibody or antigen-binding fragment.

The term "antibody" is used in the broadest sense to refer to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or combination thereof, through one or more antigen recognition sites within the variable region of the immunoglobulin molecule. Antibodies may be polyclonal or monoclonal, multichain or singlechain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. An "intact" antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibodies can be monoclonal, human, humanized, camelid, or chimeric. The antibody may be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The antibody may be an intact antibody or an antigen-binding fragment thereof.

In some embodiments, the antibodies or antibody fragments disclosed herein comprise modified or engineered amino acid residues, such as one or more cysteine residues, as conjugation sites for a drug moiety (Junutula JR, et al., Nat Biotechnol 2008, 26:925-932). In one embodiment, the present invention provides modified antibodies or antibody fragments comprising cysteine substitutions of one or more amino acids at positions described herein. Because the cysteine substitution sites are present in the constant region of the antibody or antibody fragment, they can be applied to a variety of antibodies or antibody fragments, and these sites are selected to provide stable and homogeneous conjugates. The modified antibodies or fragments may have one, two, or more cysteine substitutions, and these substitutions may be used in combination with other modifications and conjugation methods described herein. Methods for inserting cysteines into specific positions in antibodies are known in the art (see, e.g., Lyons et al., (1990) Protein Eng., 3:703-708, WO 2011/005481, WO2014/124316, WO 2015/138615). In certain embodiments, the modified antibody comprises a heavy chain of the antibody selected from positions 117, 119, 121, 124, 139, 152, 153, 155, 157, 164, 169, 171, 174, 189, 191, 195, 197, 205, 207, 246, 258, 269, 274, 286, 288, 290, 292, 293, 320, 322, 326, 333, 334, 335, 337, 344, 355, 360, 375, 382, 390, 392, 398, 400, and 422. A cysteine substitution of one or more amino acids in the constant region (positions numbered according to the EU system). In some embodiments, the modified antibody or antibody fragment comprises a cysteine substitution of one or more amino acids in the constant region selected from positions 107, 108, 109, 114, 129, 142, 143, 145, 152, 154, 156, 159, 161, 165, 168, 169, 170, 182, 183, 197, 199, and 203 of a light chain of the antibody or antibody fragment (positions numbered according to the EU system), wherein the light chain is a human kappa light chain. In a specific embodiment, the modified antibody or antibody fragment thereof comprises a combination of cysteine substitutions of two or more amino acids in the constant region, wherein the combination comprises a substitution at position 375 of the antibody heavy chain, position 152 of the antibody heavy chain, position 360 of the antibody heavy chain, or position 107 of the antibody light chain (positions numbered according to the EU system). In a specific embodiment, the modified antibody or antibody fragment thereof comprises a cysteine substitution of one amino acid in the constant region, wherein the substitution is at position 375 of the antibody heavy chain, position 152 of the antibody heavy chain, position 360 of the antibody heavy chain, position 107 of the antibody light chain, position 165 of the antibody light chain, or position 159 of the antibody light chain (positions numbered according to the EU system), wherein the light chain is a human kappa chain. In a specific embodiment, the modified antibody or antibody fragment thereof comprises a combination of two amino acid cysteine substitutions in the constant region, the combination comprising a substitution at position 375 of the antibody heavy chain and a substitution at position 152 of the antibody heavy chain (positions numbered according to the EU system). In a specific embodiment, the modified antibody or antibody fragment thereof comprises a cysteine substitution of one amino acid at position 360 of the antibody heavy chain (positions numbered according to the EU system). In another specific embodiment, the modified antibody or antibody fragment thereof comprises a cysteine substitution of one amino acid at position 107 of the antibody light chain (positions numbered according to the EU system), wherein the light chain is a kappa chain.

As used herein, the term "antibody fragment" or "antigen-binding fragment" or "functional antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution) with an epitope of an antigen (e.g., EphA2 or B7-H3 (CD276)). The antigen-binding fragment may also retain the ability to internalize into antigen-expressing cells. In some embodiments, the antigen-binding fragment also retains immune effector activity. The terms antibody, antibody fragment, antigen-binding fragment, and the like are intended to encompass the use of binding domains from antibodies in the context of larger macromolecules, such as ADCs. Fragments of full-length antibodies have been shown to be capable of performing the antigen-binding function of full-length antibodies. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , Fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multispecific antibodies formed from antibody fragments such as bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs or other epitope binding fragments of antibodies. Antigen-binding fragments may also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, bispecific or multispecific antibody constructs, ADCs, v-NARs and bis-scFvs (see, e.g., Holliger and Hudson (2005) Nat Biotechnol. 23(9):1126-36). Antigen-binding fragments may also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see, e.g., U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies). The term "scFv" refers to a fusion protein comprising at least one antigen-binding fragment comprising a variable region of a light chain and at least one antigen-binding fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and can be expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and an scFv may comprise VL-linker-VH or VH-linker-VL. Antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and these binding fragments are screened for utility (e.g., binding affinity, internalization) in the same manner as intact antibodies. For example, antigen-binding fragments can be prepared by cleavage of the intact protein, for example, by proteases or chemical cleavage.

As used herein, the term "complementarity determining region" or "CDR" refers to the sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. For example, there are typically three CDRs within each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs within each light chain variable region (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR are described in Kabat et al. (1991) "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD" ("Kabat" numbering system); Al-Lazikani et al. (1997) J Mol Biol. 273(4):927-48" ("Chothia" numbering system); ImmunoGenTics (IMGT) numbering (Lefranc (2001) Nucleic Acids Res. 29(1):207-9; Lefranc et al. (2003) Dev Comp Immunol. 27(1):55-77) (“IMGT” numbering system); or a combination thereof. For the combined Kabat and Chothia numbering system for a given CDR region (e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, or LC CDR3), in some embodiments, the CDR corresponds to the amino acid residues defined as part of a Kabat CDR together with the amino acid residues defined as part of a Chothia CDR. As used herein, CDRs defined according to the “Chothia” numbering system are sometimes referred to as “hypervariable loops.”

In some embodiments, under Kabat, the CDR amino acid residues within the heavy chain variable domain (VH) are numbered 31-35 (HCDR1) (e.g., insertion(s) after position 35), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues within the light chain variable domain (VL) are numbered 24-34 (LCDR1) (e.g., insertion(s) after position 27), 50-56 (LCDR2), and 89-97 (LCDR3). In some embodiments, under Chothia, the CDR amino acids within the VH are numbered 26-32 (HCDR1) (e.g., insertion(s) after position 31), 52-56 (HCDR2), and 95-102 (HCDR3); Amino acid residues within the VL are numbered 26-32 (LCDR1) (e.g., insertion(s) after position 30), 50-52 (LCDR2), and 91-96 (LCDR3). In some embodiments, by combining the CDR definitions of both Kabat and Chothia, the CDR comprises or consists of, for example, amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) within a human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) within a human VL. In some embodiments, under IMGT, the CDR amino acid residues within the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues within the VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). In some embodiments, under IMGT, the CDR regions of an antibody can be determined using the IMGT/DomainGap Align program.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies included in the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific antibodies directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically comprise a plurality of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention may be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be produced by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, in Clackson et al. (1991) Nature 352:624-8 and Marks et al. (1991) J Mol Biol. 222:581-97. The term also encompasses antibody molecule preparations of single molecule composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The monoclonal antibodies described herein may be non-human, human, or humanized antibodies. This term specifically includes "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as such fragments specifically bind to the target antigen and/or exhibit the desired biological activity.

As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having the amino acid sequence of an antibody produced by a human. The term includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such human sequences, e.g., a human germline sequence, or a mutated version of a human germline sequence, or an antibody containing a consensus framework sequence derived from human framework sequence analysis, as described, e.g., in Knappik et al. ((2000) J Mol Biol. 296(1):57-86). The structure and location of immunoglobulin variable domains, e.g., CDRs, can be defined using well-known numbering systems, e.g., the Kabat numbering system, the Chothia numbering system, or a combination of Kabat and Chothia, and/or ImMunoGenTics (IMGT) numbering. The human antibodies of the present invention may include amino acid residues not encoded by human sequences (e.g., conservative substitutions to enhance stability or production, or mutations introduced in vivo by somatic mutation or in vitro by random or site-specific mutagenesis). However, as used herein, the term "human antibody" is not intended to encompass antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

As used herein, the term "recombinant human antibody" refers to any human antibody prepared, expressed, created, or isolated by recombinant means, including antibodies isolated from animals (e.g., mice) that have been transgenic or transgenic for human immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed to express human antibodies, e.g., transfectomas, antibodies isolated from recombinant, combinatorial human antibody libraries, and antibodies prepared, expressed, created, or isolated by any other means that involves splicing all or part of the human immunoglobulin gene sequences to another DNA sequence. Such recombinant human antibodies have variable regions in which the framework regions and CDR regions are derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that are derived from and related to human germline VH and VL sequences, but may not be natively present within the human antibody germline repertoire in vivo.

As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. In some cases, the variable regions of both the heavy and light chains correspond to those of an antibody from one species having the desired specificity, affinity, and activity, while the constant regions are homologous to antibodies from another species (e.g., humans) to minimize an immune response in the latter species.

As used herein, the term "humanized antibody" refers to a form of an antibody that contains sequences derived from a non-human (e.g., murine) antibody and a human antibody. Such antibodies are a type of chimeric antibody that contains minimal sequence derived from a non-human immunoglobulin. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions correspond to those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. A humanized antibody may be further modified by substitution of residues within the Fv framework region and/or by replacing non-human residues to improve and optimize antibody specificity, affinity, and/or activity.

As used herein, the term "Fc region" refers to a polypeptide comprising at least a portion of the CH3, CH2, and hinge regions of the constant domains of an antibody. Optionally, the Fc region may comprise a CH4 domain present in some antibody classes. The Fc region may comprise the entire hinge region of the constant domain of an antibody. In some embodiments, the antibody or antigen-binding fragment comprises an Fc region and a CH1 region of an antibody. In some embodiments, the antibody or antigen-binding fragment comprises an Fc region CH3 region of an antibody. In some embodiments, the antibody or antigen-binding fragment comprises an Fc region, a CH1 region, and a kappa/lambda region from an antibody constant domain. In some embodiments, the antibody or antigen-binding fragment comprises a constant region, e.g., a heavy chain constant region and/or a light chain constant region. In some embodiments, such constant regions are modified compared to a wild-type constant region. That is, the polypeptide may comprise alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and/or the light chain constant region domain (CL). Examples of modifications include the addition, deletion, or substitution of one or more amino acids in one or more domains. Such changes may be included to optimize effector function, half-life, etc.

As used herein with respect to an antibody or antigen-binding fragment, “internalization” refers to an antibody or antigen-binding fragment that, upon binding to a cell, can be taken up through the lipid bilayer membrane of the cell, preferably into a catabolic compartment within the cell (i.e., “internalized”). For example, an internalizing anti-EphA2 antibody is one that can bind to EphA2 on the cell membrane and then be taken up into the cell. In some embodiments, the antibody or antigen-binding fragment used in the ADCs disclosed herein targets a cell surface antigen (e.g., EphA2 or B7-H3 (CD276)) and is an internalizing antibody or internalizing antigen-binding fragment (i.e., the ADC is transported through the cell membrane after antigen binding). In some embodiments, the internalizing antibody or antigen-binding fragment binds to a receptor on the cell surface. An internalizing antibody or internalizing antigen-binding fragment that targets a receptor on the cell membrane can induce receptor-mediated endocytosis. In some embodiments, the internalized antibody or internalized antigen-binding fragment is taken up into the cell via receptor-mediated endocytosis.

As used herein, "non-internalizing" with respect to an antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment that remains on the cell surface upon binding to the cell. In some embodiments, the antibody or antigen-binding fragment used in the ADCs disclosed herein targets a cell surface antigen and is a non-internalizing antibody or antigen-binding fragment (i.e., the ADC remains on the cell surface and is not transported through the cell membrane after antigen binding). In some embodiments, the non-internalizing antibody or antigen-binding fragment binds to a non-internalizing receptor or other cell surface antigen. Exemplary non-internalized cell surface antigens include, but are not limited to, CA125 and CEA, and antibodies that bind to non-internalized antigen targets are also known in the art (see, e.g., Bast et al. (1981) J Clin Invest. 68(5):1331-7; Scholler and Urban (2007) Biomark Med. 1(4):513-23; and Boudousq et al. (2013) PLoS One 8(7):e69613).

The terms "EPH receptor A2," "ephrin type A receptor 2," and "EphA2" are used interchangeably herein and refer to any native form of human EphA2. The term encompasses full-length human EphA2 (e.g., NCBI Reference Sequence: NP_004422.2; SEQ ID NO: 1) and any form of human EphA2 that can result from cellular processing. The term also encompasses functional variants or fragments of human EphA2, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biological functions of human EphA2 (i.e., variants and fragments are included unless the context indicates that the term is being used to refer only to the wild-type protein). EphA2 can be isolated from a human or can be produced recombinantly or by synthetic methods.

As used herein, the term "anti-EphA2 antibody" or "antibody that binds to EphA2" refers to any form of antibody or antigen-binding fragment thereof that binds, e.g., specifically, to EphA2. This term encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antigen-binding fragments thereof, so long as they bind, e.g., specifically, to EphA2. WO2007/030642 provides exemplary EphA2-binding sequences comprising exemplary anti-EphA2 antibody sequences and is incorporated herein by reference. In some embodiments, the anti-EphA2 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antigen-binding fragment. 1C1 (WO2007/030642) is an example of an exemplary anti-EphA2 antibody.

The terms "B7 homology 3 protein", "B7-H3" and "CD276" are used interchangeably herein and refer to any native form of human B7-H3 or CD276. The term encompasses full-length human B7-H3 (CD276) (e.g., NCBI Reference Sequence: NP_001019907.1) and any form of human B7-H3 (CD276) that may result from cellular processing. The term also encompasses functional variants or fragments of human B7-H3, including but not limited to splice variants, allelic variants and isoforms that retain one or more biological functions of human B7-H3 (CD276) (i.e., variants and fragments are included unless the context indicates that the term is used to refer only to the wild-type protein). B7-H3 (CD276) can be isolated from humans or produced recombinantly or by synthetic methods.

As used herein, the term "anti-B7-H3 antibody" or "antibody that binds to B7-H3 (CD276)" refers to any form of antibody or antigen-binding fragment thereof that binds, e.g., specifically, to B7-H3 (CD276). The term encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antigen-binding fragments thereof, so long as they bind, e.g., specifically, to B7-H3 (CD276). WO 2017214322 and WO 2012147713 provide exemplary B7-H3-binding sequences, including exemplary anti-B7-H3 (CD276) antibody sequences, and are incorporated herein by reference. ABBV-155 and DS-5573a are examples of exemplary anti-B7-H3 (CD276) antibodies.

As used herein, the term "binding specificity" refers to the ability of an individual antibody or antigen-binding fragment to preferentially react with one antigenic determinant over others. The degree of specificity indicates the extent to which an antibody or fragment preferentially binds to one antigenic determinant over others. Furthermore, as used herein, the terms "specific,""bindsspecifically," and "binds specifically" refer to the binding reaction between an antibody or antigen-binding fragment (e.g., an anti-EphA2 antibody or an anti-B7-H3 antibody) and a target antigen (e.g., EphA2 or B7-H3 (CD276)) in a heterogeneous population of proteins and other biological agents. An antibody can be tested for specificity of binding by comparing its binding to an appropriate antigen with its binding to an irrelevant antigen or mixture of antigens under a given set of conditions. An antibody is considered specific if it binds to an appropriate antigen with an affinity that is at least 2-fold, 5-fold, 7-fold, 10-fold, or greater than that it binds to an irrelevant antigen or mixture of antigens. A “specific antibody” or “target-specific antibody” is one that binds only to a target antigen (e.g., EphA2 or B7-H3 (CD276)) and does not bind (or exhibits minimal binding to) other antigens. In some embodiments, an antibody or antigen-binding fragment that specifically binds to a target antigen (e.g., EphA2 or B7-H3 (CD276)) has a K D of less than 1x10 ^-6 M, less than 1x10 ^-7 M, less than 1x10 ^-8 M, less than 1x10 ^-9 M, less than 1x10 ^-10 M, less than 1x10 ^-11 M, less than 1x10 ^-12 M, or less than 1x10 ^-13 M. In some embodiments, _{the K D is} from 1 pM to 500 pM. In some embodiments, the K _D is 500 pM to 1 μM, 1 μM to 100 nM, or 100 mM to 10 nM.

As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Without wishing to be bound by theory, within each antigen-binding site, the variable regions of the antibody "arms" interact with the antigen at many sites through weak noncovalent forces; the more interactions, the stronger the affinity typically is. The binding affinity of an antibody is the sum of the attractive and repulsive forces acting between the antigenic determinant and the antibody binding site.

The term "k _on " or "k _a " refers to the on-rate constant for the association of an antibody to an antigen to form an antibody/antigen complex. The rate can be determined using standard analytical methods such as surface plasmon resonance, biolayer interferometry, or ELISA assays.

The term "k _off " or "k _d " refers to the off-rate constant for the dissociation of an antibody from an antibody/antigen complex. The rate can be determined using standard analytical methods such as surface plasmon resonance, biolayer interferometry, or ELISA assays.

The term "K _D " refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. K _D is calculated as k _a /k _d . The rate can be determined using standard analytical methods such as surface plasmon resonance, biolayer interferometry, or ELISA assays.

The term "epitope" refers to a portion of an antigen that can be recognized and specifically bound by an antibody (or antigen-binding fragment). Epitope determinants typically consist of chemically active surface groups of a molecule, such as amino acids or carbohydrate or sugar side chains, and may have specific three-dimensional structural properties as well as specific charge characteristics. If the antigen is a polypeptide, the epitope may be formed from contiguous or discontinuous amino acids juxtaposed by tertiary folding of the polypeptide. Epitopes may be "linear" or "conformational." Conformational and linear epitopes are distinguished by the fact that binding to the former is lost in the presence of denaturing solvents, whereas the latter is not. The epitope bound by an antibody (or antigen-binding fragment) can be identified using any epitope mapping technique known in the art, including X-ray crystallography for epitope identification by direct visualization of the antigen-antibody complex, as well as monitoring binding of the antibody to fragments or mutant variants of the antigen or monitoring the solvent accessibility of the antibody and other portions of the antigen. Exemplary strategies used to map antibody epitopes include, but are not limited to, array-based oligopeptide scanning, limited proteolysis, site-directed mutagenesis, high-throughput mutagenesis mapping, hydrogen-deuterium exchange, and mass spectrometry (see, e.g., Gershoni et al. (2007) BioDrugs 21:145-56; and Hager-Braun and Tomer (2005) Expert Rev Proteomics 2:745-56).

Competitive binding and epitope binning can also be used to determine antibodies that share identical or overlapping epitopes. Competitive binding can be assessed using a cross-blocking assay, such as that described in "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory, Harlow and Lane ( ^1st edition 1988, ^2nd edition 2014). In some embodiments, competitive binding is identified when a test antibody or binding protein reduces binding of a reference antibody or binding protein (e.g., a binding protein comprising CDRs and/or variable domains selected from those identified in Tables 3-5) to a target antigen, such as EphA2 or B7-H3 (CD276), and/or vice versa, by at least about 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or more, or percentages therebetween) in a cross-blocking assay. In some embodiments, competitive binding may be due to shared or similar (e.g., partially overlapping) epitopes, or due to steric hindrance of the antibody or binding protein to a nearby epitope (see, e.g., Tzartos, Methods in Molecular Biology (Morris, ed. (1998) vol. 66, pp. 55-66)). In some embodiments, competitive binding may be used to group binding proteins that share similar epitopes. For example, binding proteins that compete for binding may be "binned" into a group of binding proteins with overlapping or nearby epitopes, while those that do not compete are placed into a separate group of binding proteins that do not have overlapping or nearby epitopes.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to polymers of amino acid residues. The terms encompass both naturally occurring and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, and amino acid polymers comprising two or more amino acids linked to each other by peptide bonds. The terms specifically encompass, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. The terms also encompass natural peptides, recombinant peptides, synthetic peptides, or any combination thereof. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

A "recombinant" protein refers to a protein (e.g., an antibody) made using recombinant technology, for example, by expression of a recombinant nucleic acid.

An "isolated" protein refers to a protein that is free of at least some of the materials with which it is normally associated in its natural state. For example, a naturally occurring polynucleotide or polypeptide present in a living organism is not isolated, but rather a polynucleotide or polypeptide that has been separated from some or all of the materials with which it coexists in the living organism is isolated. This definition encompasses the production of antibodies in a wide variety of organisms and/or host cells known in the art.

As used herein, an "isolated antibody" is an antibody that has been identified and separated (by weight) from one or more (e.g., a majority) components of its source environment, e.g., from components of a hybridoma cell culture or a different cell culture used for its production. In some embodiments, the separation is performed to sufficiently remove components that may interfere with the suitability of the antibody for the desired application (e.g., therapeutic use). Methods for preparing isolated antibodies are known in the art and include, without limitation, protein A chromatography, anion exchange chromatography, cation exchange chromatography, virus retention filtration, and ultrafiltration.

As used herein, the term "variant" refers to a nucleic acid sequence or amino acid sequence that differs from a reference nucleic acid sequence or amino acid sequence, respectively, but retains one or more biological properties of the reference sequence. A variant may contain one or more amino acid substitutions, deletions, and/or insertions (or corresponding substitutions, deletions, and/or insertions of codons) relative to the reference sequence. The changes in a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid sequence, or may result in amino acid substitutions, additions, deletions, fusions, and/or truncations. In some embodiments, the nucleic acid variants disclosed herein encode the same amino acid sequence as that encoded by the unmodified nucleic acid, or encode a modified amino acid sequence that retains one or more functional properties of the unmodified amino acid sequence. The sequence changes in peptide variants are typically limited or conservative, such that the sequences of the unmodified peptide and the variant are overall very similar and identical in many regions. In some embodiments, the peptide variant retains one or more functional properties of the unmodified peptide sequence. The unmodified peptide of the variant may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination.

Variants of nucleic acids or peptides may be naturally occurring variants or variants not known to occur naturally. Variants of nucleic acids and peptides may be prepared by mutagenesis techniques, direct synthesis, or other techniques known in the art. Variants do not necessarily require physical manipulation of the reference sequence. A sequence is considered a "variant" regardless of the method of synthesis as long as it contains different nucleic acids or amino acids compared to the reference sequence. In some embodiments, a variant has high sequence identity (i.e., at least 60% nucleic acid or amino acid sequence identity) compared to the reference sequence. In some embodiments, a peptide variant includes a polypeptide having amino acid substitutions, deletions, and/or insertions (e.g., variants that also retain one or more functions of the reference sequence), so long as the polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the reference sequence, or a corresponding fragment (e.g., a functional fragment) of the reference sequence. In some embodiments, a nucleic acid variant includes a polynucleotide having amino acid substitutions, deletions, and/or insertions, so long as the polynucleotide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleic acid sequence identity to a reference sequence, or a corresponding segment (e.g., a functional fragment) of a reference sequence.

The term "conservatively modified variant" applies to both amino acid sequences and nucleic acid sequences. In nucleic acid sequences, a conservatively modified variant refers to a nucleic acid that encodes the same or essentially the same amino acid sequence. Due to the degeneracy of the genetic code, multiple functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at any position where an alanine is specified by a codon, that codon can be changed to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," a type of conservatively modified variation. All nucleic acid sequences herein that encode polypeptides also describe all possible silent variations of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is typically the sole codon for methionine, and TGG, which is typically the sole codon for tryptophan) can be modified to produce a functionally identical molecule. Therefore, each silent mutation in a nucleic acid encoding a polypeptide is inherent in each described sequence. In a polypeptide sequence, conservatively modified variants include individual substitutions, deletions, or additions to the polypeptide sequence that replace a given amino acid with a chemically similar amino acid. Conservative substitutions that provide functionally similar amino acids are well known in the art.

As used herein, the term "conservative sequence modification" refers to amino acid modifications that do not significantly affect or alter, for example, the binding properties of an antibody or antigen-binding fragment comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into an antibody or antigen-binding fragment using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in some embodiments, one or more amino acid residues in an antibody can be replaced with another amino acid residue from the same side chain family, and the altered antibody can be tested using the functional assays described herein.

As used herein, the term "homology" or "identity" refers to the subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. If a subunit position in both molecules is occupied by the same monomer subunit, e.g., if one position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of positions that are matched or homologous. For example, if half of the positions in two sequences are matched or homologous (e.g., 5 positions in a polymer that is 10 subunits in length), the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, the two sequences are 90% homologous.

The percentage of "sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, wherein a fragment of the amino acid sequence in the comparison window may include additions or deletions (e.g., gaps or overhangs) compared to a reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the same amino acid residue appears in the two sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity. The result is the percent identity of the subject sequence to the query sequence. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences, and the length of each gap. In general, the amino acid identity or homology between the proteins disclosed herein and variants thereof (including variants of target antigens (e.g., EphA2 or B7-H3 (CD276)) and variants of antibody variable domains (including individual variant CDRs)) is at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, nearly 100%, or 100% identity or homology) with respect to the sequences depicted herein.

Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some embodiments, the percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch ((1970) J Mol Biol. 48:444-53), which is included in the GAP program of the GCG software package, using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In some embodiments, the percent identity between two nucleotide sequences is determined using the GAP program of the GCG software package using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. An exemplary parameter set is a Blosum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of Meyers and Miller ((1989) CABIOS 4:11-17) included in the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

The term "formulation" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, an extract prepared from a biological material, or a combination of two or more thereof. The term "therapeutic agent" or "drug" refers to an agent capable of modulating a biological process and/or possessing biological activity. PanRAS inhibitors and ADCs comprising the same, as described herein, are exemplary therapeutic agents.

The term "chemotherapeutic agent" or "anticancer agent" is used herein to refer to any agent that is effective in treating cancer (regardless of its mechanism of action). Inhibition of metastasis or angiogenesis is a frequent characteristic of chemotherapeutic agents. Chemotherapeutic agents include antibodies, biological molecules, and small molecules, including panRAS inhibitors and ADCs comprising them, as described herein. Chemotherapeutic agents may be cytotoxic or cytostatic agents. The term "cytostatic agent" refers to an agent that inhibits or suppresses cell growth and/or cell proliferation. The term "cytotoxic agent" refers to a substance that causes cell death, primarily by interfering with cell expression, activity, and/or function.

As used herein, the term "rat sarcoma virus (Ras)" or "panRAS" refers to any native form of the human Ras protein family (e.g., K-Ras (including splice variants KRAS4A and KRAS4B), H-Ras, and N-Ras). The term includes full-length human K-Ras (Kristen rat sarcoma virus) (e.g., UniProt Reference Sequence: P01116; SEQ ID NO: 64), H-Ras (Harvey rat sarcoma virus) (e.g., UniProt Reference Sequence: P01112; SEQ ID NO: 65), N-Ras (neuroblastoma rat sarcoma virus) (e.g., UniProt Reference Sequence: P01111; SEQ ID NO: 66), and any form of human Ras that can be generated by cell processing. The term also encompasses functional variants or fragments of a human Ras protein, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biological functions of the human Ras protein (i.e., variants and fragments are included unless the context indicates that the term is used to refer only to the wild-type protein). The Ras protein may be isolated from a human, or may be produced recombinantly or by synthetic methods. Exemplary Ras protein amino acid sequences are listed in Table C below.

Table C. Exemplary RAS amino acid sequences

As used herein, the terms "inhibit," "suppression," or "inhibiting" mean reducing a biological activity or process by a measurable amount, and may include, but does not require, complete prevention or inhibition. In some embodiments, "inhibition" means reducing the expression and/or activity of panRAS and/or one or more of its upstream regulators or downstream targets.

As used herein, the term “panRAS inhibitor” refers to an agent capable of reducing the expression and/or activity of panRAS (e.g., K-Ras (including splice variants KRAS4A and KRAS4B), H-Ras, and N-Ras) and/or one or more upstream regulators or downstream targets thereof. Exemplary panRAS modulators (including exemplary panRAS inhibitors) that can be incorporated as drug moieties into the disclosed ADCs are described in WO2021/091956 or WO2022/060836 (each of which is incorporated herein by reference).

As used herein, "panRAS inhibitor drug moiety," "panRAS inhibitor," and the like, refer to a component of an ADC or composition, which provides the structure of a panRAS inhibitor compound or a compound modified to be attached to an ADC, and which retains essentially the same, similar, or improved biological function or activity as compared to the original compound. In some embodiments, the panRAS inhibitor drug moiety is component (D) in an ADC of Formula 1.

As used herein, the term "cancer" refers to the presence of cells that possess typical characteristics of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic capacity, rapid growth and proliferation rate, and/or specific morphological characteristics. Cancer cells may commonly exist in the form of a tumor or mass, but such cells may also exist alone within a subject or circulate in the bloodstream as independent cells, such as leukemia cells or lymphoma cells. The term "cancer" includes all types of cancer and cancer metastases, including blood cancers, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumor cancers. Blood cancers may include B-cell malignancies, blood cancers (leukemias), plasma cell cancers (myelomas, e.g., multiple myeloma), or cancers of the lymph nodes (lymphomas). Exemplary B-cell malignancies include chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma. Leukemia may include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), and acute monocytic leukemia (AMoL). The terms "acute lymphoblastic leukemia" and "acute lymphocytic leukemia" may be used interchangeably to describe ALL. Lymphoma may include Hodgkin's lymphoma, non-Hodgkin's lymphoma, and others. Other blood cancers may include myelodysplastic syndromes (MDS). The solid tumor may include a carcinoma, such as an adenocarcinoma, for example, breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, stomach cancer or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer.

As used herein, the term "tumor" refers to any tissue mass resulting from excessive cell growth or proliferation, benign or malignant, including precancerous lesions. In some embodiments, the tumor is breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, stomach cancer or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer.

The terms "tumor cell" and "cancer cell" are used interchangeably herein and refer to individual cells or entire populations of cells derived from a tumor or cancer, including both non-tumorigenic cells and cancer stem cells. The terms "tumor cell" and "cancer cell" will be modified with the term "non-tumorigenic" when referring only to cells lacking the capacity for renewal and differentiation, to distinguish these cells from cancer stem cells.

As used herein, the terms "target-negative," "target antigen-negative," or "antigen-negative" refer to the absence of target antigen expression by a cell or tissue. The terms "target-positive," "target antigen-positive," or "antigen-positive" refer to the presence of target antigen expression. For example, a cell or cell line that does not express a target antigen may be described as target-negative, while a cell or cell line that expresses a target antigen may be described as target-positive.

The terms "subject" and "patient" are used interchangeably herein to refer to any human or non-human animal in need of treatment. Non-human animals include all vertebrates (e.g., mammals and non-mammals), including any mammal. Non-limiting examples of mammals include humans, chimpanzees, apes, monkeys, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, and guinea pigs. Non-limiting examples of non-mammals include birds and fish. In some embodiments, the subject is a human.

As used herein, the term "subject in need of treatment" refers to a subject who benefits biologically, medically, or quality of life from treatment (e.g., treatment with any one or more exemplary ADC compounds described herein).

As used herein, the terms "treat," "treating," or "treatment" refer to any improvement in any outcome of a disease, disorder, or condition, such as prolonging survival, reducing morbidity, and/or reducing side effects, resulting from an alternative treatment modality. In some embodiments, treatment comprises delaying or ameliorating the disease, disorder, or condition (i.e., slowing, arresting, or reducing the onset of the disease or at least one of its clinical symptoms). In some embodiments, treatment comprises delaying, alleviating, or ameliorating at least one physical parameter of the disease, disorder, or condition, including parameters that may not be discernible to the patient. In some embodiments, treatment comprises modulating the disease, disorder, or condition physically (e.g., stabilizing discernible symptoms), physiologically (e.g., stabilizing physical parameters), or both. In some embodiments, treatment comprises administering to a subject, e.g., a patient, an ADC compound or composition described herein to obtain a therapeutic benefit as recited herein. Treatment can be to cure, heal, alleviate, delay, prevent, reduce, alter, correct, ameliorate, alleviate, ameliorate, or influence a disease, disorder, or condition (e.g., cancer), a symptom of a disease, disorder, or condition (e.g., cancer), or a predisposition to a disease, disorder, or condition (e.g., cancer). In some embodiments, in addition to treating a subject having a disease, disorder, or condition, the compositions disclosed herein can also be provided prophylactically to prevent or reduce the likelihood of developing the disease, disorder, or condition.

As used herein, the terms "prevent," "preventing," or "prevention" of a disease, disorder, or condition refers to the prophylactic treatment of the disease, disorder, or condition; or the delay in the onset or progression of the disease, disorder, or condition.

As used herein, a "pharmaceutical composition" refers to a preparation of a composition, e.g., an ADC compound or composition, in addition to at least one other (and optionally more than one) ingredient suitable for administration to a subject, such as a pharmaceutically acceptable carrier, stabilizer, diluent, dispersant, suspending agent, thickener, and/or excipient. The pharmaceutical compositions provided herein are in a form that permits administration and subsequently provides the intended biological activity of the active ingredient(s) and/or achieves a therapeutic effect. The pharmaceutical compositions provided herein preferably do not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is to be administered.

As used herein, the terms "pharmaceutically acceptable carrier" and "physiologically acceptable carrier," which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered ADC compound or composition and/or any additional therapeutic agent in the composition. A pharmaceutically acceptable carrier may enhance or stabilize the composition, or may be used to facilitate the manufacture of the composition. Pharmaceutically acceptable carriers may include solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterials, antifungals), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, glidants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated. The carrier may be selected to minimize side effects in the subject and/or to minimize degradation of the active ingredient(s). Adjuvants may also be included in any of these formulations.

As used herein, the term "excipient" refers to a formulation material added to a pharmaceutical composition to facilitate administration of the active ingredient. Formulations for parenteral administration may contain, for example, excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, or hydrogenated naphthalenes. Other exemplary excipients include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and various types of starches, cellulose derivatives, gelatin, ethylene-vinyl acetate copolymer particles, and surfactants, including, for example, polysorbate 20.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt that does not interfere with the biological activity and properties of the compounds of the present invention and is not significantly irritating to the subject to which it is administered. Examples of such salts include, but are not limited to: (a) acid addition salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like; and salts formed with organic acids, such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (b) salts formed from anions of elements such as chlorine, bromine, and iodine. See, for example, Haynes et al., "Commentary: Occurrence of Pharmaceutically Acceptable Anions and Cations in the Cambridge Structural Database," J. Pharmaceutical Sciences, vol. 94, no. 10 (2005), and Berge et al., "Pharmaceutical Salts," J. Pharmaceutical Sciences, vol. 66, no. 1 (1977), which are incorporated herein by reference.

In some embodiments, depending on the electronic charge, the antibody-drug conjugates (ADCs), conjugate linkers, payloads, and linker-payloads described herein may contain a monovalent anionic counterion M ₁ ^- . Any suitable anionic counterion may be used. In certain embodiments, the monovalent anionic counterion is a pharmaceutically acceptable monovalent anionic counterion. In certain embodiments, the monovalent anionic counterion M ₁ ^- may be selected from bromide, chloride, iodide, acetate, trifluoroacetate, benzoate, mesylate, tosylate, triflate, formate, and the like. In some embodiments, the monovalent anionic counterion M ₁ ^- is trifluoroacetate or formate.

As used herein, the term “therapeutically effective amount” or “therapeutically effective dose” refers to an amount of a compound described herein, e.g., an ADC compound or composition described herein, that will produce a desired therapeutic result (i.e., reduction or inhibition of enzyme or protein activity, improvement of symptoms, alleviation of symptoms or conditions, delay in disease progression, reduction in tumor size, inhibition of tumor growth, prevention of metastasis). In some embodiments, a therapeutically effective amount does not induce or cause undesirable side effects. In some embodiments, a therapeutically effective amount induces or causes only those side effects that are acceptable to the treating clinician in light of the patient’s condition. In some embodiments, a therapeutically effective amount is effective in detectable killing, reduction, and/or inhibition of the growth or spread of cancer cells, the size or number of tumors, and/or other measures of the level, stage, progression, and/or severity of the cancer. The term also applies to a dose that induces a specific response in target cells, e.g., reduction, slowing, or inhibition of cell growth. A therapeutically effective amount can be determined by initially administering a low dose and then increasing the dose incrementally until the desired effect is achieved. The therapeutically effective amount may also vary depending on the intended application (in vitro or in vivo), or the subject and disease state being treated, such as the subject's body weight and age, the severity of the disease state, the mode of administration, etc., and can be readily determined by one of ordinary skill in the art. The specific amount may vary depending on, for example, the particular pharmaceutical composition, the subject and their age and pre-existing health condition or risks to the health condition, the dosing regimen to be followed, the severity of the disease, whether it is administered in combination with other agents, the timing of administration, the tissue to which it is administered, and the physical delivery system through which it is delivered. In cancer, a therapeutically effective amount of an ADC may reduce the number of cancer cells, reduce tumor size, inhibit (e.g., slow or stop) tumor metastasis, inhibit (e.g., slow or stop) tumor growth, and/or reduce one or more symptoms.

As used herein, the term "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a compound disclosed herein, e.g., an ADC compound or composition described herein, effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Generally, because a prophylactic dose is used in a subject prior to or at an early stage of disease, the prophylactically effective amount will be less than a therapeutically effective amount. In some embodiments, the prophylactically effective amount can prevent the onset of disease symptoms, including symptoms associated with cancer.

The term " p " or "drug loading" or "drug:antibody ratio" or "drug to antibody ratio" or "DAR" refers to the number of drug moieties per antibody or antigen-binding fragment, i.e., drug loading, or -LD moieties per antibody or antigen-binding fragment (Ab) in an ADC of Formula 1. In an ADC comprising a panRAS inhibitor drug moiety, " p " represents the number of panRAS inhibitor compounds linked to the antibody or antigen-binding fragment. For example, if two panRAS inhibitor compounds are linked to an antibody or antigen-binding fragment, then p = 2. In a composition comprising multiple copies of an ADC of Formula 1, "average p " refers to the average number of -LD moieties per antibody or antigen-binding fragment, also referred to as "average drug loading".

antibody-drug conjugates

Antibody-drug conjugate (ADC) compounds of the present invention include those having anticancer activity. In particular, the ADC compounds comprise an antibody or antigen-binding fragment conjugated (i.e., covalently attached via a conjugate linker) to a drug moiety (e.g., a panRAS inhibitor), wherein the drug moiety, when not conjugated to the antibody or antigen-binding fragment, has a cytotoxic or cytostatic effect. In some embodiments, the drug moiety, when not conjugated to the antibody or antigen-binding fragment, can reduce the expression and/or activity of panRAS and/or one or more of its upstream regulators or downstream targets. Without being bound by theory, in some embodiments, the ADCs disclosed herein can provide potent anticancer agents by targeting panRAS expression and/or activity. Additionally, without being bound by theory, by conjugating a drug moiety to an antibody that binds to an antigen associated with expression on tumor cells or cancer, the ADC may provide improved activity, better cytotoxic specificity, and/or reduced off-target killing compared to the drug moiety when administered alone.

Thus, in some embodiments, the components of the ADC (i) retain one or more therapeutic properties exhibited by the antibody and drug moiety in isolation; (ii) retain the specific binding properties of the antibody or antigen-binding fragment; (iii) optimize drug loading and drug-to-antibody ratio; (iv) allow delivery of the drug moiety, e.g., intracellular delivery, via stable attachment to the antibody or antigen-binding fragment; (v) maintain ADC stability as an intact conjugate until transport or delivery to the target site; (vi) minimize aggregation of the ADC prior to or after administration; (vii) allow therapeutic effects, e.g., cytotoxic effects, of the drug moiety after cleavage or other release mechanisms in the cellular environment; (viii) exhibit in vivo anticancer therapeutic efficacy comparable to or superior to the antibody and drug moiety in isolation; and/or (ix) minimize off-target killing by the drug moiety. (x) are selected to exhibit desirable pharmacokinetic and pharmacodynamic properties, formulation potential, and toxicological/immunological profiles. Each of these properties may provide improved ADCs for therapeutic use (Ab et al. (2015) Mol Cancer Ther. 14:1605-13).

The ADC compounds of the present invention can selectively deliver effective doses of cytotoxic or cytostatic agents to cancer cells or tumor tissues. In some embodiments, the cytotoxic and/or cytostatic activity of the ADC depends on the expression of the target antigen on the cell. In some embodiments, the disclosed ADCs are particularly effective in killing cancer cells expressing the target antigen while minimizing off-target killing. In some embodiments, the disclosed ADCs do not exhibit cytotoxic and/or cytostatic effects against cancer cells that do not express the target antigen.

In certain embodiments, provided herein are ADC compounds comprising an antibody or antigen-binding fragment thereof (Ab), a panRAS inhibitor drug moiety (D), and a conjugate linker moiety (L) covalently attaching the Ab to D. In some embodiments, provided herein are ADC compounds comprising an antibody or antigen-binding fragment thereof (Ab) that targets a cancer cell, a panRAS inhibitor drug moiety (D), and a conjugate linker moiety (L) covalently attaching the Ab to D. In some embodiments, the antibody or antigen-binding fragment can bind to a tumor-associated antigen (e.g., EphA2 or B7-H3 (CD276)), for example, with high specificity and high affinity. In some embodiments, the antibody or antigen-binding fragment, upon binding, is internalized into a target cell, for example, into a catabolic compartment within the cell. In some embodiments, the ADC, upon binding to a target cell, is internalized, undergoes degradation, and releases the panRAS inhibitor drug moiety, thereby killing the cancer cell. The panRAS inhibitor drug moiety can be released from the conjugate linker moiety of the ADC and/or the antibody by enzymatic action, hydrolysis, oxidation or any other mechanism.

An exemplary ADC has the chemical formula 1:

[Chemical Formula 1]

Ab-(LD) _p

(wherein Ab = antibody or antigen-binding fragment, L = conjugate linker moiety, D = panRAS inhibitor drug moiety, and p = number of panRAS inhibitor drug moieties per antibody or antigen-binding fragment).

antibodies

The antibody or antigen-binding fragment (Ab) of formula 1 includes any antibody or antigen-binding fragment within that category that specifically binds to a target antigen on a cell. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, B7-H4, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2(NaPi2b), nectin4, TROP2, LIV1, CD46, MSLN, CD142 (F3), MUC1, MUC16, SLC39A6, TFRC, TACSTD2, GPNMB, EphA2, CD56, SEZ6, CD25, CCR8, CEACAM5, CEACAM6, 4-1BB, 5AC, 5T4, alpha-fetoprotein, Angiopoietin 2, ASLG659, TCLI, BMPRIB, brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (imitation), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR) I), CD22 (B-cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B-cell differentiation antigen CD72), CD79a, CD80, CD166 (ALCAM), CDH17, CA9, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1, CFC1B), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), episialin, ERBB3, ETBR (endothelin type B receptor), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extradomain-B, frizzled receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, human scatter factor receptor kinase, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily receptor translocation-associated 2), Lewis-Y antigen, LY64 (RP105), LY6E, STEAP1, ADAM9, PTK7, MMP14, TM4SF1, ITGB6, FXYD5, MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen (PSMA), PSCA (prostate stem cell antigen precursor), PRLR (prolactin receptor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-related gene I, TAG-72, TEMI, tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel subfamily M, member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or vimentin. In some embodiments, the target antigen is EphA2 or B7-H3. In some embodiments, the antibody or antigen-binding fragment (Ab) of formula 1 comprises any antibody or antigen-binding fragment within its scope that specifically binds to a target antigen on a cancer cell. In some embodiments, the cell or the cancer cell expresses EphA2. In some embodiments, the target antigen EphA2 has an amino acid sequence set forth in Table 6.

In some embodiments, the target antigen B7-H3 (CD276) has the amino acid sequence set forth in Table 6.

The antibody or antigen-binding fragment (e.g., an anti-EphA2 or anti-B7-H3 antibody or antigen-binding fragment) can bind to a target antigen with a dissociation constant (K _D ) of 1 mM or less, 100 nM or less, or 10 nM or less, or any amount therebetween, as measured, for example, by a BIAcore ^® assay. In some embodiments, the K _D is from 1 pM to 500 pM. In some embodiments, the K _D is from 500 pM to 1 μM, from 1 μM to 100 nM, or from 100 mM to 10 nM.

In some embodiments, the antibody or antigen-binding fragment (e.g., an anti-EphA2 or anti-B7-H3 antibody or antigen-binding fragment) is a four-chain antibody (also referred to as an immunoglobulin or a full-length antibody or an intact antibody) comprising two heavy chains and two light chains. In some embodiments, the antibody or antigen-binding fragment (e.g., an anti-EphA2 or anti-B7-H3 antibody or antigen-binding fragment) is an antigen-binding fragment of an immunoglobulin. In some embodiments, the antibody or antigen-binding fragment (e.g., an anti-EphA2 or anti-B7-H3 antibody or antigen-binding fragment) is an antigen-binding fragment of an immunoglobulin that retains the ability to bind a target cancer antigen and/or provides at least one function of an immunoglobulin.

In some embodiments, the antibody or antigen-binding fragment (e.g., an anti-EphA2 or anti-B7-H3 antibody or antigen-binding fragment) is an internalizing antibody or an internalizing antigen-binding fragment thereof. In some embodiments, the internalizing antibody (e.g., an anti-EphA2 antibody or an anti-B7-H3 antibody) or an internalizing antigen-binding fragment thereof (e.g., an anti-EphA2 or anti-B7-H3 antigen-binding fragment) binds to a target cancer antigen expressed on the cell surface and enters the cell upon binding. In some embodiments, the panRAS inhibitor drug moiety of the ADC is released from the antibody or antigen-binding fragment (e.g., an anti-EphA2 or anti-B7-H3 antibody or antigen-binding fragment) of the ADC after the ADC has entered and become present in a cell expressing the target cancer antigen (i.e., after the ADC has been internalized), e.g., by cleavage, degradation of the antibody or antigen-binding fragment, or any other suitable release mechanism.

In some embodiments, the antibody (e.g., an anti-EphA2 antibody or an anti-B7-H3 antibody) comprises a mutation that mediates reduced antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), or does not mediate ADCC or CDC. In some embodiments, such mutations are known as Fc silencing, Fc silencing, or Fc silencing mutations. In some embodiments, amino acid residues L234 and L235 in the IgG1 constant region are substituted with A234 and A235 (also known as "LALA"). In some embodiments, amino acid residue N297 in the IgG1 constant region is substituted with A297 (also known as "N297A"). In some embodiments, amino acid residues D265 and P329 in the IgG1 constant region are substituted with A265 and A329 (also known as "DAPA"). Other antibody Fc silencing mutations may also be used. In some embodiments, Fc silencing mutations are used in combination (e.g., D265A, N297A, and P329A (also known as “DANAPA”)).

In addition to exemplary antigen targets, amino acid sequences of exemplary antibodies of the present invention are set forth in Tables 2-6.

Table 2. Example antibodies

Table 3. Amino acid sequences of mAb variable regions

Table 4. Amino acid sequence of mAb CDR (assembled)

Table 5. Amino acid and nucleic acid sequences of full-length mAb Ig chains

Table 6. Exemplary target antigen amino acid sequences

In some embodiments, an antibody or antigen-binding fragment of an ADC disclosed herein can comprise any set of heavy and light chain variable domains listed in the Tables above or a set of six CDRs from any set of heavy and light chain variable domains listed in the Tables above. In some embodiments, an antibody or antigen-binding fragment of an ADC disclosed herein can comprise an amino acid sequence that is conservatively modified and/or homologous to a sequence listed in the Tables above, so long as the ADC retains the ability to bind its target cancer antigen (e.g., with a K _D of less than 1 ^× 10 -8 M) and retains one or more functional properties of an ADC disclosed herein (e.g., the ability to be internalized and to bind to an antigen target, e.g., an antigen expressed on a tumor or other cancer cell).

In some embodiments, the antibody or antigen-binding fragment of the ADC disclosed herein further comprises human heavy and light chain constant domains or fragments thereof. For example, the antibody or antigen-binding fragment of the described ADC may comprise a human IgG heavy chain constant domain (e.g., IgG1) and a human kappa or lambda light chain constant domain. In some embodiments, the antibody or antigen-binding fragment of the described ADC comprises a human immunoglobulin G subtype 1 (IgG1) heavy chain constant domain together with a human Ig kappa light chain constant domain.

In some embodiments, the target cancer antigen for the ADC is EphA2.

In some embodiments, the anti-EphA2 antibody or antigen-binding fragment of the ADC disclosed herein further comprises human heavy and light chain constant domains or fragments thereof. For example, the anti-EphA2 antibody or antigen-binding fragment of the described ADC may comprise a human IgG heavy chain constant domain (e.g., IgG1) and a human kappa or lambda light chain constant domain. In some embodiments, the anti-EphA2 antibody or antigen-binding fragment of the described ADC comprises a human immunoglobulin G subtype 1 (IgG1) heavy chain constant domain together with a human Ig kappa light chain constant domain.

In some embodiments, the anti-EphA2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 17, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 18, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 19; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 26, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 27, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 28.

In some embodiments, the anti-EphA2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 20, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 21, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 19; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 29, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 30, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 31.

In some embodiments, the anti-EphA2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 22, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 23, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 24; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 32, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 27, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 31.

In some embodiments, the anti-EphA2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 25, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 21, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 19; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 29, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 30, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 31.

In some embodiments, the anti-EphA2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs, wherein the CDRs comprise additions, deletions, or substitutions of no more than one, two, three, four, five, or six amino acids of HCDR1 (SEQ ID NO: 17), HCDR2 (SEQ ID NO: 18), HCDR3 (SEQ ID NO: 19); LCDR1 (SEQ ID NO: 26), LCDR2 (SEQ ID NO: 27), and LCDR3 (SEQ ID NO: 28).

In some embodiments, the anti-EphA2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs, wherein the CDRs comprise additions, deletions, or substitutions of no more than one, two, three, four, five, or six amino acids of HCDR1 (SEQ ID NO: 20), HCDR2 (SEQ ID NO: 21), HCDR3 (SEQ ID NO: 19); LCDR1 (SEQ ID NO: 29), LCDR2 (SEQ ID NO: 30), and LCDR3 (SEQ ID NO: 31).

In some embodiments, the anti-EphA2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs, wherein the CDRs comprise additions, deletions, or substitutions of no more than one, two, three, four, five, or six amino acids of HCDR1 (SEQ ID NO: 22), HCDR2 (SEQ ID NO: 23), HCDR3 (SEQ ID NO: 24); LCDR1 (SEQ ID NO: 32), LCDR2 (SEQ ID NO: 27), and LCDR3 (SEQ ID NO: 31).

In some embodiments, the anti-EphA2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs, wherein the CDRs comprise additions, deletions, or substitutions of no more than one, two, three, four, five, or six amino acids of HCDR1 (SEQ ID NO: 25), HCDR2 (SEQ ID NO: 21), HCDR3 (SEQ ID NO: 19); LCDR1 (SEQ ID NO: 29), LCDR2 (SEQ ID NO: 30), and LCDR3 (SEQ ID NO: 31).

In some embodiments, the anti-EphA2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 11 and a light chain variable region amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-EphA2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 11 and a light chain variable region amino acid sequence of SEQ ID NO: 12 or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-EphA2 antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12.

In some embodiments, the anti-EphA2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 3 or a sequence at least 95% identical to SEQ ID NO: 3 and a light chain amino acid sequence of SEQ ID NO: 5 or a sequence at least 95% identical to SEQ ID NO: 5. In some embodiments, the anti-EphA2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 3 and a light chain amino acid sequence of SEQ ID NO: 5 or a sequence at least 95% identical to the disclosed sequences. In some embodiments, the anti-EphA2 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5.

In some embodiments, the target cancer antigen for the ADC is B7-H3 (CD276).

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment of the ADC disclosed herein further comprises human heavy and light chain constant domains or fragments thereof. For example, the anti-B7-H3 (CD276) antibody or antigen-binding fragment of the described ADC may comprise a human IgG heavy chain constant domain (e.g., IgG1) and a human kappa or lambda light chain constant domain. In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment of the described ADC comprises a human immunoglobulin G subtype 1 (IgG1) heavy chain constant domain together with a human Ig kappa light chain constant domain.

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 33, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 34, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 35; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 42, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 43, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 44.

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 36, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 37, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 35; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 45, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 46, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 47.

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 38, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 39, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 40; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 48, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 43, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 47.

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 41, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 37, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 35; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 45, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 46, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 47.

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs, wherein the CDRs comprise additions, deletions, or substitutions of no more than one, two, three, four, five, or six amino acids of HCDR1 (SEQ ID NO: 33), HCDR2 (SEQ ID NO: 34), HCDR3 (SEQ ID NO: 35); LCDR1 (SEQ ID NO: 42), LCDR2 (SEQ ID NO: 43), and LCDR3 (SEQ ID NO: 44).

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs, wherein the CDRs comprise additions, deletions, or substitutions of no more than one, two, three, four, five, or six amino acids of HCDR1 (SEQ ID NO: 36), HCDR2 (SEQ ID NO: 37), HCDR3 (SEQ ID NO: 35); LCDR1 (SEQ ID NO: 45), LCDR2 (SEQ ID NO: 46), and LCDR3 (SEQ ID NO: 47).

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs, wherein the CDRs comprise additions, deletions, or substitutions of no more than one, two, three, four, five, or six amino acids of HCDR1 (SEQ ID NO: 38), HCDR2 (SEQ ID NO: 39), HCDR3 (SEQ ID NO: 40); LCDR1 (SEQ ID NO: 48), LCDR2 (SEQ ID NO: 43), and LCDR3 (SEQ ID NO: 47).

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs, wherein the CDRs comprise additions, deletions, or substitutions of no more than one, two, three, four, five, or six amino acids of HCDR1 (SEQ ID NO: 41), HCDR2 (SEQ ID NO: 37), HCDR3 (SEQ ID NO: 35); LCDR1 (SEQ ID NO: 45), LCDR2 (SEQ ID NO: 46), and LCDR3 (SEQ ID NO: 47).

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 49, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 50, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 51; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 58, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 59, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 60.

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 52, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 53, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 51; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 61, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 62, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 63.

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 54, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 55, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 56; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 58, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 59, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 63.

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 57, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 53, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 51; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 61, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 62, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 63.

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs, wherein the CDRs comprise additions, deletions, or substitutions of no more than one, two, three, four, five, or six amino acids of HCDR1 (SEQ ID NO: 49), HCDR2 (SEQ ID NO: 50), HCDR3 (SEQ ID NO: 51); LCDR1 (SEQ ID NO: 58), LCDR2 (SEQ ID NO: 59), and LCDR3 (SEQ ID NO: 60).

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs, wherein the CDRs comprise additions, deletions, or substitutions of no more than one, two, three, four, five, or six amino acids of HCDR1 (SEQ ID NO: 52), HCDR2 (SEQ ID NO: 53), HCDR3 (SEQ ID NO: 51); LCDR1 (SEQ ID NO: 61), LCDR2 (SEQ ID NO: 62), and LCDR3 (SEQ ID NO: 63).

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs, wherein the CDRs comprise additions, deletions, or substitutions of no more than one, two, three, four, five, or six amino acids of HCDR1 (SEQ ID NO: 54), HCDR2 (SEQ ID NO: 55), HCDR3 (SEQ ID NO: 56); LCDR1 (SEQ ID NO: 58), LCDR2 (SEQ ID NO: 59), and LCDR3 (SEQ ID NO: 63).

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs, wherein the CDRs comprise additions, deletions or substitutions of no more than one, two, three, four, five or six amino acids of HCDR1 (SEQ ID NO: 57), HCDR2 (SEQ ID NO: 53), HCDR3 (SEQ ID NO: 51); LCDR1 (SEQ ID NO: 61), LCDR2 (SEQ ID NO: 62), and LCDR3 (SEQ ID NO: 63).

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 13 and a light chain variable region amino acid sequence of SEQ ID NO: 14 or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14.

In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 15 and a light chain variable region amino acid sequence of SEQ ID NO: 16 or a sequence that is at least 95% identical to the disclosed sequences. In some embodiments, the anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16.

In some embodiments, the anti-B7-H3 (CD276) antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 7 or a sequence at least 95% identical to SEQ ID NO: 7 and a light chain amino acid sequence of SEQ ID NO: 8 or a sequence at least 95% identical to SEQ ID NO: 8. In some embodiments, the anti-B7-H3 (CD276) antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 7 and a light chain amino acid sequence of SEQ ID NO: 8 or a sequence at least 95% identical to the disclosed sequences. In some embodiments, the anti-B7-H3 (CD276) antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8.

In some embodiments, the anti-B7-H3 (CD276) antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 9 or a sequence at least 95% identical to SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10 or a sequence at least 95% identical to SEQ ID NO: 10. In some embodiments, the anti-B7-H3 (CD276) antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10 or a sequence at least 95% identical to the disclosed sequences. In some embodiments, the anti-B7-H3 (CD276) antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10.

Residues within two or more polypeptides are said to be "corresponding" if they occupy similar positions in the polypeptide structures. Similar positions in two or more polypeptides can be determined by aligning the polypeptide sequences based on amino acid sequence or structural similarity. Those skilled in the art will appreciate that introducing gaps in one of the sequences may be necessary to produce a satisfactory alignment.

In some embodiments, amino acid substitutions are of a single residue. Insertions will typically be of the order of about 1 to about 20 amino acid residues, although significantly larger insertions may be tolerated as long as biological function is maintained (e.g., binding to a target antigen). Deletions typically range from about 1 to about 20 amino acid residues, although in some cases, deletions can be much larger. Substitutions, deletions, insertions, or any combination thereof can be used to arrive at the final derivative or variant. Typically, these changes are made to a few amino acids to minimize molecular alterations, particularly the immunogenicity and specificity of the antigen-binding protein. However, larger changes may be tolerated in certain circumstances. Conservative substitutions can be made according to the chart below, shown in Table 7.

Table 7

In some embodiments where variant antibody sequences are used in ADCs, the variants will typically exhibit the same qualitative biological activity and elicit the same immune response, but may also be selected to alter characteristics of the antigen binding protein, as needed. Alternatively, the variants may be designed to alter the biological activity of the antigen binding protein. For example, glycosylation sites may be altered or removed.

A variety of antibodies can be used in conjunction with the ADCs disclosed herein to target cancer cells. As shown below, the linker-payloads of the ADCs disclosed herein are surprisingly effective with a variety of tumor antigen-targeting antibodies. Suitable antigens expressed on cancer cells but not on healthy cells, or expressed at higher levels on cancer cells than on healthy cells, are known in the art, as are antibodies directed against them. Additional antibodies directed against these antigen targets can be prepared by those skilled in the art. These antibodies can be used in conjunction with the conjugate linkers and panRAS inhibitor payloads disclosed herein. In some embodiments, the antibodies or antigen-binding fragments target EphA2 or B7-H3 (CD276) and, in particular, provide improved drug:antibody ratios, aggregation levels, stability (i.e., in vitro and in vivo stability), tumor targeting (i.e., cytotoxicity, potency), minimized off-target killing, and/or therapeutic efficacy. Improved therapeutic efficacy may be measured in vitro or in vivo and may include a reduction in tumor growth rate and/or a reduction in tumor volume.

In some embodiments, alternative antibodies to the same target or antibodies to different antigen targets are used and provide at least some of the advantageous functional properties described above (e.g., improved stability, improved tumor targeting, improved therapeutic efficacy, etc.). In some embodiments, some or all of these advantageous functional properties are observed when the disclosed conjugate linker and panRAS inhibitor payload are conjugated to alternative EphA2 or B7-H3 (CD276) targeting antibodies or antigen-binding fragments. In some other embodiments, some or all of these advantageous functional properties are observed when the disclosed conjugate linker and panRAS inhibitor payload are conjugated to an EphA2-targeting antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment targets EphA2. In other embodiments, some or all of these advantageous functional properties are observed when the disclosed conjugate linker and panRAS inhibitor payload are conjugated to a B7-H3 (CD276)-targeting antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment targets B7-H3 (CD276).

conjugate linker

In some embodiments, the conjugate linker of the ADC is stable extracellularly in a manner sufficient to be therapeutically effective. In some embodiments, the conjugate linker is stable extracellularly such that the ADC remains intact when present in extracellular conditions (e.g., prior to transport or delivery into a cell). The term "intact" as used in the context of an ADC means that the antibody or antigen-binding fragment remains attached to the drug moiety (e.g., a panRAS inhibitor).

As used herein, “stable” in the context of a conjugate linker or an ADC comprising a conjugate linker means that no more than about 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% (or any percentage therebetween) of the conjugate linker in a sample of the ADC is cleaved (or, for the entire ADC, otherwise remains intact) when the ADC is present in extracellular conditions. In some embodiments, the conjugate linkers and/or ADCs disclosed herein are stable compared to alternative conjugate linkers and/or ADCs having alternative conjugate linkers and/or panRAS inhibitor payloads. In some embodiments, the ADCs disclosed herein can remain intact for greater than about 48 hours, greater than about 60 hours, greater than about 72 hours, greater than about 84 hours, or greater than about 96 hours.

Whether a conjugate linker is stable outside of the cell can be determined, for example, by incorporating the ADC into plasma for a predetermined period of time (e.g., 2, 4, 6, 8, 16, 24, 48, or 72 hours) and quantifying the amount of free drug moiety present in the plasma thereafter. Stability can ensure that the ADC time is confined to the target cancer cells and prevent premature release of the drug moiety, which can lower the therapeutic index of the ADC by indiscriminately damaging both normal and cancer tissues. In some embodiments, the conjugate linker is stable outside of the target cell and releases the drug moiety from the ADC once inside the cell, allowing the drug to bind to its target. Thus, an effective conjugate linker (i) maintains the specific binding properties of the antibody or antigen-binding fragment; (ii) allows delivery of the drug moiety, e.g., intracellular delivery, through stable attachment to the antibody or antigen-binding fragment; (iii) the ADC remains stable and intact until transported or delivered to its target site; and (iv) allows the therapeutic effect of the drug moiety, e.g., cytotoxic effect, after cleavage or alternative release mechanism.

The conjugate linker can affect the physicochemical properties of the ADC. Many cytotoxic agents are inherently hydrophobic, and linking them to antibodies with additional hydrophobic moieties can result in aggregation. ADC aggregates are insoluble and often limit the achievable drug loading onto the antibody, which can negatively impact the efficacy of the ADC. Protein aggregates in biologics are also commonly associated with increased immunogenicity. As shown below, the conjugate linker disclosed herein produces ADCs with low aggregation levels and desirable drug loading levels.

Conjugate linkers can be "cleavable" or "non-cleavable" (Ducry and Stump (2010) Bioconjugate Chem. 21:5-13). Cleavable conjugate linkers are designed to release the drug moiety (e.g., a panRAS inhibitor) upon exposure to a specific environmental factor, for example, upon internalization into a target cell, whereas non-cleavable conjugate linkers typically rely on cleavage of the antibody or antigen-binding fragment itself.

As used herein, the term "alkyl" refers to a straight or branched chain hydrocarbon radical consisting solely of carbon and hydrogen atoms and containing no unsaturation. As used herein, the term "C ₁ -C ₆ alkyl" refers to a straight or branched chain hydrocarbon radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from 1 to 6 carbon atoms, and attached to the remainder of the molecule by a single bond. Non-limiting examples of "C ₁ -C ₆ alkyl" groups include methyl (C ₁ alkyl), ethyl (C ₂ alkyl), 1-methylethyl (C ₃ alkyl), n-propyl (C ₃ alkyl), isopropyl (C ₃ alkyl), n-butyl (C ₄ alkyl), isobutyl (C ₄ alkyl), sec-butyl (C ₄ alkyl), tert-butyl (C ₄ alkyl), n-pentyl (C ₅ alkyl), isopentyl (C ₅ alkyl), neopentyl (C ₅ alkyl), and hexyl (C ₆ alkyl).

As used herein, the term "alkenyl" refers to a straight or branched chain hydrocarbon radical group consisting solely of carbon and hydrogen atoms and containing at least one double bond. As used herein, the term "C ₂ -C ₆ alkenyl" refers to a straight or branched chain hydrocarbon radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, having from 2 to 6 carbon atoms, and being attached to the remainder of the molecule by a single bond. Non-limiting examples of "C ₂ -C ₆ alkenyl" groups include ethenyl (C ₂ alkenyl), prop-1-enyl (C ₃ alkenyl), but-1-enyl (C ₄ alkenyl), pent-1-enyl (C ₅ alkenyl), pent-4-enyl (C ₅ alkenyl), penta-1,4-dienyl (C ₅ alkenyl), hexa-1-enyl (C ₆ alkenyl), hexa-2-enyl (C ₆ alkenyl), hexa-3-enyl (C ₆ alkenyl), hexa-1-,4-dienyl (C ₆ alkenyl), hexa-1-,5-dienyl (C ₆ alkenyl), and hexa-2-,4-dienyl (C ₆ alkenyl). As used herein, the term "C ₂ -C ₃ alkenyl" refers to a straight or branched chain hydrocarbon radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, having two to three carbon atoms, and being attached to the remainder of the molecule by a single bond. Non-limiting examples of "C ₂ -C ₃ alkenyl" groups include ethenyl (C ₂ alkenyl) and prop-1-enyl (C ₃ alkenyl).

As used herein, the term "alkylene" refers to a divalent straight or branched chain hydrocarbon radical consisting solely of carbon and hydrogen atoms and containing no unsaturation. As used herein, the term "C ₁ -C ₆ alkylene" refers to a divalent straight or branched chain hydrocarbon radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, and having from 1 to 6 carbon atoms. Non-limiting examples of "C ₁ -C ₆ alkylene" groups include methylene (C ₁ alkylene), ethylene (C ₂ alkylene), 1-methylethylene (C ₃ alkylene), n-propylene (C ₃ alkylene), isopropylene (C ₃ alkylene), n-butylene (C ₄ alkylene), isobutylene (C ₄ alkylene), sec-butylene (C ₄ alkylene), tert-butylene (C ₄ alkylene), n-pentylene (C ₅ alkylene), isopentylene (C ₅ alkylene), neopentylene (C ₅ alkylene), and hexylene (C ₆ alkylene).

As used herein, the term "alkenylene" refers to a divalent straight or branched chain hydrocarbon radical consisting solely of carbon and hydrogen atoms and containing at least one double bond. As used herein, the term "C ₂ -C ₆ alkenylene" refers to a divalent straight or branched chain hydrocarbon radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, and having from 2 to 6 carbon atoms. Non-limiting examples of "C ₂ -C ₆ alkenylene" groups include ethenylene (C ₂ alkenylene), prop-1-enylene (C ₃ alkenylene), but-1-enylene (C ₄ alkenylene), pent-1-enylene (C ₅ alkenylene), pent-4-enylene (C ₅ alkenylene), penta-1,4-dienylene (C ₅ alkenylene), hexa-1-enylene (C ₆ alkenylene), hexa-2-enylene (C ₆ alkenylene), hexa-3-enylene (C ₆ alkenylene), hexa-1-,4-dienylene (C ₆ alkenylene), hexa-1-,5-dienylene (C ₆ alkenylene), and hexa-2-,4-dienylene (C ₆ As used herein, the term "C ₂ -C ₆ alkenylene" refers to a divalent straight or branched chain hydrocarbon radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, and having 2 to 3 carbon atoms. Non-limiting examples of "C ₂ -C ₃ alkenylene" groups include ethenylene (C ₂ alkenylene) and prop-1-enylene (C ₃ alkenylene).

As used herein, the term "cycloalkyl" refers to a non-aromatic, monocyclic, fused bicyclic, fused tricyclic, or bridged polycyclic ring system. In some embodiments, cycloalkyl is a monocyclic or bicyclic saturated carbocyclic group containing from 3 to 10 ring members, which may include a fused, bridged, or spiro ring system. Non-limiting examples of fused bicyclic or bridged polycyclic ring systems include bicyclo[1.1.1]pentane, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, bicyclo[3.2.1]octane, bicyclo[2.2.2]octane, and adamantanyl. Non-limiting examples of monocyclic C ₃ -C ₈ cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups.

The terms heteroarylene, cycloalkylene, and heterocycloalkylene refer to divalent heteroaryl, cycloalkyl, and heterocycloalkyl.

As used herein, the term "haloalkyl" refers to a linear or branched alkyl chain in which one or more halogen groups are substituted for hydrogen along the hydrocarbon chain. Examples of halogen groups suitable for substitution in a haloalkyl group include fluorine, bromine, chlorine, and iodine. A haloalkyl group may include substitution of multiple halogen groups for hydrogen in the alkyl chain, wherein the halogen groups may be attached to the same carbon atom or to different carbon atom(s) in the alkyl chain.

As used herein, alkyl, alkenyl, alkynyl, alkoxy, amino, aryl, heteroaryl, cycloalkyl, and heterocycloalkyl groups include optionally substituted linear or branched (C ₁ -C ₆ )alkyl, optionally substituted linear or branched (C ₂ -C ₆ )alkenyl groups, optionally substituted linear or branched (C ₂ -C ₆ )alkynyl groups, optionally substituted linear or branched (C ₁ -C ₆ )alkoxy, optionally substituted (C ₁ -C ₆ )alkyl-S-, hydroxy, oxo (or N-oxide, where appropriate), nitro, cyano, -C(O)-OR ₀ ', -OC(O)-R ₀ ', -C(O)-NR ₀ 'R ₀ '', -NR ₀ 'R ₀ '', -(C=NR ₀ ')-OR ₀ '', linear or branched (C ₁ -C ₆ ) haloalkyl, trifluoromethoxy, or halogen (wherein R ₀ ' and R ₀ '' are each independently a hydrogen atom or an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group, wherein one or more of the carbon atoms of the linear or branched (C ₁ -C ₆ )alkyl group is optionally deuterated).

As used herein, the term "polyoxyethylene", "polyethylene glycol" or "PEG" refers to a linear chain, branched chain or star-shaped arrangement comprised of (OCH ₂ CH ₂ ) groups. In certain embodiments, the polyethylene or PEG groups are -(OCH ₂ CH ₂ ) _t *-, wherein t is 1-40 or 4-40, "-" indicates the terminus toward the self-immolative spacer, and "*-" indicates the point of attachment to the terminal group R', where R' is OH, OCH ₃ or OCH ₂ CH ₂ C(=O)OH. In another embodiment, the polyethylene or PEG group is -(CH ₂ CH ₂ O) _t *-, where t is 1-40 or 4-40, "-" indicates the terminus toward the self-immolative spacer, and "*-" indicates the point of attachment to the terminal group R'', where R'' is H, CH ₃ or CH ₂ CH ₂ C(=O)OH. For example, as used herein, the term "PEG12" means that t is 12.

As used herein, the term "polyalkylene glycol" refers to a linear, branched, or star-shaped arrangement of (O(CH ₂ ) _m ) _n groups. In certain embodiments, the polyethylene or PEG groups are -(O(CH ₂ ) _m ) _t *-, wherein m is 1 to 10, t is 1 to 40 or 4 to 40, "-" indicates the terminus toward the self-immolative spacer, and "*-" indicates the point of attachment to the terminal group R', where R' is OH, OCH ₃ , or OCH ₂ CH ₂ C(=O)OH. In another embodiment, the polyethylene or PEG group is (CH ₂ ) _m O) _t *-, where m is 1 to 10, t is 1 to 40 or 4 to 40, "-" indicates the terminal toward the self-immolative spacer, and "*-" indicates the point of attachment to the terminal group R'', where R'' is H, CH ₃ or CH ₂ CH ₂ C(=O)OH.

As used herein, the term "reactive group" refers to a functional group capable of forming a covalent bond with a functional group of an antibody, antibody fragment, or another reactive group attached to an antibody or antibody fragment. Non-limiting examples of such functional groups include the reactive groups set forth in Table 8 provided herein.

As used herein, the term "attachment group" or "coupling group" refers to a divalent moiety that connects a crosslinking spacer to an antibody or fragment thereof. An attachment or coupling group is a divalent moiety formed by a reaction between a reactive group and a functional group on an antibody or fragment thereof. Non-limiting examples of such 9-valent moieties include the divalent chemical moieties provided in Table 8 and Table 2 provided herein.

As used herein, the term "crosslinking spacer" refers to one or more conjugated linker components covalently attached together to form a bivalent moiety that links a bivalent peptide spacer to a reactive group, links a bivalent peptide spacer to a coupling group, or links an attachment group to at least one cleavable group. In certain embodiments, the "crosslinking spacer" comprises a carboxyl group that is attached to the N-terminus of the bivalent peptide spacer via an amide bond.

As used herein, the term "spacer moiety" refers to one or more conjugated linker components covalently attached together to form a moiety that connects a self-immolative spacer to a hydrophilic moiety.

As used herein, the term "bivalent peptide spacer" refers to a bivalent conjugate linker comprising one or more amino acid residues covalently attached together to form a moiety that links a cross-linking spacer to a self-immolative spacer. The one or more amino acid residues may be amino acid residues selected from alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), norvaline (Nva), norleucine (Nle), selenocysteine (Sec), pyrolysine (Pyl), homoserine, homocysteine, and desmethyl pyrolysine.

In certain embodiments, the "bipeptide spacer" is a combination of two to four amino acid residues, each residue independently selected from alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), norvaline (Nva), norleucine (Nle), selenocysteine (Sec), pyrolysine (Pyl), homoserine, homocysteine, and desmethyl pyrolysine. A combination of choice, for example -ValCit*; -CitVal*; -AlaAla*; -AlaCit*; -CitAla*; -AsnCit*; -CitAsn*; -CitCit*; -ValGlu*; -GluVal*; -SerCit*; -CitSer*; -LysCit*; -CitLys*; -AspCit*; -CitAsp*; -AlaVal*; -ValAla*; -PheAla*; -AlaPhe*; -PheLys*; -LysPhe*; -ValLys*; -LysVal*; -AlaLys*; -LysAla*; -PheCit*; -CitPhe*; -LeuCit*; -CitLeu*; -IleCit*; -CitIle*; -PheArg*; -ArgPhe*; -CitTrp*; -TrpCit*; -PhePheLys*; -LysPhePhe*; -DPhePheLys*; -DLysPhePhe*; -GlyPheLys*; -LysPheGly*; -GlyPheLeuGly-[SEQ ID NO: 67]; -GlyLeuPheGly-[SEQ ID NO: 68]; -AlaLeuAlaLeu-[SEQ ID NO: 69], -GlyGlyGly*; -GlyGlyGlyGly-[SEQ ID NO: 70]; -GlyPheValGly-[SEQ ID NO: 71]; and -GlyValPheGly-[SEQ ID NO: 72], where "-" indicates an attachment point to a bridging spacer and "*" indicates an attachment point to a self-immolative spacer.

As used herein, the term "conjugate linker component" refers to a chemical moiety that is part of a conjugate linker. Examples of conjugate linker components include: an alkylene group: -(CH ₂ ) _n - (which may be linear or branched) (wherein n is 1-18); an alkenylene group; an alkynylene group; an alkenyl group; an alkynyl group; an ethylene glycol unit: -OCH ₂ CH ₂ - or -CH ₂ CH ₂ O-; a polyethylene glycol unit: (-CH ₂ CH ₂ O-) _x (wherein x is 2-20); -O-; -S-; a carbonyl: -C(=O); an ester: C(=O)-O or OC(=O); a carbonate: -OC(=O)O-; an amine: -NH-; a tertiary amine; Amide: -C(=O)-NH-, -NH-C(=O)- or -C(=O)N(C _1-6 alkyl); Carbamate: -OC(=O)NH- or -NHC(=O)O; Urea: -NHC(=O)NH; Sulfonamide: -S(O) ₂ NH- or -NHS(O) ₂ ; Ether: -CH ₂ O- or -OCH ₂ -; Alkylene substituted with one or more groups independently selected from carboxy, sulfonate, hydroxyl, amine, amino acid, sugar, phosphate and phosphonate; Alkenylene substituted with one or more groups independently selected from carboxy, sulfonate, hydroxyl, amine, amino acid, sugar, phosphate and phosphonate; Alkynylene substituted with one or more groups independently selected from carboxy, sulfonate, hydroxyl, amine, amino acid, sugar, phosphate and phosphonate; C ₁ -C ₁₀ alkylene wherein one or more methylene groups are replaced with one or more -S-, -NH- or -O- moieties; a divalent ring selected from ring systems having two available points of attachment, such as phenyl (including 1,2-, 1,3- and 1,4-disubstituted phenyl), C ₅ -C ₆ heteroaryl, C ₃ -C ₈ cycloalkyl (including 1,1-disubstituted cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, and 1,4-disubstituted cyclohexyl) and C ₄ -C ₈ heterocycloalkyl; A residue of an amino acid selected from alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), norvaline (Nva), norleucine (Nle), selenocysteine (Sec), pyrolysine (Pyl), homoserine, homocysteine, and desmethyl pyrolysine; A combination of two or more amino acid residues, wherein each residue is independently selected from amino acid residues selected from alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), norvaline (Nva), norleucine (Nle), selenocysteine (Sec), pyrolysine (Pyl), homoserine, homocysteine and desmethyl pyrolysine), for example, Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; Val-Ala; Phe-Lys; Lys-Phe; Val-Lys; Lys-Val; Ala-Lys; Lys-Ala; Phe-Cit; Cit-Phe; Leu-Cit; Cit-Leu; Ile-Cit; Cit-Ile; Phe-Arg; Arg-Phe; Cit-Trp; and Trp-Cit; and a self-immolative spacer, wherein the self-immolative spacer comprises one or more protecting (triggering) groups that are sensitive to acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase-induced cleavage, phosphodiesterase-induced cleavage, phosphatase-induced cleavage, protease-induced cleavage, lipase-induced cleavage, or disulfide bond cleavage.

Non-limiting examples of such self-immolative spacers include:

[Here,

PG is the triggering device;

X _a is O, NH or S;

X _b is O, NH, NCH ₃ or S;

X _c is O or NH;

Y _a is CH ₂ , CH ₂ O or CH ₂ NH;

Y _b is CH ₂ , O or NH;

Y _c is a bond, CH ₂ , O or NH;

LG is a leaving group such as the drug moiety (D) of the linker-drug group of the present invention.

Additional non-limiting examples of such self-immolative spacers are described in the literature [Angew. Chem. Int. Ed. 2015, 54, 7492 - 7509].

Additionally, the conjugate linker component may be a chemical moiety readily formed by a reaction between two reactive groups. Non-limiting examples of such chemical moieties are provided in Table 8.

Table 8.

Here, R ³² in Table 8 is H, C _1-4 alkyl, phenyl, pyrimidine or pyridine; R ³⁵ in Table 8 is H, C _1-6 alkyl, phenyl, or C _1-4 alkyl substituted with 1 to 3 -OH groups; each R ⁷ in Table 8 is independently selected from H, C _1-6 alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C _1-4 alkoxy substituted with -C(=O)OH, and C _1-4 alkyl substituted with -C(=O)OH; R ³⁷ in Table 8 is independently selected from H, phenyl and pyridine; q in Table 8 is 0, 1, 2 or 3; R ⁸ or R ¹³ in Table 8 is H or methyl; In Table 8, R ⁹ or R ¹⁴ is H, -CH ₃ or phenyl; in Table 8, R is H or any suitable substituent; in Table 8, R ⁵⁰ is H.

Additionally, the conjugate linker component may be a group listed in Table 9 below.

Table 9.

As used herein, when a partial structure of a compound is illustrated, a wavy line ( ) represents the attachment point of the substructure to the rest of the molecule.

As used herein, the terms "self-immolative spacer" and "self-immolative group" refer to a moiety comprising one or more triggering groups (TGs) that are activated by acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase-induced cleavage, phosphodiesterase-induced cleavage, phosphatase-induced cleavage, protease-induced cleavage, lipase-induced cleavage, or disulfide bond cleavage, wherein after activation, the protecting group is removed, which generates a cascade of cleavage reactions resulting in the temporally sequential release of the leaving group. Such reaction cascade may be, but is not limited to, 1,4-, 1,6-, or 1,8-elimination reactions.

Non-limiting examples of self-immolative spacers or gi include:

(Here, these groups can be optionally substituted,

TG is the triggering device;

X _a is O, NH or S;

X _b is O, NH, NCH ₃ or S;

X _c is O or NH;

Y _a is CH ₂ , CH ₂ O or CH ₂ NH;

Y _b is CH ₂ , O or NH;

Y _c is a bond, CH ₂ , O or NH;

LG is a leaving group such as the drug moiety (D) of the linker-drug group of the present invention.

Additional non-limiting examples of self-immolative spacers are described in Angew. Chem. Int. Ed. 2015, 54, 7492 - 7509.

In certain embodiments, the self-sacrificing spacer is structurally

A moiety having, wherein Lp is an enzymatically cleavable divalent peptide spacer, and A, D, L ₃ and R ² are as defined herein.

In a preferred embodiment, the self-sacrificing spacer is structurally

A moiety having, wherein Lp is an enzymatically cleavable divalent peptide spacer, and D, L ₃ and R ² are as defined herein. In some embodiments, D is a quaternized tertiary amine-containing panRAS inhibitor.

In another preferred embodiment, the self-sacrificing spacer is structurally

A moiety having, wherein Lp is an enzymatically cleavable divalent peptide spacer, and D, L ₃ and R ² are as defined herein.

As used herein, the term "hydrophilic moiety" refers to a moiety having hydrophilicity that increases the water solubility of a drug moiety (D) when the drug moiety (D) is attached to a conjugate linker group of the present invention. Examples of such hydrophilic groups include polyethylene glycol, polyalkylene glycol, sugars, oligosaccharides, polypeptides, 1 to 3 Including, but not limited to, C ₂ -C ₆ alkyl substituted with a group.

drug moiety

In some embodiments, an intermediate that is a precursor of a conjugate linker moiety reacts with a drug moiety (e.g., a panRAS inhibitor) under appropriate conditions. In some embodiments, a reactive group is used on the drug and/or the intermediate or conjugate linker. The reaction product between the drug and the intermediate or the derivatized drug (drug+conjugate linker) is subsequently reacted with the antibody or antigen-binding fragment under conditions that facilitate conjugation of the drug and the intermediate or derivatized drug with the antibody or antigen-binding fragment. Alternatively, the intermediate or conjugate linker may first be reacted with the antibody or antigen-binding fragment or the derivatized antibody or antigen-binding fragment, and then reacted with the drug or derivatized drug.

A number of different reactions are available for covalently attaching drug moieties and/or conjugate linker moieties to antibodies or antigen-binding fragments. This is often accomplished by reacting one or more amino acid residues of the antibody or antigen-binding fragment, including the amine group of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl group of cysteine, and various moieties of aromatic amino acids. For example, nonspecific covalent attachment can be initiated using a carbodiimide reaction to link a carboxyl (or amino) group on the drug moiety to an amino (or carboxyl) group on the antibody or antigen-binding fragment. Bifunctional agents, such as dialdehydes or imidoesters, can also be used to link an amino group on the drug moiety to an amino group on the antibody or antigen-binding fragment. Another option for attaching drugs (e.g., panRAS inhibitors) to binding agents is the Schiff base reaction. This method involves periodate oxidation of a drug containing a glycol or hydroxyl group to form an aldehyde, which is then reacted with a binding agent. Attachment occurs by forming a Schiff base with the amino group of the binding agent. Isothiocyanates can also be used as coupling agents to covalently attach the drug to the binding agent. Other techniques are known to those skilled in the art and are within the scope of the present invention. Examples of drug moieties that can be generated using various chemistries known in the art and linked to antibodies or antigen-binding fragments include panRAS inhibitors, such as the panRAS inhibitors described and exemplified herein.

Suitable drug moieties may include compounds of formula Ia, I, Ic, If, Ig, Ih, Ij, Ik, Im, or In, or enantiomers, diastereomers, and/or addition salts thereof with pharmaceutically acceptable acids or bases. Furthermore, the drug moiety may include any of the panRAS inhibitors (D) described herein.

In some embodiments, the drug moiety (D) comprises a chemical formula selected from Table A2.

In some embodiments, the drug moiety (D) comprises a panRAS inhibitor known in the art, for example as disclosed in WO2021/091956 or WO2022/060836, which are incorporated herein by reference in their entirety.

In some embodiments, the drug moiety (D) comprises a panRAS inhibitor selected from:

In some embodiments, the linker-drug (or “linker-payload”) moiety -(L-D) may comprise an enantiomer, diastereomer, deuterated derivative and/or pharmaceutically acceptable salt of a compound of Table B or any of the foregoing.

Definition of terms in drug moieties

Those skilled in the art will appreciate that certain compounds described herein may exist in one or more different isomers (e.g., stereoisomers, geometric isomers, atropisomers, tautomers) or isotopic forms (e.g., where one or more atoms are replaced with a different isotope of that atom, e.g., where hydrogen is replaced with deuterium). Unless otherwise indicated or clear from context, the depicted structures are to be understood to represent any such isomeric or isotopic forms, individually or in combination.

The compounds described herein may be asymmetric (e.g., having more than one stereocenter). Unless otherwise indicated, all stereoisomers, such as enantiomers and diastereomers, are intended.

Compounds of the present invention containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically active starting materials are well known in the art, such as by resolution of racemic mixtures or stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, etc., may also exist in the compounds described herein, and all such stable isomers are contemplated by the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and can be isolated (either as mixtures of isomers or as separate isomeric forms).

In some embodiments, one or more of the compounds depicted herein may exist in different tautomeric forms. Unless explicitly excluded, reference to such compounds encompasses all such tautomeric forms, as the context clearly indicates. In some embodiments, a tautomeric form is formed by the exchange of a single bond with an adjacent double bond, thereby transferring a proton. In certain embodiments, a tautomeric form may be a protic tautomer, which is an isomeric protonation state having the same empirical formula and net charge as the reference form. Examples of moieties having protic tautomeric forms include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-imine pairs, and ring forms in which protons can occupy two or more positions of a heterocyclic system, such as 1H- and 3H-imidazoles, 1H-, 2H-, and 4H-1,2,4-triazoles, 1H- and 2H-isoindoles, 1H- and 2H-pyrazoles. In some embodiments, the tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution. In a particular embodiment, the tautomeric forms are produced by acetal interconversion.

Unless otherwise specified, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. Exemplary isotopes that may be incorporated into the compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ³² P, ³³ P, ³⁵ S, ¹⁸ F, ³⁶ Cl, ¹²³ I, and ¹²⁵ I. Isotopically labeled compounds (e.g., compounds labeled with ³ H and ¹⁴ C) may be useful for compound or substrate tissue distribution analysis. Tritium (i.e., ³ H) and carbon-14 (i.e., ¹⁴ C) isotopes may be useful because of their detectability and ease of preparation. Additionally, substitution with heavier isotopes, such as deuterium (i.e., ² H), may provide certain therapeutic advantages (e.g., increased in vivo half-life or reduced dosing requirements) due to greater metabolic stability. In some embodiments, one or more hydrogen atoms are replaced with ² H or ³ H, or one or more carbon atoms are replaced with a ¹³ C- or ¹⁴ C-enriched carbon. Positron-emitting isotopes such as ¹⁵ O, ¹³ N, ¹¹ C, and ¹⁸ F are useful in positron emission tomography (PET) studies to investigate substrate receptor occupancy.

The preparation of isotopically labeled compounds is well known to those skilled in the art. For example, isotopically labeled compounds can generally be prepared by substituting an isotopically labeled reagent for a non-isotopically labeled reagent, following procedures similar to those disclosed for the compounds of the invention described herein.

As is known in the art, many chemical entities can take on a variety of different solid forms, such as amorphous or crystalline forms (e.g., polymorphs, hydrates, solvates). In some embodiments, the compounds of the present invention can be utilized in any such form (including any solid form). In some embodiments, the compounds described or depicted herein can be provided or utilized in the form of hydrates or solvates.

In several places throughout this specification, substituents of the compounds of the invention are disclosed in groups or ranges. The invention is specifically intended to encompass each and every individual subcombination of members of such ranges and groups. For example, the term "C ₁ -C ₆ alkyl" is specifically intended to individually disclose methyl, ethyl, C ₃ alkyl, C ₄ alkyl, C ₅ alkyl, and C ₆ alkyl. Furthermore, when a compound includes multiple positions where substituents are disclosed in groups or ranges, the invention is intended to encompass individual compounds and groups of compounds (e.g., genera and subgenera) containing each and every individual subcombination of members at each position, unless otherwise indicated.

The term "optionally substituted X" (e.g., "optionally substituted alkyl") is intended to be equivalent to "X, wherein X is optionally substituted" (e.g., "alkyl, wherein said alkyl is optionally substituted"). This is not intended to imply that the feature "X" (e.g., alkyl) itself is optional. As described herein, a particular compound of interest may contain one or more "optionally substituted" moieties. In general, the term "substituted," whether preceded by the term "optionally," means that one or more hydrogens of the designated moiety are replaced by a suitable substituent, e.g., any of the substituents or groups described herein. Unless otherwise indicated, an optionally substituted group may have a suitable substituent at each substitutable position of the group, and where more than one position in any given structure may be substituted with one or more substituents selected from a particular group, the substituents may be the same or different at all positions. For example, in the term "optionally substituted C ₁ -C ₆ alkyl-C ₂ -C ₉ heteroaryl", the alkyl moiety, the heteroaryl moiety, or both, may be optionally substituted. Combinations of substituents contemplated by the present invention are preferably those that form stable or chemically feasible compounds. As used herein, the term "stable" refers to a compound that is substantially unchanged when subjected to conditions that permit the production, detection, and in certain embodiments, recovery, purification, and use thereof for one or more of the purposes disclosed herein.

Suitable monovalent substituents on the replaceable carbon atoms of the "optionally substituted" group are independently deuterium; halogen; -(CH ₂ ) _0-4 R°; -(CH ₂ ) _0-4 OR°; -O(CH ₂ ) _0-4 R ^o ; -O-(CH ₂ ) _0-4 C(O)OR°; -(CH ₂ ) _0-4 CH(OR°) ₂ ; -(CH ₂ ) _0-4 SR°; -(CH ₂ ) _0-4 Ph which may be substituted with R°; -(CH ₂ ) _0-4 O(CH ₂ )0-1Ph which may be substituted with R°; -CH=CHPh which may be substituted with R°; -(CH ₂ ) _0-4 O(CH ₂ )0-1-pyridyl which may be substituted with R°; 4-8 membered saturated or unsaturated heterocycloalkyl (e.g., pyridyl); 3-8 membered saturated or unsaturated cycloalkyl (e.g., cyclopropyl, cyclobutyl, or cyclopentyl); -NO ₂ ; -CN; -N ₃ ; -(CH ₂ ) _0-4 N(R°) ₂ ; -(CH ₂ ) _0-4 N(R°)C(O)R°; -N(R°)C(S)R°; -(CH 2 ) 0-4 N(R°)C(O)NR° 2 ; -N(R°)C(S)NR° ₂ ; -(CH _{2 ) 0-4 N} (R°)C(O)OR°; - N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR° _{2 ;} -N(R°)N(R°)C(O)OR°; -(CH ₂ ) _0-4 C(O)R°; -C(S)R°; -(CH ₂ ) _0-4 C(O)OR°; -(CH ₂ ) _0-4 -C(O)-N(R°) ₂ ; -(CH ₂ ) _0-4 -C(O)-N(R°)-S(O) ₂ -R°; -C(NCN)NR° ₂ ; -(CH ₂ ) _0-4 C(O)SR°; -(CH ₂ ) _0-4 C(O)OsiR° ₃ ; -(CH ₂ ) _0-4 OC(O)R°; -OC(O)(CH ₂ ) _0-4 SR°; -SC(S)SR°; -(CH ₂ ) _0-4 SC(O)R°; -(CH ₂ ) _0-4 C(O)NR° ₂ ; -C(S)NR° ₂ ; -C(S)SR°; -(CH ₂ ) _0-4 OC(O)NR° ₂ ; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH ₂ C(O)R°; -C(NOR°)R°; -(CH ₂ ) _0-4 SSR°; -(CH ₂ ) _0-4 S(O) ₂ R°; -( CH ₂ ) _0-4 S(O) ₂ OR°; -(CH ₂ ) _0-4 OS(O) ₂ R°; -S(O) ₂ NR° ₂ ; -(CH ₂ ) _0-4 S(O)R°; -N(R°)S(O) ₂ NR° ₂ ; -N(R°)S(O) ₂ R°; -N(OR°)R°; -C(NOR°)NR° ₂ ; -C(NH)NR° ₂ ; -P(O) ₂ R°; -P(O)R° ₂ ; -P(O)(OR°) ₂ ; -OP(O)R° ₂ ; -OP(O)(OR°) ₂ ; -OP(O)(OR°)R°, -SiR° ₃ ; -(C _1-4 straight or branched alkylene)ON(R°) ₂ ; or -(C _1-4 straight or branched alkylene)C(O)ON(R°) ₂ (wherein each R° may be substituted as defined below and may independently be hydrogen, -C _1-6 aliphatic, -CH ₂ Ph, -O(CH ₂ ) _0-1 Ph, -CH ₂ -(5-6 membered heteroaryl ring), or a 3-6 membered saturated, partially unsaturated, or aryl ring (having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur), or, notwithstanding the above definition, two independent R° may be taken together with their intervening atom(s) to form a 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring (having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur), which may be substituted as defined below).

Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include: =O, =S, =NNR ^* 2, =NNHC(O)R ^* , =NNHC(O)OR ^* , =NNHS(O) ₂ R ^* , =NR ^* , =NOR ^* , -O(C(R ^* 2)) ₂ -3O-, or -S(C(R ^* 2)) ₂ -3S- (wherein each independent R ^* is selected from hydrogen, C _1-6 aliphatic (which may be substituted as defined below), or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur). Suitable divalent substituents bonded to adjacent substitutable carbons of an "optionally substituted" group include: -O(CR ^* 2) _2-3O- , wherein each independent R ^* is selected from hydrogen, _C1-6 aliphatic (which may be substituted as defined below), or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring (having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur).

Suitable substituents on the aliphatic group of R ^* include: halogen, - , -(Hello ), -OH, -O , -O(halo ), -CN, -C(O)OH, -C(O)O , -NH2, -NH , -N ₂ , or -NO ₂ (wherein, each is unsubstituted or, if preceded by "halo", substituted by one or more halogens, independently C _1-4 aliphatic, -CH ₂ Ph, -O(CH ₂ )0-1Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring (having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur).

Suitable substituents on the substitutable nitrogen of the "optional substitution" group include: - , -N 2, -C(O) , -C(O)O , -C(O)C(O) , -C(O)CH ₂ C(O) , -S(O) ₂ , -S(O) ₂ N 2, -C(S)N 2, -C(NH)N 2, or -N( )S(O) ₂ (Here, each is independently hydrogen, C _1-6 aliphatic (which may be substituted as defined below), unsubstituted -Oph, or an unsubstituted 3-6 membered saturated, partially unsaturated, or aryl ring (having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur), or, notwithstanding the above definition, two independent , taken together with its intervening atom(s) to form an unsubstituted 3- to 12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring (having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur).

Suitable substituents on the aliphatic group are independently halogen, - , -(Hello ), -OH, -O , -O(halo ), -CN, -C(O)OH, -C(O)O , -NH2, -NH , -N ₂ , or -NO ₂ , where each is unsubstituted or, if preceded by "halo", substituted with one or more halogens, independently C _1-4 aliphatic, -CH ₂ Ph, -O(CH ₂ )0-1Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring (having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur). Suitable divalent substituents on the saturated carbon atom of R ^† include =O and =S.

As used herein, the term “acetyl” refers to the group —C(O)CH ₃ .

As used herein, the term “alkoxy” refers to a —OC ₁ -C ₂₀ alkyl group, wherein the alkoxy group is attached to the remainder of the compound through an oxygen atom.

As used herein, the term "alkyl" refers to a saturated, straight-chain, or branched monovalent hydrocarbon group containing 1 to 20 carbons (e.g., 1 to 10 or 1 to 6). In some embodiments, the alkyl group is unbranched (i.e., linear); and in some embodiments, the alkyl group is branched. Alkyl groups are exemplified by, but not limited to, methyl, ethyl, n- and iso-propyl, n-, sec-, iso-, and tert-butyl, and neopentyl.

As used herein, the term "heteroalkyl" refers to an "alkyl" group, as defined herein, in which at least one carbon atom is replaced by a heteroatom (e.g., an O, N, or S atom). The heteroatom may appear in the middle or at the end of the radical.

As used herein, the term "alkylene" refers to a saturated divalent hydrocarbon group derived from a straight or branched chain saturated hydrocarbon by the removal of two hydrogen atoms, and is exemplified by methylene, ethylene, isopropylene, and the like. The term "Cx-Cy alkylene" refers to an alkylene group having x to y carbons. Exemplary values of x include 1, 2, 3, 4, 5, and 6, and exemplary values of y include 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or ₂₀ (e.g., C ₁ -C ₆ , C ₁ -C ₁₀ , C 2 -C ₂₀ , C ₂ -C ₆ , C ₂ -C ₁₀ , or C ₂ -C ₂₀ alkylene). In some embodiments, the alkylene may be further substituted with 1, 2, 3 or 4 substituents as defined herein.

As used herein, unless otherwise specified, the term "alkenyl" refers to a monovalent straight or branched chain group having 2 to 20 carbon atoms (e.g., 2 to 6 or 2 to 10 carbon atoms) containing one or more carbon-carbon double bonds, and is exemplified by ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, and 2-butenyl. Alkenyl includes both cis and trans isomers. As used herein, the term "alkenylene" refers to a divalent straight or branched chain group having 2 to 20 carbon atoms (e.g., 2 to 6 or 2 to 10 carbon atoms) containing one or more carbon-carbon double bonds, unless otherwise specified.

As used herein, the term "alkynyl" refers to a monovalent straight or branched chain group having 2 to 20 carbon atoms (e.g., 2 to 4, 2 to 6, or 2 to 10 carbon atoms) containing a carbon-carbon triple bond, exemplified by ethynyl and 1-propynyl.

As used herein, the term "amino" means -N( ) ₂ , for example, -NH ₂ and -N(CH ₃ ) ₂ .

As used herein, the term “aminoalkyl” refers to an alkyl moiety substituted on one or more carbon atoms with one or more amino moieties.

As used herein, the term "amino acid" refers to a molecule having a side chain, an amino group, and an acid group (e.g., -CO ₂ H or -SO ₃ H), wherein the amino acid is attached to a parent molecular group by the side chain, the amino group, or the acid group (e.g., the side chain). As used herein, the term "amino acid" in its broadest sense refers to any compound or substance that can be incorporated into a polypeptide chain, for example, via the formation of one or more peptide bonds. In some embodiments, the amino acid has the general structure H ₂ NC(H)(R ^A* )-COOH, wherein R ^A* is any chemically feasible substituent described herein. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid; in some embodiments, the amino acid is a D-amino acid; and in some embodiments, the amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, optionally substituted hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrolysine, selenocysteine, serine, taurine, threonine, tryptophan, tyrosine, and valine.

As used herein, the term "aryl" refers to a monovalent monocyclic, bicyclic, or polycyclic ring system formed by carbon atoms, wherein the ring attached to the pendant group is aromatic. Examples of aryl groups include phenyl, naphthyl, phenanthrenyl, and anthracenyl. An aryl ring may be attached to its pendant group at any heteroatom or carbon ring atom (which produces a stable structure), and any ring atom may be optionally substituted unless otherwise specified. In some embodiments, aryl refers to a phenyl, naphthyl, biphenyl, or indenyl group.

As used herein, the term "C ₀ " represents a bond. For example, part of the term -N(C(O)-(C ₀ -C ₅ alkylene-H)- includes -N(C(O)-(C ₀ alkylene-H)-, which is also represented as -N(C(O)-H)-.

As used herein, the terms "carbocyclic" and "carbocyclyl" refer to a monovalent optionally substituted C ₃ -C ₁₂ monocyclic, bicyclic, or tricyclic ring structure (which may be bridged, fused, or spirocyclic), wherein all rings are formed of carbon atoms and at least one ring is non-aromatic.

Carbocyclic structures include cycloalkyl, cycloalkenyl, and cycloalkynyl groups. Examples of carbocyclyl groups include cyclohexyl, cyclohexenyl, cyclooctynyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indenyl, indanyl, and decalinyl. The carbocyclic ring may be attached to its pendant group at any ring atom (which produces a stable structure), and any ring atom may be optionally substituted unless otherwise specified.

As used herein, the term “carbonyl” refers to a C(O) group, which may also be represented as C=O.

As used herein, the term "carboxyl" means -CO ₂ H, (C=O)(OH), COOH, or C(O)OH or the unprotonated counterpart.

As used herein, the term “cyano” refers to a -CN group.

As used herein, the term "diastereoisomer" refers to stereoisomers that are not mirror images of each other and are not superimposable.

As used herein, the term "enantiomer" means each individual optically active form of a compound of the present invention, which has an optical purity or enantiomeric excess (as determined by standard methods in the art) of at least 80% (i.e., at least 90% of one enantiomer and no more than 10% of the other enantiomer), preferably at least 90%, more preferably at least 98%.

As used herein, the term “haloalkyl” refers to an alkyl moiety substituted on one or more carbon atoms with one or more same or different halogen moieties.

As used herein, the term “halogen” refers to a halogen selected from bromine, chlorine, iodine or fluorine.

As used herein, the term "heteroalkyl" refers to an "alkyl" group, as defined herein, in which at least one carbon atom is replaced by a heteroatom (e.g., an O, N, or S atom). The heteroatom may appear in the middle or at the end of the radical.

As used herein, the term "heteroaryl" refers to a monovalent, monocyclic, or polycyclic ring structure containing at least one fully aromatic ring (i.e., containing 4n+2 pi electrons within the monocyclic or polycyclic ring system) and containing at least one ring heteroatom selected from N, O, or S within said aromatic ring. Exemplary unsubstituted heteroaryl groups have from 1 to 12 carbon atoms (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9). The term "heteroaryl" includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heteroaromatic rings are fused to one or more aryl or carbocyclic rings, for example, a phenyl ring, or a cyclohexane ring. Examples of heteroaryl groups include, but are not limited to, pyridyl, pyrazolyl, benzoxazolyl, benzoimidazolyl, benzothiazolyl, imidazolyl, thiazolyl, quinolinyl, tetrahydroquinolinyl, and 4-azaindolyl. The heteroaryl ring can be attached to its pendant group at any ring atom (which produces a stable structure), and any ring atom can be optionally substituted unless otherwise specified. In some embodiments, the heteroaryl is substituted with 1, 2, 3, or 4 substituents. In some embodiments, the heteroaryl is any monocyclic or bicyclic group consisting of 5 to 10 ring members, having at least one aromatic moiety, and containing 1 to 4 heteroatoms selected from oxygen, sulfur, and nitrogen (including quaternary nitrogen).

As used herein, the term "heterocycloalkyl" refers to a monovalent monocyclic, bicyclic, or polycyclic ring system which may be bridged, fused, or spirocyclic, wherein at least one ring is non-aromatic, and wherein said non-aromatic ring contains 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. Five-membered rings have 0 to 2 double bonds, and six- and seven-membered rings have 0 to 3 double bonds. Exemplary unsubstituted heterocycloalkyl groups are those having 1 to 12 carbon atoms (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9).

The term "heterocycloalkyl" also denotes heterocyclic compounds having a bridged polycyclic structure, wherein one or more carbon or heteroatoms bridge two non-adjacent members of a monocyclic ring, for example a quinuclidinyl group. The term "heterocycloalkyl" includes bicyclic, tricyclic and tetracyclic groups in which any of the above heterocyclic rings is fused to one or more aromatic, carbocyclic, heteroaromatic or heterocyclic rings, for example an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, a pyridine ring or a pyrrolidine ring.

Examples of heterocycloalkyl groups include pyrrolidinyl, piperidinyl, 1,2,3,4-tetrahydroquinolinyl, decahydroquinolinyl, dihydropyrrolopyridine, and decahydronaphthyridinyl. The heterocycloalkyl ring may be attached to its pendant group at any ring atom (which produces a stable structure), and any ring atom may be optionally substituted unless otherwise specified.

As used herein, the term “hydroxy” refers to an -OH group.

As used herein, the term “hydroxyalkyl” refers to an alkyl moiety substituted on one or more carbon atoms with one or more —OH moieties.

As used herein, the term "isomer" means a tautomer, stereoisomer, atropisomer, enantiomer, or diastereomer of any compound of the invention. It is recognized that the compounds of the invention may have one or more chiral centers or double bonds, and thus may exist as stereoisomers, such as double bond isomers (i.e., geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis/trans isomers). In accordance with the invention, the chemical structures depicted herein, and thus the compounds of the invention, include all corresponding stereoisomers, i.e., stereoisomerically pure forms (e.g., geometrically pure forms, enantiomerically pure forms, or diastereomerically pure forms), and enantiomeric mixtures and stereoisomer mixtures, e.g., racemates. Enantiomeric mixtures and stereoisomeric mixtures of the compounds of the present invention can typically be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral phase gas chromatography, chiral phase high performance liquid chromatography, crystallizing the compounds as chiral salt complexes, or crystallizing the compounds in chiral solvents. Enantiomers and stereoisomers can also be obtained from stereoisomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

As used herein, the term "drug linker" refers to a divalent organic moiety that connects moiety B D to moiety W ^D in a compound of formula I such that the resulting compound achieves an Ic50 of 2 μM or less in the Ras- ^RAF disruption assay protocol provided in the Examples below and provided herein.

The objective of this biochemical assay is to determine the ability of test compounds to promote the formation of a ternary complex between nucleotide-loaded Ras isoforms and cyclophilin A; the resulting ternary complex inhibits Ras signaling through RAF effectors by interfering with the binding of the BRAF ^RBD construct.

Untagged cyclophilin A, His6-K-Ras-GMPPNP (or other Ras mutant), and GST-BRAF _RBD are combined in an assay buffer containing 25 mM HEPES pH 7.3, 0.002% Tween 20, 0.1% BSA, 100 mM NaCl, and 5 mM MgCl ² in a 384-well assay plate at final concentrations of 25 μM, 12.5 nM, and 50 nM, respectively. Compounds are present in the plate wells as a 10-point 3-fold dilution series starting at a final concentration of 30 μM. After incubation at 25°C for 3 h, a mixture of anti-His Eu-W1024 and anti-GST allophycocyanin is then added to the assay sample wells at final concentrations of 10 nM and 50 nM, respectively, and the reactions are incubated for an additional 1.5 h. The TR-FRET signal is read on a microplate reader (Ex 320 nm, Em 665/615 nm). Compounds that promote the disruption of the Ras:RAF complex are identified as compounds that decrease the TR-FRET ratio compared to the DMSO control wells.

In some embodiments, the drug linker comprises 20 or fewer linear atoms. In some embodiments, the drug linker comprises 15 or fewer linear atoms. In some embodiments, the drug linker comprises 10 or fewer linear atoms. In some embodiments, the drug linker has a molecular weight less than 500 g/mol. In some embodiments, the drug linker has a molecular weight less than 400 g/mol. In some embodiments, the drug linker has a molecular weight less than 300 g/mol. In some embodiments, the drug linker has a molecular weight less than 200 g/mol. In some embodiments, the drug linker has a molecular weight less than 100 g/mol. In some embodiments, the drug linker has a molecular weight less than 50 g/mol.

As used herein, the term "stereoisomer" refers to all possible isomeric forms and configurational forms that a compound may have (e.g., a compound of any of the formulae described herein), specifically all possible stereochemical and conformational isomeric forms of the basic molecular structure, all diastereomers, enantiomers, or conformational isomers (including atropisomers). Some compounds of the present invention may exist in different tautomeric forms, all of which are within the scope of the present invention.

As used herein, the term “sulfonyl” refers to the group —S(O) ₂ —.

As used herein, the term “thiocarbonyl” refers to the group —C(S)—.

Drug loading

Drug loading is denoted by p and is also referred to herein as drug-to-antibody ratio (DAR). Drug loading can range from 1 to 16 drug moieties per antibody or antigen-binding fragment. In some embodiments, p is an integer from 1 to 16. In some embodiments, p is an integer from 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is an integer from 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments, p is an integer from 1 to 16. In some embodiments, p is an integer from 1 to 8. In some embodiments, p is an integer from 1 to 5. In some embodiments, p is an integer from 2 to 4. In some embodiments, p is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 4.

Drug loading may be limited by the number of attachment sites on the antibody or antigen-binding fragment. In some embodiments, the conjugate linker moiety (L) of the ADC is attached to the antibody or antigen-binding fragment via a chemically active group on one or more amino acid residues on the antibody or antigen-binding fragment. For example, the conjugate linker may be attached to the antibody or antigen-binding fragment via a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., at the N or C terminus, to the epsilon amino group of one or more lysine residues, to the free carboxylic acid group of one or more glutamic or aspartic acid residues, or to the sulfhydryl group of one or more cysteine residues). The site to which the conjugate linker is attached may be a natural residue in the amino acid sequence of the antibody or antigen-binding fragment, or it may be introduced into the antibody or antigen-binding fragment, for example, by DNA recombinant techniques (for example, by introduction of a cysteine residue into the amino acid sequence) or by protein biochemistry (for example, by reduction, pH adjustment or hydrolysis).

In some embodiments, the number of drug moieties that can be conjugated to an antibody or antigen-binding fragment is limited by the number of free cysteine residues. For example, if attachment is to a cysteine thiol group, the antibody may have only one or a few cysteine thiol groups, or only one or a few sufficiently reactive thiol groups to which the conjugate linker can be attached. Typically, antibodies do not contain many free and reactive cysteine thiol groups that can be linked to a drug moiety. In fact, most cysteine thiol residues in antibodies are involved in interchain or intrachain disulfide bonds. Therefore, in some embodiments, conjugation to cysteine may require at least partial reduction of the antibody. Excessive attachment of the conjugate linker-toxin to the antibody may destabilize the antibody by reducing the cysteine residues available for disulfide bond formation. Therefore, the optimal drug:antibody ratio should increase the potency of the ADC (by increasing the number of drug moieties attached per antibody) without destabilizing the antibody or antigen-binding fragment. In some embodiments, the optimal ratio may be 2, 4, 6, or 8. In some embodiments, the optimal ratio may be 2 or 4.

In some embodiments, the antibody or antigen-binding fragment is exposed to reducing conditions prior to conjugation to generate one or more free cysteine residues. In some embodiments, the antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) under partial or complete reducing conditions to generate reactive cysteine thiol groups. Unpaired cysteines can be generated through partial reduction with limited molar equivalents of TCEP, which can reduce the interchain disulfide bonds connecting the two heavy chains in the hinge region (two pairs per H-H pairing in human IgG1) and the light and heavy chains (one pair per H-L pairing) while leaving the intrachain disulfide bonds intact (Stefano et al. (2013) Methods Mol Biol. 1045:145-71). In embodiments, disulfide bonds within the antibody are electrochemically reduced, for example, by using a working electrode that applies alternating reduction and oxidation voltages. This approach may allow online coupling of the disulfide bond reduction to an analytical device (e.g., an electrochemical detection device, NMR spectrometer, or mass spectrometer) or a chemical separation device (e.g., a liquid chromatograph (e.g., HPLC) or an electrophoretic device (see, e.g., US 2014/0069822)). In some embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups on amino acid residues, such as cysteine.

Drug loading of ADCs can be controlled in different ways, for example, by: (i) limiting the molar excess of drug-linker intermediate or conjugated linker reagent relative to the antibody; (ii) limiting the conjugation reaction time or temperature; (iii) by partial or limited reducing conditions for cysteine thiol modification; and/or (iv) by recombinantly engineering the amino acid sequence of the antibody to vary the number and position of cysteine residues to control the number and/or position of linker-drug attachments.

In some embodiments, a free cysteine residue is introduced into the amino acid sequence of an antibody or antigen-binding fragment. For example, a cysteine-engineered antibody can be prepared, in which one or more amino acids of the parent antibody are replaced with a cysteine amino acid. Any type of antibody can be engineered (i.e., mutated) in this manner. For example, a parent Fab antibody fragment can be engineered to form a cysteine-engineered Fab, referred to as a "ThioFab." Similarly, a parent monoclonal antibody can be engineered to form a "ThioMab." A single-site mutation results in a single engineered cysteine residue in a ThioFab, whereas a single-site mutation results in two engineered cysteine residues in a ThioMab due to the dimeric nature of IgG antibodies. DNA encoding amino acid sequence variants of a parent polypeptide can be prepared by various methods known in the art (see, e.g., methods described in WO 2006/034488). Such methods include, but are not limited to, site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared DNA encoding the polypeptide. Variants of recombinant antibodies can also be constructed by restriction enzyme fragment manipulation or by overlap extension PCR using synthetic oligonucleotides. ADCs of formula 1 include, but are not limited to, antibodies having one, two, three, or four engineered cysteine amino acids (Lyon et al. (2012) Methods Enzymol. 502:123-38). In some embodiments, one or more free cysteine residues are already present in the antibody or antigen-binding fragment without the use of engineering, in which case the existing free cysteine residues can be used to conjugate the antibody or antigen-binding fragment to a drug moiety.

When more than one nucleophilic group reacts with a drug-linker intermediate or a conjugate linker moiety reagent, followed by a drug moiety reagent, in a reaction mixture comprising multiple copies of an antibody or antigen-binding fragment and a conjugate linker moiety, the resulting product can be a mixture of ADC compounds having a distribution of one or more drug moieties attached to each copy of the antibody or antigen-binding fragment in the mixture. In some embodiments, the drug loading in the ADC mixture resulting from the conjugation reaction ranges from 1 to 16 drug moieties attached per antibody or antigen-binding fragment. The average number of drug moieties per antibody or antigen-binding fragment (i.e., average drug loading or average p) can be calculated by any conventional method known in the art, such as by mass spectrometry (e.g., liquid chromatography-mass spectrometry (LC-MS)) and/or high performance liquid chromatography (e.g., HIC-HPLC). In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is determined by liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 1.5 to about 3.5, about 2.5 to about 4.5, about 3.5 to about 5.5, about 4.5 to about 6.5, about 5.5 to about 7.5, about 6.5 to about 8.5, or about 7.5 to about 9.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 2 to about 4, about 3 to about 5, about 4 to about 6, about 5 to about 7, about 6 to about 8, about 7 to about 9, about 2 to about 8, or about 4 to about 8.

In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 2. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is 2.

In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 4. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, or about 4.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is 4.

In some embodiments, the term "about" as used in reference to the average number of drug moieties per antibody or antigen-binding fragment means ± 20%, 15%, 10%, 5%, or 1%. In one embodiment, the term "about" refers to a range of values that are 10% more or less than a stated value. In another embodiment, the term "about" refers to a range of values that are 5% more or less than a stated value. In yet another embodiment, the term "about" refers to a range of values that are 1% more or less than a stated value.

Individual ADC compounds or "species" can be identified in the mixture by mass spectrometry and separated, for example, by UPLC or HPLC, such as hydrophobic interaction chromatography (HIC-HPLC). In some embodiments, a homogeneous or nearly homogeneous ADC product having a single loading value can be isolated from the conjugation mixture, for example, by electrophoresis or chromatography.

In some embodiments, higher drug loadings (e.g., p > 16) may cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. Higher drug loadings may also negatively impact the pharmacokinetics (e.g., clearance) of certain ADCs. In some embodiments, lower drug loadings (e.g., p < 2) may reduce the potency of certain ADCs against target expressing cells. In some embodiments, the drug loading for the ADCs of the invention ranges from about 2 to about 16, from about 2 to about 10, from about 2 to about 8; from about 2 to about 6; from about 2 to about 5; from about 3 to about 5; from about 2 to about 4; or from about 4 to about 8.

In some embodiments, a drug loading and/or average drug loading of about 2 is achieved, for example, using partial reduction of intrachain disulfides of the antibody or antigen-binding fragment, and provides beneficial properties. In some embodiments, a drug loading and/or average drug loading of about 4 or about 6 or about 8 is achieved, for example, using partial reduction of intrachain disulfides of the antibody or antigen-binding fragment, and provides beneficial properties. In some embodiments, a drug loading and/or average drug loading of less than about 2 may result in unacceptably high levels of unconjugated antibody species, which may compete with the ADC for binding to the target antigen and/or may provide reduced therapeutic efficacy. In some embodiments, a drug loading and/or average drug loading of greater than about 16 may result in unacceptably high levels of product heterogeneity and/or ADC aggregation. Drug loadings exceeding about 16 and/or average drug loadings may also affect the stability of the ADC due to the loss of one or more chemical bonds required to stabilize the antibody or antigen-binding fragment.

The present invention encompasses methods for producing the described ADCs. Briefly, the ADCs comprise an antibody or antigen-binding fragment (e.g., an anti-EphA2 or anti-B7-H3 antibody or antigen-binding fragment), a drug moiety (e.g., a panRAS inhibitor), and a conjugate linker connecting the drug moiety and the antibody or antigen-binding fragment. In some embodiments, the ADCs can be prepared using a conjugate linker having a reactive functional group for covalently attaching the drug moiety to the antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is functionalized to produce a functional group that is reactive with the conjugate linker or drug-linker intermediate. For example, in some embodiments, a cysteine thiol of the antibody or antigen-binding fragment can form a bond with a reactive functional group of the conjugate linker or drug-linker intermediate to produce the ADC. In some embodiments, the antibody or antigen-binding fragment is prepared using bacterial transglutaminase (BTG) (a reactive glutamine specifically functionalized with an amine containing a cyclooctyne BCN ( N -[(1 R ,8 S ,9 s )-bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl]-1,8-diamino-3,6-dioxaoctane) moiety). In some embodiments, site-specific conjugation of a conjugate linker or drug-linker intermediate to the BCN moiety of the antibody or antigen-binding fragment is performed, for example, as described and exemplified herein. Production of ADCs can be accomplished by techniques known to the skilled artisan.

In some embodiments, the ADC is produced by sequentially contacting an antibody or antigen-binding fragment (e.g., an anti-EphA2 or anti-B7-H3 antibody or antigen-binding fragment) with a conjugate linker and a drug moiety (e.g., a panRAS inhibitor), such that the antibody or antigen-binding fragment is first covalently linked to the conjugate linker, and then a preformed antibody-linker intermediate is reacted with the drug moiety. The antibody-linker intermediate may or may not undergo a purification step prior to contacting the drug moiety. In other embodiments, the ADC is produced by contacting the antibody or antigen-binding fragment with a linker-drug compound preformed by reaction of a conjugate linker and a drug moiety. The preformed linker-drug compound may or may not undergo a purification step prior to contacting with the antibody or antigen-binding fragment. In another embodiment, the antibody or antigen-binding fragment is contacted with a conjugate linker and a drug moiety in a single reaction mixture, thereby allowing simultaneous formation of covalent bonds between the antibody or antigen-binding fragment and the conjugate linker, and between the conjugate linker and the drug moiety. The method for producing an ADC may comprise a reaction wherein the antibody or antigen-binding fragment is contacted with the antibody or antigen-binding fragment before adding the conjugate linker to the reaction mixture, or vice versa. In some embodiments, the ADC is produced by reacting the antibody or antigen-binding fragment with a conjugate linker linked to a drug moiety, such as a panRAS inhibitor, under conditions that permit conjugation.

The ADC prepared according to the method described above may be subjected to a purification step. The purification step may include any biochemical method known in the art for purifying proteins, or any combination thereof. These include, but are not limited to, tangential flow filtration (TFF), affinity chromatography, ion exchange chromatography, any charge- or isoelectric-point-based chromatography, mixed-mode chromatography, e.g., CHT (ceramic hydroxyapatite), hydrophobic interaction chromatography, size exclusion chromatography, dialysis, filtration, selective precipitation, or any combination thereof.

Therapeutic uses and compositions

Disclosed herein are compositions, e.g., disclosed ADC compounds and methods of using the compositions, described herein, for treating a subject with a disorder, e.g., cancer. The compositions, e.g., ADCs, may be administered alone or in combination with at least one additional inert and/or active agent, e.g., at least one additional therapeutic agent, and may be administered in any pharmaceutically acceptable formulation, dosage, and administration regimen. Therapeutic efficacy may be assessed for toxicity and efficacy indicators and adjusted accordingly. Efficacy measures include, but are not limited to, cytostatic and/or cytotoxic effects, reduced tumor volume, tumor growth inhibition, and/or prolonged survival observed in vitro or in vivo.

Methods for determining whether an ADC exerts a cytostatic and/or cytotoxic effect on cells are known. For example, the cytotoxic or cytostatic activity of an ADC can be measured by, for example, exposing mammalian cells expressing the target antigen of the ADC in cell culture medium; culturing the cells for a period of about 6 hours to about 6 days; and measuring cell viability (e.g., using a CellTiter-Glo® (CTG) or MTT cell viability assay). Cell-based in vitro assays can also be used to measure apoptosis induction (caspase activation), cytotoxicity, and viability (proliferation) of an ADC.

To determine cytotoxicity, necrosis or apoptosis (programmed cell death) can be measured. Necrosis is typically accompanied by increased plasma membrane permeability, cell swelling, and plasma membrane rupture. Apoptosis can be quantified, for example, by measuring DNA fragmentation. Commercial photometric methods for quantitative in vitro measurement of DNA fragmentation are available. Examples of such assays, including TUNEL (which detects the incorporation of labeled nucleotides into fragmented DNA) and ELISA-based assays, are described in the literature [Biochemica (1999) 2:34-7 (Roche Molecular Biochemicals)].

Apoptosis can also be determined by measuring morphological changes in cells. For example, as with necrosis, loss of plasma membrane integrity can be determined by measuring the uptake of specific dyes (e.g., fluorescent dyes such as acridine orange or ethidium bromide). Methods for determining the number of apoptotic cells are described in Duke and Cohen, Current Protocols in Immunology (Coligan et al., eds. (1992) pp. 3.17.1-3.17.16). Cells can also be labeled with DNA dyes (e.g., acridine orange, ethidium bromide, or propidium iodide), and cells can be observed for chromatin condensation and marginal tropism along the inner nuclear membrane. Apoptosis can also be determined, in some embodiments, by screening for caspase activity. In some embodiments, the Caspase-Glo® Assay can be used to measure the activity of caspase-3 and caspase-7. In some embodiments, the assay provides a luminescent caspase-3/7 substrate in a reagent optimized for caspase activity, luciferase activity, and cell lysis. In some embodiments, addition of the Caspase-Glo® 3/7 reagent in an "add-mix-measure" format can result in cell lysis, followed by substrate cleavage by caspase and a "glow-type" luminescent signal generated by luciferase. In some embodiments, luminescence can be proportional to the amount of caspase activity present and can serve as an indicator of apoptosis. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane blebbing, and cell shrinkage. Determination of any of these effects on cancer cells indicates that the ADC is useful in cancer treatment.

Cell viability can be measured by determining the uptake of a dye, such as neutral red, trypan blue, crystal violet, or ALAMAR™ blue, by the cells (see, e.g., Page et al. (1993) Intl J Oncology 3:473-6). In such assays, cells are incubated in medium containing the dye, the cells are washed, and the remaining dye, which reflects cellular uptake of the dye, is measured spectrophotometrically.

Cell viability can also be measured, for example, by quantifying ATP, an indicator of metabolically active cells. In some embodiments, the in vitro potency and/or cell viability of the prepared ADC or panRAS inhibitor compound can be assessed using the CellTiter-Glo® (CTG) cell viability assay, as described in the Examples provided herein. In this assay, in some embodiments, a single reagent (CellTiter-Glo® reagent) is added directly to cells cultured in serum-supplemented medium. Addition of the reagent causes cell lysis and generates a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture.

Cell viability can also be measured, for example, by measuring the reduction of a tetrazolium salt. In some embodiments, the in vitro potency and/or cell viability of the prepared ADC or panRAS inhibitor compound can be assessed using the MTT cell viability assay, as described in the Examples provided herein. In this assay, in some embodiments, the yellow tetrazolium salt MTT (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to produce reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan can then be dissolved and quantified spectrophotometrically.

In certain embodiments, the invention features a method of modulating the growth of, inhibiting, or killing a cancer cell or tissue by interfering with the expression and/or activity of a panRAS (e.g., K-Ras (including splice variants KRAS4A and KRAS4B), H-Ras, and N-Ras) and/or one or more upstream regulators or downstream targets thereof. The method can be used in any subject in which interfering with the expression and/or activity of a panRAS (e.g., K-Ras (including splice variants KRAS4A and KRAS4B), H-Ras, and N-Ras) provides a therapeutic benefit. Subjects that may benefit from interfering with the expression and/or activity of a panRAS (e.g., K-Ras (including splice variants KRAS4A and KRAS4B), H-Ras, and N-Ras) include, but are not limited to, subjects having or at risk for having a cancer, such as a tumor or a hematological malignancy. In some embodiments, the cancer is breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, stomach cancer or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer.

In some embodiments, the disclosed ADCs can be administered to any cell or tissue that expresses EphA2, such as an EphA2-expressing cancer cell or tissue. An exemplary embodiment includes a method for killing an EphA2-expressing cancer cell or tissue. The method can be used to treat any cell or tissue that expresses EphA2, such as a cancerous cell or a metastatic lesion. Non-limiting examples of EphA2-expressing cancers include breast cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, head and neck cancer, gastric cancer or gastrointestinal cancer, bladder cancer, and colorectal cancer.

In some embodiments, the disclosed ADCs can be administered to any cell or tissue that expresses B7-H3 (CD276), such as a B7-H3 (CD276)-expressing cancer cell or tissue. Exemplary embodiments include a method of killing a B7-H3 (CD276)-expressing cancer cell or tissue. The method can be used to treat any cell or tissue that expresses B7-H3 (CD276), such as a cancerous cell or metastatic lesion. Non-limiting examples of B7-H3 (CD276)-expressing cancers include colorectal cancer, pancreatic cancer, lymphoma, non-small cell lung cancer, small cell lung cancer, breast cancer including ER-positive breast cancer, metastatic castration-resistant prostate cancer, melanoma, bladder urothelial carcinoma, head and neck cancer, and leukemia (e.g., acute myeloid leukemia).

An exemplary method comprises contacting a cell with an ADC, as described herein, in an effective amount, i.e., an amount sufficient to kill the cell. This method can be used in cultured cells, e.g., in vitro, in vivo, ex vivo, or in situ. For example, cells expressing EphA2 (e.g., cells collected by biopsy of a tumor or metastatic lesion; cells from an established cancer cell line; or recombinant cells) can be cultured in vitro in culture medium, and the contacting step can be effected by adding the ADC to the culture medium. This method will kill cells expressing EphA2 (including, in particular, cancer cells expressing EphA2). Alternatively, the ADC can be administered to a subject by any suitable route of administration (e.g., intravenously, subcutaneously, or by direct contact with tumor tissue) to produce an effect in vivo. This approach can be used with antibodies targeting other cell surface antigens (e.g., B7-H3 (CD276)).

The in vivo efficacy of the disclosed ADC therapeutic compositions can be assessed in suitable animal models. For example, xenograft cancer models can be used, in which cancer explants or passaged xenograft tissues are introduced into immunocompromised animals, such as nude or SCID mice (Klein et al. (1997) Nature Med. 3:402-8). Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression, or metastasis.

In vivo assays assessing the promotion of tumor death by mechanisms such as apoptosis may also be used. In some embodiments, xenografts from tumor-bearing mice treated with the therapeutic composition are examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The degree to which apoptotic foci are found in the tumors of treated mice is indicative of the therapeutic efficacy of the composition.

Also provided herein are methods for treating disorders, such as cancer. Compositions described herein, such as the ADCs disclosed herein, can be administered to a non-human mammal or human subject for therapeutic purposes. The method comprises administering to a subject having or suspected of having cancer a therapeutically effective amount of a composition comprising a panRAS inhibitor, such as an ADC, wherein the inhibitor is linked to a targeting antibody that binds to an antigen that is (1) expressed on a cancer cell, (2) capable of binding to, and/or (3) localized or preferentially expressed on the surface of a cancer cell compared to a non-cancerous cell.

An exemplary embodiment is a method of treating a subject having or suspected of having cancer, comprising administering to the subject a therapeutically effective amount of a composition disclosed herein, e.g., an ADC, composition, or pharmaceutical composition (e.g., any exemplary ADC, composition, or pharmaceutical composition disclosed herein). In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, B7-H4, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2(NaPi2b), nectin4, TROP2, LIV1, CD46, MSLN, CD142 (F3), MUC1, MUC16, SLC39A6, TFRC, TACSTD2, GPNMB, EphA2, CD56, SEZ6, CD25, CCR8, CEACAM5, CEACAM6, 4-1BB, 5AC, 5T4, alpha-fetoprotein, Angiopoietin 2, ASLG659, TCLI, BMPRIB, brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (imitation), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR) I), CD22 (B-cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B-cell differentiation antigen CD72), CD79a, CD80, CD166 (ALCAM), CDH17, CA9, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1, CFC1B), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), episialin, ERBB3, ETBR (endothelin type B receptor), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extradomain-B, frizzled receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, human scatter factor receptor kinase, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily receptor translocation-associated 2), Lewis-Y antigen, LY64 (RP105), LY6E, STEAP1, ADAM9, PTK7, MMP14, TM4SF1, ITGB6, FXYD5, MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen (PSMA), PSCA (prostate stem cell antigen precursor), PRLR (prolactin receptor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-related gene I, TAG-72, TEMI, tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel subfamily M, member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or vimentin. In some embodiments, the target antigen is EphA2 or B7-H3 (CD276). In some embodiments, the cancer is a tumor or a hematological malignancy. In some embodiments, the cancer is breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, stomach cancer or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer.

Another exemplary embodiment is a method for delivering a panRAS inhibitor to an EphA2-expressing cell, comprising conjugating the panRAS inhibitor to an antibody or antigen-binding fragment that immunospecifically binds to an EphA2 epitope and exposing the cell to the ADC. Exemplary cancer cells expressing EphA2 for which the ADC of the present invention is recommended include breast cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, head and neck cancer, gastric or gastrointestinal cancer, bladder cancer, and colorectal cancer cells.

Another exemplary embodiment is a method for delivering a panRAS inhibitor to a B7-H3 (CD276) expressing cell, comprising conjugating the panRAS inhibitor to an antibody or antigen-binding fragment that immunospecifically binds to a B7-H3 (CD276) epitope and exposing the cell to the ADC. Exemplary cancer cells expressing B7-H3 (CD276) for which the ADC of the present invention is recommended include colorectal cancer, pancreatic cancer, lymphoma, and leukemia cells.

In certain embodiments, the invention also provides a method of reducing or inhibiting the growth of a tumor (e.g., an EphA2-expressing tumor, a B7-H3(CD276)-expressing tumor), comprising administering a therapeutically effective amount of an ADC or a composition comprising an ADC. In some embodiments, the treatment is sufficient to reduce or inhibit the growth of the tumor, reduce the number or size of metastatic lesions, reduce the tumor load, reduce the primary tumor load, reduce invasiveness, prolong survival time, and/or maintain or improve the quality of life of the patient. In some embodiments, the tumor is resistant or resistant to treatment with an antibody or antigen-binding fragment of the ADC (e.g., an anti-EphA2 antibody or antigen-binding fragment, an anti-B7-H3(CD276) antibody or antigen-binding fragment) when administered alone, and/or the tumor is resistant or resistant to treatment with a panRAS inhibitor drug moiety when administered alone.

An exemplary embodiment is a method of reducing or inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of an ADC, composition, or pharmaceutical composition (e.g., any exemplary ADC, composition, or pharmaceutical composition disclosed herein). In some embodiments, the tumor expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, B7-H4, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2(NaPi2b), nectin4, TROP2, LIV1, CD46, MSLN, CD142 (F3), MUC1, MUC16, SLC39A6, TFRC, TACSTD2, GPNMB, EphA2, CD56, SEZ6, CD25, CCR8, CEACAM5, CEACAM6, 4-1BB, 5AC, 5T4, alpha-fetoprotein, Angiopoietin 2, ASLG659, TCLI, BMPRIB, brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (imitation), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR) I), CD22 (B-cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B-cell differentiation antigen CD72), CD79a, CD80, CD166 (ALCAM), CDH17, CA9, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1, CFC1B), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), episialin, ERBB3, ETBR (endothelin type B receptor), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extradomain-B, frizzled receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, human scatter factor receptor kinase, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily receptor translocation-associated 2), Lewis-Y antigen, LY64 (RP105), LY6E, STEAP1, ADAM9, PTK7, MMP14, TM4SF1, ITGB6, FXYD5, MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen (PSMA), PSCA (prostate stem cell antigen precursor), PRLR (prolactin receptor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-related gene I, TAG-72, TEMI, tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel subfamily M, member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or vimentin. In some embodiments, the target antigen is EphA2 or B7-H3 (CD276). In some embodiments, the tumor is breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, stomach cancer or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer. In some embodiments, the tumor is stomach cancer. In some embodiments, administration of the ADC, composition, or pharmaceutical composition reduces or inhibits tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% compared to growth in the absence of treatment.

Another exemplary embodiment is a method of delaying or slowing the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of an ADC, composition, or pharmaceutical composition (e.g., any exemplary ADC, composition, or pharmaceutical composition disclosed herein). In some embodiments, the target antigen is EphA2 or B7-H3 (CD276). In some embodiments, the tumor is breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, stomach cancer or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer. In some embodiments, the tumor is stomach cancer. In some embodiments, administration of the ADC, composition, or pharmaceutical composition delays or slows tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% compared to growth in the absence of treatment.

In certain embodiments, the invention also provides a method of reducing or slowing the expansion of a cancer cell population (e.g., an EphA2-expressing cancer cell population, a B7-H3(CD276)-expressing cancer cell population), comprising administering a therapeutically effective amount of an ADC or a composition comprising an ADC.

An exemplary embodiment is a method of reducing or slowing the expansion of a cancer cell population in a subject, comprising administering to the subject a therapeutically effective amount of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein). In some embodiments, the target antigen is EphA2 or B7-H3 (CD276). In some embodiments, the cancer cell population is derived from a tumor or a hematological malignancy. In some embodiments, the cancer cell population is breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, gastric or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer. In some embodiments, administration of the ADC, composition, or pharmaceutical composition reduces the cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% compared to the cancer cell population in the absence of treatment. In some embodiments, administration of the ADC, composition, or pharmaceutical composition slows the expansion of the cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% compared to the expansion in the absence of treatment.

Also provided herein are methods for determining whether a subject having cancer or suspected of having cancer is responsive to treatment with the disclosed ADCs and compositions. An exemplary embodiment is a method of determining whether a subject having or suspected of having cancer is responsive to treatment with an ADC, composition, or pharmaceutical composition (e.g., any exemplary ADC, composition, or pharmaceutical composition disclosed herein), comprising: providing a biological sample from the subject; contacting the sample with an ADC; and detecting binding of the ADC to cancer cells in the sample. In some embodiments, the sample is a tissue biopsy sample, a blood sample, or a bone marrow sample. In some embodiments, the method comprises providing a biological sample from the subject; contacting the sample with an ADC; and detecting one or more markers of cancer cell death in the sample (e.g., increased expression of one or more apoptosis markers, decreased expansion of a cancer cell population in culture, etc.).

Also provided herein are therapeutic uses of the disclosed ADCs and compositions. An exemplary embodiment is an ADC, composition, or pharmaceutical composition (e.g., any exemplary ADC, composition, or pharmaceutical composition disclosed herein) for use in treating a subject having or suspected of having cancer (e.g., an EphA2-expressing cancer, a B7-H3(CD276)-expressing cancer). Another exemplary embodiment is a use of an ADC, composition, or pharmaceutical composition (e.g., any exemplary ADC, composition, or pharmaceutical composition disclosed herein) for treating a subject having or suspected of having cancer (e.g., an EphA2-expressing cancer, a B7-H3(CD276)-expressing cancer). Another exemplary embodiment is the use of an ADC, composition, or pharmaceutical composition (e.g., any exemplary ADC, composition, or pharmaceutical composition disclosed herein) in a method of preparing a medicament for treating a subject having or suspected of having a cancer (e.g., an EphA2-expressing cancer, a B7-H3(CD276)-expressing cancer). Methods for identifying a subject having a cancer that expresses a target antigen (e.g., EphA2 or B7-H3(CD276)) are known in the art and can be used to identify patients suitable for treatment with the disclosed ADC compounds or compositions.

Furthermore, the ADCs of the present invention may be administered to non-human mammals expressing an antigen to which the ADCs can bind, either for veterinary purposes or as animal models of human diseases. In the latter context, such animal models may be useful for evaluating the therapeutic efficacy of the disclosed ADCs (e.g., testing dosage and time course of administration).

The therapeutic compositions used in the practice of the methods described above can be formulated as pharmaceutical compositions comprising a pharmaceutically acceptable carrier suitable for the desired delivery method. An exemplary embodiment is a pharmaceutical composition comprising an ADC of the present invention and a pharmaceutically acceptable carrier, e.g., a carrier suitable for a selected means of administration, e.g., intravenous administration. The pharmaceutical composition may also include one or more additional inert agents and/or therapeutic agents (e.g., standard-of-care agents, etc.), suitable for, e.g., treating or preventing cancer. The pharmaceutical composition may also include one or more carrier, excipient, and/or stabilizer components, etc. Methods for formulating such pharmaceutical compositions and suitable formulations are known in the art (see, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA).

Suitable carriers include any substance that, when combined with a therapeutic composition, maintains the antitumor activity of the therapeutic composition and is generally non-reactive with the patient's immune system. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, mesylate salts, and the like, and combinations thereof. In many cases, isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, are included in the composition. Pharmaceutically acceptable carriers may additionally include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the ADC.

The pharmaceutical composition of the present invention can be administered by various methods known in the art. The route and/or method of administration may vary depending on the desired result. In some embodiments, the therapeutic formulation is administered via any route that allows dissolution and delivery of the therapeutic composition to the cancer site. Potentially effective routes of administration include, but are not limited to, parenteral (e.g., intravenous, subcutaneous), intraperitoneal, intramuscular, intratumoral, intradermal, intraorgan, and stereotactic administration. In some embodiments, administration is intravenous, subcutaneous, intraperitoneal, or intramuscular. The pharmaceutically acceptable carrier must be compatible with the route of administration, e.g., intravenous or subcutaneous (e.g., by injection or infusion). Depending on the route of administration, the active compound(s), i.e., the ADC and/or any additional therapeutic agent, may be coated with a material to protect the compound(s) from the action of acids and other natural conditions that may inactivate the compound(s). Administration may be systemic or topical.

The therapeutic compositions disclosed herein may be sterile and stable under the conditions of manufacture and storage, and may exist in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The form depends on the intended route of administration and therapeutic application. In some embodiments, the disclosed ADCs may be incorporated into pharmaceutical compositions suitable for parenteral administration. Injectable solutions may be comprised of liquid or lyophilized dosage forms in flint or amber vials, ampoules, or prefilled syringes, or other known delivery or storage devices. In some embodiments, one or more ADCs or pharmaceutical compositions are supplied as dry, sterile, lyophilized powders or anhydrous concentrates in hermetically sealed containers, which may be reconstituted (e.g., with water or saline) to a concentration suitable for administration to a subject.

Typically, a therapeutically effective amount or effective amount of the disclosed composition, e.g., the disclosed ADC, is used in the pharmaceutical composition of the present invention. The composition, e.g., a composition comprising the ADC, can be formulated into a pharmaceutically acceptable dosage form by conventional methods known in the art. The dosage and administration protocol for treating cancer using the aforementioned methods will vary depending on the method and the target cancer, as well as a number of other factors generally recognized in the art.

Dosage regimens for compositions disclosed herein, e.g., compositions comprising ADCs alone or in combination with at least one additional inactive and/or active therapeutic agent, can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus of one or both agents may be administered at once, multiple divided doses may be administered over a given period of time, or the dose of one or both agents may be proportionally increased or decreased as indicated by the exigencies of the therapeutic situation. In some embodiments, treatment comprises a single bolus or repeated administrations of the ADC preparation via an acceptable route of administration. In some embodiments, the ADC is administered to the patient daily, weekly, monthly, or any period in between. For any particular subject, a specific dosage regimen may be adjusted over time according to the individual's need and the professional judgment of the treating clinician. Parenteral compositions may be formulated in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units designed as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Dosage values for compositions comprising ADC and/or any additional therapeutic agent(s) may be selected based on the unique properties of the active compound(s) and the particular therapeutic effect to be achieved. A physician or veterinarian may initiate the dosage of the ADC employed in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and may gradually increase the dosage until the desired effect is achieved. In general, the effective dosage of the composition of the present invention for the treatment of cancer may vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. The selected dosage level may also vary depending on various pharmacodynamic factors, including the activity of the particular composition of the present invention employed or its ester, salt, or amide, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, compounds, and/or substances concomitantly administered with the particular composition employed, the age, sex, weight, condition, general health and past medical history of the patient being treated, and similar factors. Therapeutic dosage may be titrated to optimize safety and efficacy.

The toxicity and therapeutic efficacy of the compounds provided herein can be determined using standard pharmaceutical procedures in cell culture or animal models. For example, the LD50, ED50, EC50, and IC50 can be determined, and the dose ratio between toxic and therapeutic effects (LD50/ED50) can be calculated as a therapeutic index. Data obtained from in vitro and in vivo assays can be used to estimate or formulate dosage ranges for human use. For example, the compositions and methods disclosed herein can be initially evaluated in xenograft cancer models (e.g., the NCI-H929 multiple myeloma mouse model).

In some embodiments, the ADC or a composition comprising the ADC is administered as a single dose. In other embodiments, the ADC or a composition comprising the ADC is administered as multiple doses. The interval between single doses may be, for example, daily, weekly, monthly, or yearly. The interval may also be irregular, based on measurements of the patient's blood levels of the administered agent (e.g., ADC), to maintain a relatively consistent plasma concentration of the agent. The dosage and frequency of administration of the ADC or a composition comprising the ADC may also vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses may be administered at relatively infrequent intervals over an extended period of time. Some patients continue to receive treatment for the remainder of their lives. In therapeutic applications, relatively higher doses at shorter intervals are sometimes necessary until disease progression is reduced or terminated, preferably until the patient demonstrates partial or complete improvement in one or more symptoms of the disease. Afterwards, the patient may receive less, for example, prophylactic therapy.

The above therapeutic approach may be combined with any of a wide variety of additional surgical, chemotherapy, or radiotherapy regimens. In some embodiments, the ADCs or compositions disclosed herein are co-formulated and/or co-administered with one or more additional therapeutic agents, such as one or more chemotherapeutic agents, one or more standard-of-care agents (specific to the specific condition being treated).

Kits for use in the therapeutic and/or diagnostic applications described herein are also provided. Such kits may comprise a carrier, package, or container compartmentalized to accommodate one or more containers, such as vials, tubes, or the like, each of which comprises one of the distinct elements to be used in the methods disclosed herein. A label may be present on or with the container to indicate that the ADC or composition in the kit is used for a particular therapeutic or non-therapeutic application, such as a prognostic application, a prophylactic application, a diagnostic application, or a laboratory application. The label may also indicate directions for in vivo or in vitro use as described herein. Directions and/or other information may also be included on insert(s) or label(s) included with or on the kit. The label may be on or associated with the container. The label may be present on the container if letters, numbers, or other characters forming the label are molded or etched into the container itself. The label may be associated with the container, if present within a receptacle or carrier that also holds the container (e.g., as a package insert). The label may indicate that the ADC or composition within the kit is used to diagnose or treat a condition, such as cancer, described herein.

In some embodiments, the kit comprises an ADC or a composition comprising an ADC. In some embodiments, the kit further comprises one or more additional components, including but not limited to: instructions for use; other reagents, e.g., therapeutic agents (e.g., standard-of-care formulations); a device, container, or other material for preparing the ADC for administration; a pharmaceutically acceptable carrier; and a device, container, or other material for administering the ADC to a subject. The instructions for use may include instructions for therapeutic applications, including suggested dosages and/or administration regimens (e.g., in patients with or suspected of having cancer). In some embodiments, the kit comprises an ADC and instructions for using the ADC in treating, preventing, and/or diagnosing cancer.

Increased expression of panRAS (e.g., K-Ras (including splice variants KRAS4A and KRAS4B), H-Ras, and N-Ras) is known to correlate with resistance to radiotherapy and chemotherapy. Antibody-drug conjugates (ADCs) that may not be sufficiently effective as monotherapy for treating cancer may provide therapeutic benefit when administered in combination with other therapeutics (including non-targeted and targeted therapies) or radiotherapy (including radioligand therapy). Without wishing to be bound by theory, it is believed that the ADCs described herein sensitize tumor cells to treatment with other therapeutics (including standard-of-care chemotherapeutics to which the tumor cells may be resistant) and/or radiotherapy. In some embodiments, the antibody-drug conjugates described herein are administered to a subject having cancer in an amount effective to sensitize the tumor cells. As used herein, the term "sensitize" means that treatment with an ADC increases the potency or efficacy of treatment with other therapeutic agents and/or radiotherapy against tumor cells.

combination therapy

In some embodiments, the present invention provides a method of treatment in which an antibody-drug conjugate disclosed herein is administered in combination with one or more (e.g., one or two) additional therapeutic agents. Exemplary combination partners are disclosed herein.

In certain embodiments, the combinations described herein include a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is selected from PDR001 (Novartis), nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck & Co), pidilizumab (CureTech), MEDI0680 (Medimmune), REGN2810 (Regeneron), TSR-042 (Tesaro), PF-06801591 (Pfizer), BGB-A317 (Beigene), BGB-108 (Beigene), INCSHR1210 (Incyte), or AMP-224 (Amplimmune). In some embodiments, the PD-1 inhibitor is PDR001. PDR001 is also known as spartalizumab.

In certain embodiments, the combinations described herein comprise a LAG-3 inhibitor. In some embodiments, the LAG-3 inhibitor is selected from LAG525 (Novartis), BMS-986016 (Bristol-Myers Squibb), or TSR-033 (Tesaro).

In certain embodiments, the combinations described herein comprise a TIM-3 inhibitor. In some embodiments, the TIM-3 inhibitor is MBG453 (Novartis), TSR-022 (Tesaro), LY-3321367 (Eli Lilly), Sym23 (Symphogen), BGB-A425 (Beigene), INCAGN-2390 (Agenus), BMS-986258 (BMS), RO-7121661 (Roche), or LY-3415244 (Eli Lilly).

In certain embodiments, the combinations described herein comprise a PDL1 inhibitor. In one embodiment, the PDL1 inhibitor is selected from FAZ053 (Novartis), atezolizumab (Genentech), durvalumab (Astra Zeneca), or avelumab (Pfizer).

In certain embodiments, the combinations described herein comprise a GITR agonist. In some embodiments, the GITR agonist is selected from GWN323 (NVS), BMS-986156, MK-4166 or MK-1248 (Merck), TRX518 (Leap Therapeutics), INCAGN1876 (Incyte/Agenus), AMG 228 (Amgen), or INBRX-110 (Inhibrx).

In some embodiments, the combinations described herein include an IAP inhibitor. In some embodiments, the IAP inhibitor comprises LCL161 or a compound disclosed in International Application Publication No. WO 2008/016893.

In one embodiment, the combination comprises an mTOR inhibitor, e.g., RAD001 (also known as everolimus).

In one embodiment, the combination comprises an HDAC inhibitor, e.g., LBH589. LBH589 is also known as panobinostat.

In one embodiment, the combination comprises an IL-17 inhibitor, e.g., CJM112.

In certain embodiments, the combinations described herein include an estrogen receptor (ER) antagonist. In some embodiments, the estrogen receptor antagonist is used in combination with a PD-1 inhibitor, a CDK4/6 inhibitor, or both. In some embodiments, the combination is used to treat ER-positive (ER+) cancer or breast cancer (e.g., ER+ breast cancer).

In some embodiments, the estrogen receptor antagonist is a selective estrogen receptor degrader (SERD). SERDs are estrogen receptor antagonists that bind to the receptor, resulting in, for example, degradation or downregulation of the receptor (Boer K. et al. , (2017) Therapeutic Advances in Medical Oncology 9(7): 465-479). ER is a hormone-activated transcription factor that is important, for example, in the growth, development, and physiology of the human reproductive system. ER is activated, for example, by the hormone estrogen (17beta-estradiol). ER expression and signaling are associated with cancer, for example, breast cancer, such as ER-positive (ER+) breast cancer. In some embodiments, the SERD is selected from LSZ102, fulvestrant, brilanestrant, or elacestrant.

In some embodiments, the SERD comprises a compound disclosed in International Application Publication No. WO 2014/130310, which is incorporated herein by reference in its entirety.

In some embodiments, the SERD comprises LSZ102. LSZ102 has the chemical name (E)-3-(4-((2-(2-(1,1-difluoroethyl)-4-fluorophenyl)-6-hydroxybenzo[b]thiophen-3-yl)oxy)phenyl)acrylic acid. In some embodiments, the SERD comprises fulvestrant (CAS Reg. No.: 129453-61-8), or a compound disclosed in International Application Publication No. WO 2001/051056, which is incorporated herein by reference in its entirety. In some embodiments, the SERD comprises elacestrant (CAS Reg. No.: 722533-56-4), or a compound disclosed in U.S. Pat. No. 7,612,114, which is incorporated herein by reference in its entirety. Elacestrant is also known as RAD1901, ER-306323, or (6R)-6-{2-[ethyl({4-[2-(ethylamino)ethyl]phenyl}methyl)amino]-4-methoxyphenyl}-5,6,7,8-tetrahydronaphthalen-2-ol. Elacestrant is an orally bioavailable nonsteroidal compound selective estrogen receptor modulator (SERM) and SERD. Elacestrant is also disclosed, for example, in Garner F et al., (2015) Anticancer Drugs 26(9):948-56. In some embodiments, the SERD is brilanestrant (CAS Registry Number: 1365888-06-7), or a compound disclosed in International Application Publication No. WO 2015/136017, which is incorporated by reference in its entirety.

In some embodiments, the SERD is selected from RU 58668, GW7604, AZD9496, bazedoxifene, pipendoxifene, arzoxifene, OP-1074, or acolbifene, as disclosed, for example, in McDonell et al. (2015) Journal of Medicinal Chemistry 58(12) 4883-4887.

Other exemplary estrogen receptor antagonists are disclosed, for example, in WO 2011/156518, WO 2011/159769, WO 2012/037410, WO 2012/037411, and US 2012/0071535, all of which are incorporated herein by reference in their entireties.

In certain embodiments, the combinations described herein comprise an inhibitor of cyclin-dependent kinase 4 or 6 (CDK4/6). In some embodiments, the CDK4/6 inhibitor is used in combination with a PD-1 inhibitor, an estrogen receptor (ER) antagonist, or both. In some embodiments, the combination is used to treat ER-positive (ER+) cancer or breast cancer (e.g., ER+ breast cancer). In some embodiments, the CDK4/6 inhibitor is selected from ribociclib, abemaciclib (Eli Lilly), or palbociclib.

In some embodiments, the CDK4/6 inhibitor comprises ribociclib (CAS Registry Number: 1211441-98-3) or a compound disclosed in U.S. Patent Nos. 8,415,355 and 8,685,980, which are incorporated by reference in their entireties.

In some embodiments, the CDK4/6 inhibitor comprises a compound disclosed in International Application Publication No. WO 2010/020675 and U.S. Patent Nos. 8,415,355 and 8,685,980, which are incorporated by reference in their entireties.

In some embodiments, the CDK4/6 inhibitor comprises ribociclib (CAS reg. no.: 1211441-98-3). Ribociclib is also known as LEE011, KISQALI®, or 7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide.

In some embodiments, the CDK4/6 inhibitor comprises abemaciclib (CAS reg. no.: 1231929-97-7). Abemaciclib is also known as LY835219 or N-[5-[(4-ethyl-1-piperazinyl)methyl]-2-pyridinyl]-5-fluoro-4-[4-fluoro-2-methyl-1-(1-methylethyl)-1H-benzimidazol-6-yl]-2-pyrimidinamine. Abemaciclib is a CDK inhibitor that is selective for CDK4 and CDK6 and is disclosed, for example, in Torres-Guzman R et al. (2017) Oncotarget 10.18632/oncotarget.17778.

In some embodiments, the CDK4/6 inhibitor comprises palbociclib (CAS reg. no.: 571190-30-2). Palbociclib is also known as PD-0332991, IBRANCE®, or 6-acetyl-8-cyclopentyl-5-methyl-2-{[5-(1-piperazinyl)-2-pyridinyl]amino}pyrido[2,3-d]pyrimidin-7(8H)-one. Palbociclib inhibits CDK4 with an IC50 of 11 nM and CDK6 with an IC50 of 16 nM, and is disclosed, for example, in Finn et al. (2009) Breast Cancer Research 11(5):R77.

In certain embodiments, the combinations described herein comprise an inhibitor of chemokine (C-X-C motif) receptor 2 (CXCR2). In some embodiments, the CXCR2 inhibitor is selected from 6-chloro-3-((3,4-dioxo-2-pentan-3-ylamino)cyclobut-1-en-1-yl)amino)-2-hydroxy-N-methoxy-N-methylbenzenesulfonamide, danirixin, reparixin, or navarixin.

In some embodiments, the CSF-1/1R binding agent is selected from an inhibitor of macrophage colony-stimulating factor (M-CSF), such as a monoclonal antibody or Fab against M-CSF (e.g., MCS110), a CSF-1R tyrosine kinase inhibitor (e.g., 4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide or BLZ945), a receptor tyrosine kinase inhibitor (RTK) (e.g., pexidartinib), or an antibody targeting CSF-1R (e.g., emactuzumab or FPA008). In some embodiments, the CSF-1/1R inhibitor is BLZ945. In some embodiments, the CSF-1/1R binding agent is MCS110. In another embodiment, the CSF-1/1R binding agent is pexidartinib.

In certain embodiments, the combinations described herein include a c-MET inhibitor. c-MET, a receptor tyrosine kinase overexpressed or mutated in many tumor cell types, plays a key role in tumor cell proliferation, survival, invasion, metastasis, and tumor angiogenesis. Inhibition of c-MET can induce apoptosis in tumor cells that overexpress c-MET protein or express constitutively activated c-MET protein. In some embodiments, the c-MET inhibitor is selected from capmatinib (INC280), JNJ-3887605, AMG 337, LY2801653, MSC2156119J, crizotinib, tivantinib, or golvatinib.

In certain embodiments, the combinations described herein include a transforming growth factor beta (TGF-β, also known as TGFβ, TGFb, or TGF-beta, used interchangeably herein) inhibitor. In some embodiments, the TGF-β inhibitor is selected from fresolimumab or XOMA 089 (Xoma).

In certain embodiments, the combinations described herein include an adenosine A2a receptor (A2aR) antagonist (e.g. , an inhibitor of the A2aR pathway, e.g., an adenosine inhibitor, e.g., an inhibitor of A2aR or CD-73). In some embodiments, the A2aR antagonist is used in combination with a PD-1 inhibitor and one or more (e.g., two, three, four, five, or all) of a CXCR2 inhibitor, a CSF-1/1R tyrosine kinase inhibitor, a LAG-3 inhibitor, a GITR agonist, a c-MET inhibitor, or an IDO inhibitor. In some embodiments, the combination is used to treat pancreatic cancer, colorectal cancer, gastric cancer, or melanoma (e.g., refractory melanoma). In some embodiments, the A2aR antagonist is selected from PBF509 (NIR178) (Palobiofarma/Novartis), CPI444/V81444 (Corvus/Genentech), AZD4635/HTL-1071 (AstraZeneca/Heptares), bifadenant (Redox/Juno), GBV-2034 (Globavir), AB928 (Arcus Biosciences), theophylline, istradefylline (Kyowa Hakko Kogyo), tozadenant/SYN-115 (Acorda), KW-6356 (Kyowa Hakko Kogyo), ST-4206 (Leadiant Biosciences), or preladenant/SCH 420814 (Merck/Schering). Without wishing to be bound by theory, in some embodiments, inhibition of A2aR leads to upregulation of IL-1b.

In certain embodiments, the combinations described herein comprise an inhibitor of indoleamine 2,3-dioxygenase (IDO) and/or tryptophan 2,3-dioxygenase (TDO). In some embodiments, the IDO inhibitor is used in combination with a PD-1 inhibitor and one or more (e.g., two, three, four, or all) of a TGF-β inhibitor, an A2aR antagonist, a CSF-1/1R tyrosine kinase (CSF-1/1R) tyrosine kinase (C-MET) inhibitor, or a GITR agonist. In some embodiments, the combination is used to treat pancreatic cancer, colorectal cancer, gastric cancer, or melanoma (e.g., refractory melanoma). In some embodiments, the IDO inhibitor is selected from (4E)-4-[(3-chloro-4-fluoroanilino)-nitrosomethylidene]-1,2,5-oxadiazol-3-amine (also known as epacadostat or INCB24360), indoximod (NLG8189), (1-methyl-D-tryptophan), α-cyclohexyl-5H-imidazo[5,1-a]isoindole-5-ethanol (also known as NLG919), indoximod, BMS-986205 (formerly F001287).

In certain embodiments, the combinations described herein include a galectin inhibitor, e.g., galectin-1 or galectin-3. In some embodiments, the combination includes a galectin-1 inhibitor and a galectin-3 inhibitor. In some embodiments, the combination includes a bispecific inhibitor (e.g., a bispecific antibody molecule) that targets both galectin-1 and galectin-3. In some embodiments, the galectin inhibitor is used in combination with one or more therapeutic agents described herein. In some embodiments, the galectin inhibitor is selected from an anti-galectin antibody molecule, GR-MD-02 (Galectin Therapeutics), Galectin-3C (Mandal Med), Anginex, or OTX-008 (OncoEthix, Merck).

In some embodiments, the combinations described herein comprise MAP kinase pathway inhibitors, including ERK inhibitors, MEK inhibitors, and RAF inhibitors.

In some embodiments, the combinations described herein comprise a MEK inhibitor. In some embodiments, the MEK inhibitor is selected from trametinib, selumetinib, AS703026, BIX 02189, BIX 02188, CI-1040, PD0325901, PD98059, U0126, XL-518, G-38963, or G02443714.

In some embodiments, the MEK inhibitor is trametinib. Trametinib is also known as JTP-74057, TMT212, N-(3-{3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl}phenyl)acetamide, or mekinist (CAS No. 871700-17-3).

In some embodiments, the MEK inhibitor comprises selumetinib, which has the chemical name (5-[(4-bromo-2-chlorophenyl)amino]-4-fluoro-N-(2-hydroxyethoxy)-1-methyl-1H-benzimidazole-6-carboxamide. Selumetinib is also known as AZD6244 or ARRY 142886, for example, as described in PCT Publication No. WO 2003077914.

In some embodiments, the MEK inhibitor comprises AS703026, BIX 02189, or BIX 02188.

In some embodiments, the MEK inhibitor comprises 2-[(2-chloro-4-iodophenyl)amino]-N-(cyclopropylmethoxy)-3,4-difluoro-benzamide (also known as CI-1040 or PD184352), for example as described in PCT Publication No. WO 2000035436.

In some embodiments, the MEK inhibitor comprises N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (also known as PD0325901), as described, for example, in PCT Publication No. WO 2002006213.

In some embodiments, the MEK inhibitor comprises 2'-amino-3'-methoxyflavone (also known as PD98059) available from Biaffin GmbH & Co., KG (Germany).

In some embodiments, the MEK inhibitor comprises 2,3-bis[amino[(2-aminophenyl)thio]methylene]-butandinitrile (also known as U0126), as described, for example, in U.S. Patent No. 2,779,780.

In some embodiments, the MEK inhibitor comprises XL-518 (also known as GDC-0973) having CAS number 1029872-29-4 and available from ACC Corp.

In some embodiments, the MEK inhibitor comprises G-38963.

In some embodiments, the MEK inhibitor comprises G02443714 (also known as AS703206).

Additional examples of MEK inhibitors are disclosed in WO 2013/019906, WO 03/077914, WO 2005/121142, WO 2007/04415, WO 2008/024725, and WO 2009/085983, the contents of which are incorporated herein by reference. Additional examples of MEK inhibitors include 2,3-bis[amino[(2-aminophenyl)thio]methylene]-butandinitrile (also known as U0126 and described in U.S. Patent No. 2,779,780); (3S,4R,5Z,8S,9S,11E)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecin-1,7(8H)-dione (also known as E6201 and described in PCT Publication No. WO 2003076424); vemurafenib (PLX-4032, CAS 918504-65-1); (R)-3-(2,3-dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (TAK-733, CAS 1035555-63-5); Pimasertib (AS-703026, CAS 1204531-26-9); 2-(2-fluoro-4-iodophenylamino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide (AZD 8330); and 3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)-5-[(3-oxo-[1,2]oxazinan-2-yl)methyl]benzamide (CH 4987655 or Ro 4987655).

In some embodiments, the combinations described herein comprise a RAF inhibitor.

RAF inhibitors include, but are not limited to, vemurafenib (or Zelboraf®, PLX-4032, CAS 918504-65-1), GDC-0879, PLX-4720 (available from Symansis), dabrafenib (or GSK2118436), LGX 818, CEP-32496, UI-152, RAF 265, regorafenib (BAY 73-4506), CCT239065, or sorafenib (or sorafenib tosylate, or Nexavar®).

In some embodiments, the RAF inhibitor is dabrafenib.

In some embodiments, the RAF inhibitor is LXH254.

In some embodiments, the combinations described herein comprise an ERK inhibitor.

ERK inhibitors include, but are not limited to, LTT462, ulixertinib (BVD-523), LY3214996, GDC-0994, KO-947, and MK-8353.

In some embodiments, the ERK inhibitor is LTT462. LTT462 is 4-(3-amino-6-((1S,3S,4S)-3-fluoro-4-hydroxycyclohexyl)pyrazin-2-yl)-N-((S)-1-(3-bromo-5-fluorophenyl)-2-(methylamino)ethyl)-2-fluorobenzamide and has the following structure:

.

The method for preparing LTT462 is described in PCT Patent Application Publication No. WO2015/066188. LTT462 is an inhibitor of extracellular signal-regulated kinase 1 and 2 (ERK 1/2).

In some embodiments, the combinations described herein comprise a taxane, a vinca alkaloid, a MEK inhibitor, an ERK inhibitor, or a RAF inhibitor.

In some embodiments, the combinations described herein comprise two or more inhibitors independently selected from a MEK inhibitor, an ERK inhibitor, and a RAF inhibitor.

In some embodiments, the combinations described herein comprise an antimitotic drug.

In some embodiments, the combinations described herein comprise a taxane.

Taxanes include, but are not limited to, docetaxel, paclitaxel, or cabazitaxel. In some embodiments, the taxane is docetaxel.

In some embodiments, the combinations described herein comprise vinca alkaloids.

Vinca alkaloids include, but are not limited to, vincristine, vinblastine, and leurosine.

In some embodiments, the combinations described herein comprise a topoisomerase inhibitor.

Topoisomerase inhibitors include, but are not limited to, topotecan, irinotecan, camptothecin, diplomotecan, lamellarin D, ellipticine, etoposide (VP-16), teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, aurintricarboxylic acid, and HU-331.

In one embodiment, the combination described herein comprises an interleukin-1 beta (IL-1β) inhibitor. In some embodiments, the IL-1β inhibitor is selected from canakinumab, gevocizumab, anakinra, or rilonacept.

In certain embodiments, the combinations described herein comprise an IL-15/IL-15Ra complex. In some embodiments, the IL-15/IL-15Ra complex is selected from NIZ985 (Novartis), ATL-803 (Altor), or CYP0150 (Cytune).

In certain embodiments, the combinations described herein comprise a mouse double minute 2 homolog (MDM2) inhibitor. The human homolog of MDM2 is also known as HDM2. In some embodiments, the MDM2 inhibitors described herein are also known as HDM2 inhibitors. In some embodiments, the MDM2 inhibitor is selected from HDM201 or CGM097.

In one embodiment, the MDM2 inhibitor comprises (S)-1-(4-chlorophenyl)-7-isopropoxy-6-methoxy-2-(4-(methyl(((1r,4S)-4-(4-methyl-3-oxopiperazin-1-yl)cyclohexyl)methyl)amino)phenyl)-1,2-dihydroisoquinolin-3(4H)-one (also known as CGM097), or a compound disclosed in PCT Publication No. WO 2011/076786, for treating a disorder (e.g., a disorder described herein). In one embodiment, a therapeutic agent disclosed herein is used in combination with CGM097.

In some embodiments, the combinations described herein include a hypomethylating agent (HMA). In some embodiments, the HMA is selected from decitabine or azacitidine.

In some embodiments, the combinations described herein comprise a glucocorticoid. In some embodiments, the glucocorticoid is dexamethasone.

In some embodiments, the combination described herein comprises asparaginase.

In certain embodiments, the combinations described herein include an inhibitor that acts on any pro-survival protein of the Bcl2 family. In certain embodiments, the combinations described herein include a Bcl-2 inhibitor. In some embodiments, the Bcl-2 inhibitor is venetoclax (also known as ABT-199):

(Venetoclax).

In one embodiment, the Bcl-2 inhibitor is selected from the compounds described in WO 2013/110890 and WO 2015/011400. In some embodiments, the Bcl-2 inhibitor comprises navitoclax (ABT-263), ABT-737, BP1002, SPC2996, APG-1252, obatoclax mesylate (GX15-070MS), PNT2258, Zn-d5, BGB-11417, or oblimersen (G3139). In some embodiments, the Bcl-2 inhibitor is N-(4-hydroxyphenyl)-3-[6-[(3S)-3-(morpholinomethyl)-3,4-dihydro-1H-isoquinoline-2-carbonyl]-1,3-benzodioxol-5-yl]-N-phenyl-5,6,7,8-tetrahydroindolizine-1-carboxamide, Compound A1:

(Compound A1).

In some embodiments, the Bcl-2 inhibitor is (S)-5-(5-chloro-2-(3-(morpholinomethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)phenyl)-N-(5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)-N-(4-hydroxyphenyl)-1,2-dimethyl-1H-pyrrole-3-carboxamide), compound A2:

(Compound A2).

In one embodiment, the antibody-drug conjugate or combination disclosed herein is suitable for the treatment of cancer in vivo. For example, the combination may be used to inhibit the growth of a cancerous tumor. The combination may also be used in combination with one or more of the following to treat a disorder herein: (e.g., For example, standard care treatments (for cancer or infectious disorders), vaccines (for example) For example, therapeutic cancer vaccines), cell therapy, hormone therapy (e.g., using anti-estrogens or anti-androgens), radiation therapy, surgery, or any other therapeutic or treatment modality. For example, to achieve antigen-specific immune enhancement, the combination may be administered together with the antigen of interest. The combinations disclosed herein may be administered sequentially or simultaneously.

Additional implementation forms

The present invention provides the following additional embodiments of linker-drug groups, antibody-drug conjugates, conjugate linker groups, and conjugation methods.

linker-drug group

In some embodiments, the linker-drug group of the present invention can be a compound having the structure of Formula A' below, or a pharmaceutically acceptable salt thereof:

[Chemical Formula A']

[Here,

R ¹ is a reactor;

L ₁ is a crosslinking spacer;

Lp is a bivalent peptide spacer;

GL ₂ -A is a self-immolative spacer;

R ² is a hydrophilic moiety;

L ₂ is a bond, methylene, neopentylene or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

L ₃ is a spacer moiety;

D is a drug moiety capable of inhibiting the activity of panRAS (e.g., K-Ras (including splice variants KRAS4A and KRAS4B), H-Ras, and N-Ras) when released from, for example, an antibody drug conjugate or immunoconjugate disclosed herein.

Specific embodiments and examples of the linker-drug group of the present invention are provided in the list of embodiments below. It will be appreciated that the features specified in each embodiment may be combined with other specified features to provide additional embodiments of the present invention.

Embodiment 1.

R ¹ is a reactor;

L ₁ is a crosslinking spacer;

Lp is a bivalent peptide spacer containing 2 to 4 amino acid residues;

GL ₂ -A is a self-immolative spacer;

R ² is a hydrophilic moiety;

L ₂ is a bond, methylene, neopentylene or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

L ₃ is a spacer moiety;

D is a drug moiety as defined herein, e.g., a compound of formula A', or a pharmaceutically acceptable salt thereof, which is a panRAS inhibitor.

Embodiment 2.

R ¹ is a reactor;

L ₁ is a crosslinking spacer;

Lp is a bivalent peptide spacer containing 2 to 4 amino acid residues;

Ki-ga

As selected from,

* indicates an attachment point to D (e.g., to N or O of the drug moiety), *** indicates the attachment point to Lp;

R ² is a hydrophilic moiety;

L ₂ is a bond, methylene, neopentylene or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

L ₃ is a spacer moiety;

D is a drug moiety as defined herein, e.g., a compound of formula A', or a pharmaceutically acceptable salt thereof, which is a panRAS inhibitor.

Embodiment 3. A compound of formula A' having a structure of formula B' or a pharmaceutically acceptable salt thereof:

[Chemical Formula B']

(Here,

R ¹ is a reactor;

L ₁ is a crosslinking spacer;

Lp is a bivalent peptide spacer containing 2 to 4 amino acid residues;

R ² is a hydrophilic moiety;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

L ₃ is a spacer moiety;

D is a drug moiety as defined herein and comprising N, wherein D is linked to A via a direct bond from A to N of the drug moiety.

Embodiment 4.

R ¹ is

and;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)(CH ₂ ) _m -**; *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)O(CH ₂ ) _m SSC(R ³ ) ₂ (CH ₂ ) _m C(=O)NR ³ (CH ₂ ) _m NR ³ C(=O)(CH ₂ ) _m -**;

*-C(=O)O(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**;

*-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**;

_{* -C} ( ₌ O)(CH ₂ ) _{m *} -C(=O)(( _{CH 2 ) m} O) _t (CH ₂ ) _n

*-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n -**;

* _-C (=O) _{( CH 2} ) _m NHC(=O)(CH ₂ ) _n

*-C(=O)(( _{CH 2} ) _m O _{) t (} CH ₂ ) _n NHC(=O)(CH ₂ ) _n

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n C(=O)NH(CH ₂ ) _m -**;

*-C(=O)(CH ₂ ) _m C(R ³ ) ₂ -**; or

*-C(=O)(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**,

* of L ₁ indicates an attachment point to Lp, and ** of L ₁ indicates an attachment point to R ¹ ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 A hydrophilic moiety selected from (substituted with a group);

Each R ³ is independently selected from H and C ₁ -C ₆ alkyl;

R ⁴ is 2-pyridyl or 4-pyridyl;

Each R ⁵ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl, and -OH;

Each R ⁶ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl, -NH ₂ , -OCH ₃ , -OCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -CN, -NO ₂ and -OH;

Each R ⁷ is independently selected from H, C _1-6 alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C _1-4 alkoxy substituted with -C(=O)OH, and C _1-4 alkyl substituted with -C(=O)OH;

X ₁ is and;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is a bivalent peptide spacer comprising amino acid residues selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan and tyrosine;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)C(R ^b ) ₂ NHC(=O)O-**,

-NHC(=O)C(R ^b ) ₂ NH-**, -NHC(=O)C(R ^b ) ₂ NHC(=O)-**,

-CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**,

-C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, and each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

* in L ₃ indicates an attachment point to R ² ;

A compound of formula A' or a compound of any one of embodiments 1 to 3, or a pharmaceutically acceptable salt thereof, wherein D is a drug moiety as defined herein and comprises N or O, wherein D is linked to A via a direct bond from A to N or O of the drug moiety.

Embodiment 5.

R ¹ is and;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * in L ₁ indicates the point of attachment to Lp, and ** in L ₁ indicates the point of attachment to R ¹ ;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is As a divalent peptide spacer selected from, * of Lp indicates an attachment point to L ₁ and ** of Lp indicates an attachment point to the -NH- group of G;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -NHC(=O)CH ₂ NH-**, -NHC(=O)CH ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, and each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH2-triazolyl-*, the *** of X represents the point of attachment to W and the * of X represents the point of attachment to R ² ;

* in L ₃ indicates an attachment point to R ² ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, C ₂ -C ₆ alkyl (1 to 3 is a hydrophilic moiety selected from (substituted with);

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

A compound of formula A' or any one of embodiments 1 to 4, or a pharmaceutically acceptable salt thereof, wherein D is a drug moiety as defined herein and comprises N or O, wherein D is linked to A via a direct bond from A to N or O of the drug moiety.

Embodiment 6.

R ¹ is and;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * in L ₁ indicates the point of attachment to Lp, and ** in L ₁ indicates the point of attachment to R ¹ ;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is As a divalent peptide spacer selected from, * of Lp indicates an attachment point to L ₁ and ** of Lp indicates an attachment point to the -NH- group of G;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, and each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

* in L ₃ indicates an attachment point to R ² ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 A hydrophilic moiety selected from (substituted with a group);

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

A compound of formula A' or any one of embodiments 1 to 5, or a pharmaceutically acceptable salt thereof, wherein D is a drug moiety as defined herein and comprises N or O, wherein D is linked to A via a direct bond from A to N or O of the drug moiety.

Embodiment 7.

R ¹ is and;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * in L ₁ indicates the point of attachment to Lp, and ** in L ₁ indicates the point of attachment to R ¹ ;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is As a divalent peptide spacer selected from, * of Lp indicates an attachment point to L ₁ and ** of Lp indicates an attachment point to the -NH- group of G;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, or -NHC(=O)NH-**, each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH2-triazolyl-*, the *** of X represents the point of attachment to W and the * of X represents the point of attachment to R ₂ ;

* in L ₃ indicates an attachment point to R ² ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide, C ₂ -C ₆ alkyl (1 to 3 is a hydrophilic moiety selected from (substituted with);

A is a bond or -OC(=O)*, where * indicates the point of attachment to D;

A compound of formula A' or any one of embodiments 1 to 6, or a pharmaceutically acceptable salt thereof, wherein D is a drug moiety as defined herein and comprises N or O, wherein D is linked to A via a direct bond from A to N or O of the drug moiety.

Embodiment 8.

R ¹ is and;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * in L ₁ indicates the point of attachment to Lp, and ** in L ₁ indicates the point of attachment to R ¹ ;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is As a divalent peptide spacer selected from, * of Lp indicates an attachment point to L ₁ and ** of Lp indicates an attachment point to the -NH- group of G;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, or -C(=O)N(XR ² )-**, wherein each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl and the ** of W represents the point of attachment to X;

X is ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

* in L ₃ indicates an attachment point to R ² ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 A hydrophilic moiety selected from (substituted with a group);

A is a bond or -OC(=O)*, where * indicates the point of attachment to D;

A compound of formula A' or any one of embodiments 1 to 7, or a pharmaceutically acceptable salt thereof, wherein D is a drug moiety as defined herein and comprises N or O, wherein D is linked to A via a direct bond from A to N or O of the drug moiety.

Embodiment 9. A compound of formula A', or any one of Embodiments 1 to 8, or a pharmaceutically acceptable salt thereof, wherein R ¹ is a reactive group selected from Table 8.

Embodiment 10.

R ¹ is A compound of formula A' or any one of embodiments 1 to 9, or a pharmaceutically acceptable salt thereof.

Embodiment 11.

R ¹ is A compound of formula A' or any one of embodiments 1 to 9, or a pharmaceutically acceptable salt thereof.

Embodiment 12.

R ¹ is A compound of formula A' or any one of embodiments 1 to 9, or a pharmaceutically acceptable salt thereof.

Embodiment 13.

R ¹ is A compound of formula A' or any one of embodiments 1 to 9, or a pharmaceutically acceptable salt thereof.

Embodiment 14. R ¹ is A compound of formula A' or any one of embodiments 1 to 9, or a pharmaceutically acceptable salt thereof.

Embodiment 15. A compound of formula A', wherein R ¹ is -ONH ₂ , or a compound of any one of Embodiments 1 to 9, or a pharmaceutically acceptable salt thereof.

Embodiment 16. R ¹ is A compound of formula A' or any one of embodiments 1 to 9, or a pharmaceutically acceptable salt thereof.

Embodiment 17. R ¹ is A compound of formula A' or any one of embodiments 1 to 9, or a pharmaceutically acceptable salt thereof.

Embodiment 18. A compound of formula A' having the following structure, or a compound of any one of Embodiments 1 to 9, or a pharmaceutically acceptable salt thereof:

(Here,

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH).

Embodiment 19. A compound of formula A' having the following structure, or a compound of any one of Embodiments 1 to 9, or a pharmaceutically acceptable salt thereof:

(Here,

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH).

Embodiment 20. A compound of formula A' having the following structure, or a compound of any one of Embodiments 1 to 9, or a pharmaceutically acceptable salt thereof:

(Here,

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH).

Embodiment 21. A compound of formula A' having the following structure, or a compound of any one of Embodiments 1 to 9, or a pharmaceutically acceptable salt thereof:

(Here,

Each R is independently selected from H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH).

Embodiment 22. A compound of formula A' having the following structure, or a compound of any one of Embodiments 1 to 9, or a pharmaceutically acceptable salt thereof:

(Here,

Each R is independently selected from H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH).

Embodiment 23. A compound of formula A' having the following structure, or a compound of any one of Embodiments 1 to 9, or a pharmaceutically acceptable salt thereof:

(Here,

Xa is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ -, and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH).

Embodiment 24. A compound of formula A' having the following structure, or a compound of any one of Embodiments 1 to 9, or a pharmaceutically acceptable salt thereof:

(Here,

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH).

Embodiment 25. A compound of formula A' having the following structure, or a compound of any one of Embodiments 1 to 9, or a pharmaceutically acceptable salt thereof:

(Here,

Xb is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ -, and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH).

Embodiment 26. A compound of formula A' having the following structure, or a compound of any one of Embodiments 1 to 9, or a pharmaceutically acceptable salt thereof:

Refers to the compound of Example 58 as described in .

Embodiment 27. A compound of formula A' having the following structure, or a compound of any one of Embodiments 1 to 9, or a pharmaceutically acceptable salt thereof:

.

Embodiment 28. A compound of formula A' having the following structure, or a compound of any one of Embodiments 1 to 9, or a pharmaceutically acceptable salt thereof:

.

Embodiment 29. A compound of formula A' having the following structure, or a compound of any one of Embodiments 1 to 9, or a pharmaceutically acceptable salt thereof:

.

Embodiment 30. A compound of formula A' having the following structure, or a compound of any one of Embodiments 1 to 9, or a pharmaceutically acceptable salt thereof:

.

Embodiment 31. A compound of formula A' having the following structure, or a compound of any one of Embodiments 1 to 9, or a pharmaceutically acceptable salt thereof:

(where n is an integer from 2 to 24).

Embodiment 32. A compound of formula A' having the structure of the compound of Table B or any one of Embodiments 1 to 9 or a pharmaceutically acceptable salt thereof.

Embodiment 33. A conjugate linker of a linker-drug group of formula A' having a structure of formula C':

[Chemical formula C']

(Here,

L ₁ is a crosslinking spacer;

Lp is a bivalent peptide spacer;

GL ₂ -A is a self-immolative spacer;

R ² is a hydrophilic moiety;

L ₂ is a bond, methylene, neopentylene or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

L ₃ is a spacer moiety).

Embodiment 34.

L ₁ is a crosslinking spacer;

Lp is a bivalent peptide spacer containing 2 to 4 amino acid residues;

GL ₂ -A is a self-immolative spacer;

R ² is a hydrophilic moiety;

L ₂ is a bond, methylene, neopentylene or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

The conjugate linker of embodiment 33, wherein L ₃ is a spacer moiety.

Embodiment 35.

L ₁ is a crosslinking spacer;

Lp is a bivalent peptide spacer containing 2 to 4 amino acid residues;

Ki-ga

As selected from,

* indicates an attachment point to D (e.g., to N or O of the drug moiety), *** indicates the attachment point to Lp;

R ² is a hydrophilic moiety;

L ₂ is a bond, methylene, neopentylene or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

The conjugate linker of embodiment 33 or 34, wherein L ₃ is a spacer moiety.

Embodiment 36.

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**;

*-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)O(CH ₂ ) _m SSC(R ³ ) ₂ (CH ₂ ) _m C(=O)NR ³ (CH ₂ ) _m NR ³ C(=O)(CH ₂ ) _m -**;

*-C(=O)O(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**;

*-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**; _{* -C} ( ₌ O)(CH ₂ ) _m

_* -C(=O)(( _{CH 2 ) m} O) _t (CH ₂ ) _n *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n -**;

* _-C (=O) _{( CH 2} ) _m NHC(=O)(CH ₂ ) _n

*-C(=O)(( _{CH 2} ) _m O _{) t (} CH ₂ ) _n NHC(=O)(CH ₂ ) _n

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n C(=O)NH(CH ₂ ) _m -**;

*-C(=O)(CH ₂ ) _m C(R ³ ) ₂ -** or *-C(=O)(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**, where * in L ₁ indicates the point of attachment to Lp;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 is a hydrophilic moiety selected from (substituted with a group);

Each R ³ is independently selected from H and C ₁ -C ₆ alkyl;

X ₁ is and;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is a bivalent peptide spacer comprising amino acid residues selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan and tyrosine;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)C(R ^b ) ₂ NHC(=O)O-**,

-NHC(=O)C(R ^b ) ₂ NH-**, -NHC(=O)C(R ^b ) ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**,

-C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, and each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

A conjugate linker according to any one of embodiments 33 to 35, wherein * of L ₃ represents an attachment point to R ² .

Embodiment 37.

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * in L ₁ indicates the point of attachment to Lp;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is As a divalent peptide spacer selected from, * of Lp indicates an attachment point to L ₁ ;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -NHC(=O)CH ₂ NH-**, -NHC(=O)CH ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, and each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

* in L ₃ indicates an attachment point to R ² ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 is a hydrophilic moiety selected from (substituted with a group);

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D, the conjugate linker of any one of embodiments 33 to 36.

Embodiment 38.

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * in L ₁ indicates the point of attachment to Lp;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is As a divalent peptide spacer selected from, * of Lp indicates an attachment point to L ₁ and ** of Lp indicates an attachment point to the -NH- group of G;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH2N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, and each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

* in L ₃ indicates an attachment point to R ² ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 A hydrophilic moiety selected from (substituted with a group);

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or

A conjugate linker according to any one of embodiments 33 to 37, wherein -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D.

Embodiment 39.

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * in L ₁ indicates the point of attachment to Lp;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is As a divalent peptide spacer selected from, * of Lp indicates an attachment point to L ₁ ;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH 2 N(R b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, or -NHC(=O)NH-**, each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

* in L ₃ indicates an attachment point to R ² ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 is a hydrophilic moiety selected from (substituted with a group);

A conjugate linker according to any one of embodiments 33 to 38, wherein A is a bond or -OC(=O)*, wherein * represents an attachment point to D.

Embodiment 40.

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * in L ₁ indicates the point of attachment to Lp;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is As a divalent peptide spacer selected from, * of Lp indicates an attachment point to L ₁ ;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH 2 N(R b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, or -C(=O)N(XR ² )-**, each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

* in L ₃ indicates an attachment point to R ² ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 A hydrophilic moiety selected from (substituted with a group);

A conjugate linker according to any one of embodiments 33 to 39, wherein A is a bond or -OC(=O)*, wherein * represents an attachment point to D.

Embodiment 41. A conjugate linker of formula C' having a structure having a structure of formula D':

[Chemical formula D']

(Here,

L ₁ is a crosslinking spacer;

Lp is a bivalent peptide spacer;

R ² is a hydrophilic moiety;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

L ₃ is a spacer moiety).

Embodiment 42.

L ₁ is a crosslinking spacer;

Lp is a bivalent peptide spacer containing 2 to 4 amino acid residues;

R ² is a hydrophilic moiety;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

The conjugate linker of embodiment 41, wherein L ₃ is a spacer moiety.

Embodiment 43.

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**;

*-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)O(CH ₂ ) _m SSC(R ³ ) ₂ (CH ₂ ) _m C(=O)NR ³ (CH ₂ ) _m NR ³ C(=O)(CH ₂ ) _m -**;

*-C(=O)O(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**;

*-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**; _{* -C} ( ₌ O)(CH ₂ ) _m

_* -C(=O)(( _{CH 2 ) m} O) _t (CH ₂ ) _n *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n -**;

* _-C (=O) _{( CH 2} ) _m NHC(=O)(CH ₂ ) _n

*-C(=O)(( _{CH 2} ) _m O _{) t (} CH ₂ ) _n NHC(=O)(CH ₂ ) _n

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m C(R ³ ) ₂ -** or

*-C(=O)(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**, where * in L ₁ indicates the attachment point to Lp;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 A hydrophilic moiety selected from (substituted with a group);

Each R ³ is independently selected from H and C ₁ -C ₆ alkyl;

X ₁ is and;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is a bivalent peptide spacer comprising amino acid residues selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan and tyrosine;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH2N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, and each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

The conjugate linker of embodiment 41 or 42, wherein * of L ₃ indicates an attachment point to R ² .

Embodiment 44.

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * in L ₁ indicates the point of attachment to Lp;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is As a divalent peptide spacer selected from, * of Lp represents an attachment point to L ₁ and ** of Lp represents an attachment point to the -NH- group;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH2N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, and each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

* in L ₃ indicates an attachment point to R ² ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 A hydrophilic moiety selected from (substituted with a group);

A is a bond, -OC(=O)-*, , OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or

A conjugate linker according to any one of embodiments 41 to 43, wherein -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D.

Embodiment 45.

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * in L ₁ indicates the point of attachment to Lp;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is As a divalent peptide spacer selected from, * of Lp indicates an attachment point to L ₁ and ** of Lp indicates an attachment point to the -NH- group of G;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, and each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH2-triazolyl-*, the *** of X represents the point of attachment to W and the * of X represents the point of attachment to R ₂ ;

* in L ₃ indicates an attachment point to R ² ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 A hydrophilic moiety selected from (substituted with a group);

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or

A conjugate linker according to any one of embodiments 41 to 44, wherein -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl and the * of A represents the point of attachment to D.

Embodiment 46.

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * in L ₁ indicates the point of attachment to Lp;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is As a divalent peptide spacer selected from, * of Lp indicates an attachment point to L ₁ and ** of Lp indicates an attachment point to the -NH- group of G;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, or -NHC(=O)NH-**, each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

* in L ₃ indicates an attachment point to R ² ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 A hydrophilic moiety selected from (substituted with a group);

A conjugate linker according to any one of embodiments 41 to 45, wherein A is a bond or -OC(=O)*, wherein * represents an attachment point to D.

Embodiment 47.

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * in L ₁ indicates the point of attachment to Lp;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is As a bivalent peptide spacer selected from, * of Lp indicates the point of attachment to L ₁ and ** of Lp indicates the point of attachment to the -NH- group of G;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, or -C(=O)N(XR ² )-**, each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

* in L ₃ indicates an attachment point to R ² ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 is a hydrophilic moiety selected from (substituted with a group);

A conjugate linker according to any one of embodiments 41 to 46, wherein A is a bond or -OC(=O)*, wherein * represents an attachment point to D.

Embodiment 48. A conjugate linker of any one of Embodiments 33 to 47 having the following structure:

(Here,

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH).

Embodiment 49. A conjugate linker of any one of Embodiments 33 to 47 having the following structure:

(Here,

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH).

Embodiment 50. A conjugate linker of any one of Embodiments 33 to 47 having the following structure:

(Here,

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH).

Embodiment 51. A conjugate linker of any one of Embodiments 33 to 47 having the following structure:

(Here,

Each R is independently selected from H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH).

Embodiment 52. A conjugate linker of any one of Embodiments 37 to 47 having the following structure:

(Here,

Each R is independently selected from H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH).

Embodiment 53. A conjugate linker of any one of Embodiments 33 to 47 having the following structure:

(Here,

Xa is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ -, and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH).

Embodiment 54. A conjugate linker of any one of Embodiments 33 to 47 having the following structure:

(Here,

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH).

Embodiment 55. A conjugate linker of any one of Embodiments 33 to 47 having the following structure:

(Here,

Xb is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ -, and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH).

Embodiment 56. A conjugate linker of any one of Embodiments 33 to 47 having the following structure:

Embodiment 57. A conjugate linker of any one of Embodiments 33 to 47 having the following structure:

Embodiment 58. A conjugate linker of any one of Embodiments 37 to 47 having the following structure:

Embodiment 59. A conjugate linker of any one of Embodiments 33 to 47 having the following structure:

Embodiment 60. A conjugate linker of any one of Embodiments 33 to 47 having the following structure:

Embodiment 61. A conjugate linker of any one of Embodiments 33 to 47 having the following structure:

(where n is an integer from 2 to 24).

For illustrative purposes, the general reaction schemes presented herein provide potential routes for synthesizing the compounds and key intermediates of the present invention. For a more detailed description of individual reaction steps, see the Examples section below. While specific starting materials and reagents are indicated in the schemes and discussed below, other starting materials and reagents can be readily substituted to provide a variety of derivatives and/or reaction conditions. Furthermore, many of the compounds prepared by the methods described below can be further modified in light of the present disclosure using conventional chemistry well known to those skilled in the art.

As an example, a general synthesis of a compound of formula B' is illustrated in Scheme 1 below.

Reaction Scheme 1

Antibody drug conjugate of the present invention

The present invention provides an antibody drug conjugate, also referred to herein as an immunoconjugate, comprising a conjugate linker comprising one or more hydrophilic moieties.

The antibody drug conjugate of the present invention has a structure represented by chemical formula E':

[Chemical formula E']

[Here,

Ab is an antibody (e.g., anti-EphA2 antibody or anti-B7-H3 antibody) or a fragment thereof;

R ¹⁰⁰ is a coupling group;

L ₁ is a crosslinking spacer;

Lp is a bivalent peptide spacer;

GL ₂ -A is a self-immolative spacer;

R ² is a hydrophilic moiety;

L ₂ is a bond, methylene, neopentylene or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

L ₃ is a spacer moiety;

D may comprise a drug moiety, e.g., a panRAS inhibitor, as defined herein, and N, wherein D may be linked to A via a direct bond from A to N of the drug moiety;

y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16].

Specific embodiments and examples of antibody drug conjugates of the present invention are provided in the list of embodiments below. It will be appreciated that the features specified in each embodiment may be combined with other specified features to provide additional embodiments of the present invention.

Embodiment 62.

Ab is an antibody (e.g., anti-EphA2 antibody or anti-B7-H3 antibody) or a fragment thereof;

R ¹⁰⁰ is a coupling group;

L ₁ is a crosslinking spacer;

Lp is a bivalent peptide spacer containing 2 to 4 amino acid residues;

GL ₂ -A is a self-immolative spacer;

R ² is a hydrophilic moiety;

L ₂ is a bond, methylene, neopentylene or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

L ₃ is a spacer moiety;

D is a drug moiety as defined herein, wherein D is linked to A via a direct bond from A to D (e.g., N of the drug moiety);

An immunoconjugate of formula E', wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 63.

Ab is an antibody (e.g., anti-EphA2 antibody or anti-B7-H3 antibody) or a fragment thereof;

R ¹⁰⁰ is a coupling group;

L ₁ is a crosslinking spacer;

Lp is a bivalent peptide spacer containing 2 to 4 amino acid residues;

Ki-ga

As selected from,

* indicates an attachment point to D (e.g., to N or O of the drug moiety), *** indicates the attachment point to Lp;

R ² is a hydrophilic moiety;

L ₂ is a bond, methylene, neopentylene or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

L ₃ is a spacer moiety;

D is a drug moiety as defined herein and comprising N, wherein D is linked to A via a direct bond from A to N of the drug moiety;

An immunoconjugate of formula E' or embodiment 62, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 64. An immunoconjugate of formula E' having the structure of formula F' or any one of embodiments 62 to 63:

[Chemical formula F']

[Here,

Ab is an antibody (e.g., anti-EphA2 antibody or anti-B7-H3 antibody) or a fragment thereof;

R ¹⁰⁰ is a coupling group;

L ₁ is a crosslinking spacer;

Lp is a bivalent peptide spacer containing 2 to 4 amino acid residues;

R ² is a hydrophilic moiety;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

L ₃ is a spacer moiety;

D is a drug moiety as defined herein and comprising N, wherein D is linked to A via a direct bond from A to N of the drug moiety;

y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16].

Embodiment 65.

Ab is an antibody (e.g., anti-EphA2 antibody or anti-B7-H3 antibody) or a fragment thereof;

R ¹⁰⁰ is -S-, -C(=O)-, -ON=***, -NHC(=O)CH ₂ -***, -S(=O) ₂ CH ₂ CH ₂ -***, -(CH ₂ ) ₂ S(=O) ₂ CH ₂ CH ₂ -***, -NHS(=O) ₂ CH ₂ CH _2- ***,

-NHC(=O)CH ₂ CH ₂ -***, -CH ₂ NHCH ₂ CH ₂ -***, -NHCH ₂ CH ₂ -***, As, *** of R ¹⁰⁰ represents the attachment point to Ab;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**;

*-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)O(CH ₂ ) _m SSC(R ³ ) ₂ (CH ₂ ) _m C(=O)NR ³ (CH ₂ ) _m NR ³ C(=O)(CH ₂ ) _m -**;

*-C(=O)O(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**;

*-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**; _{* -C} ( ₌ O)(CH ₂ ) _m

_* -C(=O)(( _{CH 2 ) m} O) _t (CH ₂ ) _n *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n -**;

* _-C (=O) _{( CH 2} ) _m NHC(=O)(CH ₂ ) _n

*-C(=O)(( _{CH 2} ) _m O _{) t (} CH ₂ ) _n NHC(=O)(CH ₂ ) _n

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m C(R ³ ) ₂ -** or

*-C(=O)(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**, where * of L ₁ indicates the point of attachment to Lp, and ** of L ₁ indicates the point of attachment to R ¹⁰⁰ ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 is a hydrophilic moiety selected from (substituted with a group);

Each R ³ is independently selected from H and C ₁ -C ₆ alkyl;

R ⁴ is 2-pyridyl or 4-pyridyl;

Each R ⁵ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl, and -OH;

Each R ⁶ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl, -NH ₂ , -OCH ₃ , -OCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -CN, -NO ₂ and -OH;

Each R ⁷ is independently selected from H, C _1-6 alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C _1-4 alkoxy substituted with -C(=O)OH, and C _1-4 alkyl substituted with -C(=O)OH;

X ₁ is and;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is a bivalent peptide spacer comprising amino acid residues selected from valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan and tyrosine;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(Rb)C(=O)O-**, -NHC(=O)C(R ^b ) ₂ NHC(=O)O-**, -NHC(=O)C(R ^b ) ₂ NH-**, -NHC(=O)C(R ^b ) ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, and each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

* in L ₃ indicates an attachment point to R ² ;

D is a drug moiety as defined herein and comprising N, wherein D is linked to A via a direct bond from A to N of the drug moiety;

An immunoconjugate of formula D' or any one of embodiments 62 to 64, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 66.

Ab is an antibody (e.g., anti-EphA2 antibody or anti-B7-H3 antibody) or a fragment thereof;

R ¹⁰⁰ is As, *** of R ¹⁰⁰ represents the attachment point to Ab;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * in L ₁ indicates the point of attachment to Lp, and ** in L ₁ indicates the point of attachment to R ¹⁰⁰ ;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is As a divalent peptide spacer selected from, * of Lp represents an attachment point to L ₁ and ** of Lp represents an attachment point to the -NH- group;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**,

-NHC(=O)CH ₂ NH-**, -NHC(=O)CH ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**,

-C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**,

-OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, and each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

* in L ₃ indicates an attachment point to R ² ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 A hydrophilic moiety selected from (substituted with a group);

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a drug moiety as defined herein and comprising N or O, wherein D is linked to A via a direct bond from A to the N or O of the drug moiety;

An immunoconjugate of formula D' or any one of embodiments 62 to 65, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 67.

Ab is an antibody (e.g., anti-EphA2 antibody or anti-B7-H3 antibody) or a fragment thereof;

R ¹⁰⁰ is As, *** of R ¹⁰⁰ represents the attachment point to Ab;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * in L ₁ indicates the point of attachment to Lp, and ** in L ₁ indicates the point of attachment to R ¹⁰⁰ ;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is As a divalent peptide spacer selected from, * of Lp indicates an attachment point to L ₁ and ** of Lp indicates an attachment point to the -NH- group of G;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, and each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

* in L ₃ indicates an attachment point to R ² ;

R ² is a hydrophilic moiety selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (substituted with 1 to 3), and A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, wherein each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * of A represents the point of attachment to D;

D is a drug moiety as defined herein and comprising N or O, wherein D is linked to A via a direct bond from A to the N or O of the drug moiety;

An immunoconjugate of formula E' or any one of embodiments 62 to 66, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 68.

Ab is an antibody (e.g., anti-EphA2 antibody or anti-B7-H3 antibody) or a fragment thereof;

R ¹⁰⁰ is As, *** of R ¹⁰⁰ represents the attachment point to Ab;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * in L ₁ indicates the point of attachment to Lp, and ** in L ₁ indicates the point of attachment to R ¹⁰⁰ ;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is As a divalent peptide spacer selected from, * of Lp indicates an attachment point to L ₁ and ** of Lp indicates an attachment point to the -NH- group of G;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, or -NHC(=O)NH-**, each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

* in L ₃ indicates an attachment point to R ² ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 A hydrophilic moiety selected from (substituted with a group);

A is a bond or -OC(=O)*, where * indicates the point of attachment to D;

D is a drug moiety as defined herein and comprising N or O, wherein D is linked to A via a direct bond from A to the N or O of the drug moiety;

An immunoconjugate of formula E' or any one of embodiments 62 to 67, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 69.

Ab is an antibody (e.g., anti-EphA2 antibody or anti-B7-H3 antibody) or a fragment thereof;

R ¹⁰⁰ is As, *** of R ¹⁰⁰ represents the attachment point to Ab;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * in L ₁ indicates the point of attachment to Lp, and ** in L ₁ indicates the point of attachment to R ¹⁰⁰ ;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is As a divalent peptide spacer selected from, * of Lp indicates an attachment point to L ₁ and ** of Lp indicates an attachment point to the -NH- group of G;

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, or -C(=O)N(XR ² )-**, each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

* in L ₃ indicates an attachment point to R ² ;

R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 is a hydrophilic moiety selected from (substituted with a group);

A is a bond or -OC(=O)*, where * indicates the point of attachment to D;

D is a drug moiety as defined herein and comprising N or O, wherein D is linked to A via a direct bond from A to the N or O of the drug moiety;

An immunoconjugate of formula E' or any one of embodiments 62 to 68, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 70.

R ¹⁰⁰ is -S-, -C(=O)-, -ON=***, -NHC(=O)CH ₂ -***, -S(=O) ₂ CH ₂ CH ₂ -***, -(CH ₂ ) ₂ S(=O) ₂ CH ₂ CH ₂ -***, -NHS(=O) ₂ CH ₂ CH _2- ***, -NHC(=O)CH ₂ CH ₂ -***, -CH ₂ NHCH ₂ CH ₂ -***, -NHCH ₂ CH ₂ -***, As, *** of R ¹⁰⁰ represents an attachment point to Ab, an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 65.

Embodiment 71.

R ¹⁰⁰ is As, *** of R ¹⁰⁰ represents an attachment point to Ab, an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 60 to 63.

Embodiment 72.

R ¹⁰⁰ is As, *** of R ¹⁰⁰ represents an attachment point to Ab, an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 65.

Embodiment 73. An immunoconjugate of formula E' having the following structure or an immunoconjugate of any one of Embodiments 62 to 72:

(Here,

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH, and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 74. An immunoconjugate of formula E' having the following structure or an immunoconjugate of any one of Embodiments 62 to 72:

(Here,

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH, and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 75. An immunoconjugate of formula E' having the following structure or an immunoconjugate of any one of Embodiments 62 to 72:

(Here,

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH, and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 76. An immunoconjugate of formula E' having the following structure or an immunoconjugate of any one of Embodiments 62 to 72:

(Here,

Each R is independently selected from H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH, and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 77. An immunoconjugate of formula E' having the following structure or an immunoconjugate of any one of Embodiments 62 to 72:

(Here,

Each R is independently selected from H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH, and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 78. An immunoconjugate of formula E' having the following structure or an immunoconjugate of any one of Embodiments 62 to 72:

(Here,

Xa is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ -, each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH, and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

Embodiment 79. An immunoconjugate of formula E' having the following structure or an immunoconjugate of any one of Embodiments 62 to 72:

(Here,

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH, and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 80. An immunoconjugate of formula E' having the following structure or an immunoconjugate of any one of Embodiments 62 to 72:

(Here,

Xb is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ -, each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH, and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

Embodiment 81. An immunoconjugate of formula E' having the following structure or an immunoconjugate of any one of Embodiments 62 to 72:

(where y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16).

Embodiment 82. An immunoconjugate of formula E' having the following structure or an immunoconjugate of any one of Embodiments 62 to 72:

Here, y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16).

Embodiment 83. An immunoconjugate of formula E' having the following structure or an immunoconjugate of any one of Embodiments 62 to 72:

(where y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16).

Embodiment 84. An immunoconjugate of formula E' having the following structure or an immunoconjugate of any one of Embodiments 62 to 72:

(where y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16).

Embodiment 85. An immunoconjugate of formula E' having the following structure or an immunoconjugate of any one of Embodiments 62 to 72:

(where y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16).

Embodiment 86. An immunoconjugate of formula E' having the following structure or an immunoconjugate of any one of Embodiments 62 to 72:

(where y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16).

Specific embodiments and examples of the linker-drug groups, conjugate linkers, and antibody-drug conjugates of the present invention are provided in the list of additional embodiments below. It will be appreciated that the features specified in each embodiment may be combined with other specified features to provide additional embodiments of the present invention.

Embodiment 87.

G is A compound of formula A' or a compound of any one of embodiments ₁ to ₂ , or a pharmaceutically acceptable salt thereof, a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 40, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 63, wherein * of G represents an attachment point to L 2 , ** of G represents an attachment point to L 3 , and *** of G represents an attachment point to Lp.

Embodiment 88.

G is A compound of formula A' or a compound of any one of embodiments ₁ to ₂ , or a pharmaceutically acceptable salt thereof, a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 40, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 63, wherein * of G represents an attachment point to L 2 , ** of G represents an attachment point to L 3 , and *** of G represents an attachment point to Lp.

Embodiment 89.

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**;

*-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)O(CH ₂ ) _m SSC(R ³ ) ₂ (CH ₂ ) _m C(=O)NR ³ (CH ₂ ) _m NR ³ C(=O)(CH ₂ ) _m -**;

*-C(=O)O(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**;

*-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**; _{* -C} ( ₌ O)(CH ₂ ) _m

_* -C(=O)(( _{CH 2 ) m} O) _t (CH ₂ ) _n *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n -**; * _-C (=O) _{( CH 2} ) _m NHC(=O)(CH ₂ ) _n

*-C(=O)(( _{CH 2} ) _m O _{) t (} CH ₂ ) _n NHC(=O)(CH ₂ ) _n

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m C(R ³ ) ₂ -** or

*-C(=O)(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**, wherein * of L ₁ represents the point of attachment to Lp, ** of L ₁ represents the point of attachment to R ¹ if present, or ** of L ₁ represents the point of attachment to R ¹⁰⁰ if present, a compound of Formula A' or a compound of any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a conjugate linker of Formula C' or a conjugate linker of any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or an immunoconjugate of any one of Embodiments 62 to 72.

Embodiment 90.

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**;

*-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**;

*-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**; *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n -**; *C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n C(=O)NH(CH ₂ ) _m -**;

A compound of Formula A' or any one of Embodiments 1 to 17, or _a pharmaceutically acceptable salt thereof, a conjugate linker of _Formula C' or any one of Embodiments ^{33 to 47} _{, and} an immunoconjugate of _Formula E' or any one of Embodiments 62 to 72, wherein *-C(=O)(CH ₂ ) _m C(R ₃ ⁾ ₂ -** or *-C(=O)(CH 2 ) m C(=O)NH(CH 2 ) m -**, wherein * of L ₁ represents the point of attachment to ^Lp , ** of L 1 , if present, represents the point of attachment to R 1 , or ** of L 1 , if present, represents the point of attachment to R 100 .

Embodiment 91.

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**; or *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**, wherein * of L ₁ represents the point of attachment to Lp, ** of L ₁ , if present, represents the point of attachment to R ¹ , or ** of L ₁ , if present, represents the point of attachment to R ¹⁰⁰ , a compound of Formula A' or a compound of any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a conjugate linker of Formula C' or a conjugate linker of any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or an immunoconjugate of any one of Embodiments 62 to 72.

Embodiment 92.

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; A compound _of Formula A' _or any one of Embodiments 1 _{to 17} , or a _{pharmaceutically} acceptable salt thereof, a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula _E ' or any one of Embodiments ⁶² to 72, wherein *-C(=O)(CH ₂ ) _m - _{** or} *-C(= _O )NH((CH 2 ) m O) t (CH 2 ) n -**, wherein * of L ^{1 represents the point of attachment to Lp, ** of L 1} , if present, represents the point of attachment to R 1 , or ** of L 1 , if present, represents the point of attachment to R 100 .

Embodiment 93. A compound of Formula ^A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein L ₁ is *-C(= _O )(CH ₂ ) _m O(CH ₂ ) _m -**, wherein * of L ₁ represents the point of attachment to Lp, ** of L ₁ , if present, represents the point of attachment to R ¹ , or ** of L 1, if present, represents the point of attachment to R 100 , a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72.

Embodiment 94. A compound of Formula A' or any one of Embodiments ₁ to 17, or a pharmaceutically acceptable salt thereof, wherein L ₁ is *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**, wherein * of L ₁ represents the point of attachment to Lp, ** of L ¹ , if present, represents the point of attachment to R ¹ , or ** of L ₁ , if present, represents the point of attachment to R 100 , a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72.

Embodiment 95. A compound of Formula A _' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein L ₁ is *-C(=O)(CH ₂ ) _m -**, wherein * of L ₁ represents a point of attachment to Lp, ** of L ₁ , if present, represents a point of attachment to R ^{1 ,} or ** of L 1, if present, represents a point of attachment to R ¹⁰⁰ , a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72.

Embodiment 96. A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein L ₁ is *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**, wherein * of L ₁ represents the point of attachment to ^Lp , ** of L ₁ , if present, represents the point of attachment to R ¹ , or ** of L ₁ , if present, represents the point of attachment to R 100 , a conjugate linker of Formula C' or any one of Embodiments 32 to 46, and an immunoconjugate of Formula E' or any one of Embodiments 60 to 70.

Embodiment 97. A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein Lp is an enzymatically cleavable divalent peptide spacer, a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or any one of Embodiments 84 to 93.

Embodiment 98. A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein Lp is a divalent peptide spacer comprising an amino acid residue selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan and tyrosine, a conjugate linker of Formula C' or any one of Embodiments 32 to 46, and an immunoconjugate of Formula E' or any one of Embodiments 60 to 70, or an immunoconjugate of any one of Embodiments 87 to 97.

Embodiment 99. A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein Lp is a divalent peptide spacer comprising 2 to 4 amino acid residues, a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or any one of Embodiments 87 to 98.

Embodiment 100. A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein Lp is a divalent peptide spacer comprising 2 to 4 amino acid residues each independently selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine, a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 99.

Embodiment 101.

Lp is A compound of formula A' or a compound of any one of embodiments ₁ to 17, or a pharmaceutically acceptable salt thereof, a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 100, wherein * of Lp represents a point of attachment to L 1, ** of Lp represents a point of attachment to the -NH- group of formula B', or ** of Lp represents a point of attachment to G of formula A'.

Embodiment 102.

Lp is A compound of formula A' or a compound of any one of embodiments ₁ to 17, or a pharmaceutically acceptable salt thereof, a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 101, wherein * of Lp represents a point of attachment to L 1, ** of Lp represents a point of attachment to the -NH- group of formula B', or ** of Lp represents a point of attachment to G of formula A'.

Embodiment 103.

Lp is A compound of formula A' or a compound of any one of embodiments ₁ to 17, or a pharmaceutically acceptable salt thereof, a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 101, wherein * of Lp represents a point of attachment to L 1, ** of Lp represents a point of attachment to the -NH- group of formula B', or ** of Lp represents a point of attachment to G of formula A'.

Embodiment 104.

Lp is A compound of formula A' or a compound of any one of embodiments ₁ to 17, or a pharmaceutically acceptable salt thereof, a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 101, wherein * of Lp represents a point of attachment to L 1, ** of Lp represents a point of attachment to the -NH- group of formula B', or ** of Lp represents a point of attachment to G of formula A'.

Embodiment 105.

Lp is A compound of formula A' or a compound of any one of embodiments ₁ to 17, or a pharmaceutically acceptable salt thereof, a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 101, wherein * of Lp represents a point of attachment to L 1, ** of Lp represents a point of attachment to the -NH- group of formula B', or ** of Lp represents a point of attachment to G of formula A'.

Embodiment 106.

Lp is A compound of formula A' or a compound of any one of embodiments ₁ to 17, or a pharmaceutically acceptable salt thereof, a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 101, wherein * of Lp represents a point of attachment to L 1, ** of Lp represents a point of attachment to the -NH- group of formula B', or ** of Lp represents a point of attachment to G of formula A'.

Embodiment 107. A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein L ₂ is a bond, methylene, or C ₂ -C ₃ alkenylene, a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 106.

Embodiment 108. A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein L ₂ is a bond or methylene, a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or any one of Embodiments 87 to 107.

Embodiment 109. A compound of Formula A' or a compound of any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein L ₂ is a bond, a conjugate linker of Formula C' or a conjugate linker of any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or an immunoconjugate of any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 108.

Embodiment 110. A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein L ₂ is methylene, a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or any one of Embodiments 87 to 108.

Embodiment 111.

A compound of Formula A' or any one of Embodiments 1 to 30, _or a _{pharmaceutically} acceptable salt _thereof , a conjugate linker of Formula C' or any one of Embodiments 33 _to 47, and an immunoconjugate of Formula _E ' or any one of Embodiments 62 to ⁸⁵ , or an immunoconjugate of any one of Embodiments 87 to ₁₁₀ , wherein ^A is a bond, _-OC (=O ₎ -, -OC(=O)N(CH ₃ ) ^CH ₂ CH ₂ N(CH 3 )C(=O)-, or -OC(=O)N(CH ₃ )C(R a ) 2 C(R a ) 2 N(CH 3 )C(=O)-, wherein each R a is independently selected from H, C 1 -C 6 alkyl, or C 3 -C 8 cycloalkyl.

Embodiment 112. A compound of Formula A' or any one of Embodiments 1 to 32, or a pharmaceutically acceptable salt thereof, wherein A is a bond or -OC(=O), a conjugate linker of Formula C' or any one of Embodiments 33 to 61, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 86, or any one of Embodiments 87 to 111.

Embodiment 113. A compound of formula A' or a compound of any one of embodiments 1 to 32, or a pharmaceutically acceptable salt thereof, a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 61, and an immunoconjugate of formula E' or any one of embodiments 62 to 86, or an immunoconjugate of any one of embodiments 87 to 112, wherein A is a bond.

Embodiment 114. A compound of formula A', or a compound of any one of embodiments 1 to 32, or a pharmaceutically acceptable salt thereof, wherein A is -OC(=O), a conjugate linker of formula C', or a conjugate linker of any one of embodiments 33 to 61, and an immunoconjugate of formula E', or an immunoconjugate of any one of embodiments 62 to 86, or an immunoconjugate of any one of embodiments 87 to 112.

Embodiment 115.

A is A compound of formula A' or a compound of any one of embodiments 1 to 32, or a pharmaceutically acceptable salt thereof, a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 61, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 86, or an immunoconjugate of any one of embodiments 87 to 110.

Embodiment 116.

A compound of Formula A' _or any one of Embodiments 1 to 32, or _a pharmaceutically acceptable salt thereof, wherein A is -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)- or -OC(= ^O )N(CH ₃ )C(R ^a _{) 2} C(R a ) 2 N(CH 3 )C ₍ =O)-, wherein each R ^a is independently selected from H, C 1 -C 6 alkyl or C ₃ -C ₈ cycloalkyl, a conjugate linker of Formula C' or any one of Embodiments 33 to 61, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 85, or an immunoconjugate of any one of Embodiments 86 to 110.

Embodiment 117.

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)C(R ^b ) ₂ NHC(=O)O-**,

-NHC(=O)C(R ^b ) ₂ NH-**, -NHC(=O)C(R ^b ) ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**,

-C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**,

-CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**,

-NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**,

-C(=O)-, -C(=O)O-** or -NH-, and each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

A compound of formula A' or a compound of any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein * of L ₃ represents an attachment point to R ² , a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 49, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 116.

Embodiment 118.

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**,

-NHC(=O)CH ₂ NH-**, -NHC(=O)CH ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**,

-C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**,

-CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**,

-NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**,

-C(=O)-, -C(=O)O-** or -NH-, and each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond;

A compound of formula A' or a compound of any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein * of L ₃ represents an attachment point to R ² , a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 117.

Embodiment 119.

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**,

-NHC(=O)CH ₂ NH-**, -NHC(=O)CH ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**,

-C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**,

-NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**,

-C(=O)-, -C(=O)O-** or -NH-, and each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is triazolyl, *** of X indicates the point of attachment to W, * of X indicates the point of attachment to R ² ;

A compound of formula A' or a compound of any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein * of L ₃ represents an attachment point to R ² , a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 118.

Embodiment 120.

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**,

-NHC(=O)CH ₂ NH-**, -NHC(=O)CH ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**,

-C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**,

-CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**,

-NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**,

-C(=O)-, -C(=O)O-** or -NH-, and each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

A compound of formula A' or a compound of any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein * of L ₃ represents an attachment point to R ² , a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 118.

Embodiment 121.

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond, triazolyl or ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

A compound of formula A' or a compound of any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein * of L ₃ represents an attachment point to R ² , a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 118.

Embodiment 122.

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is a bond;

A compound of formula A' or a compound of any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein * of L ₃ represents an attachment point to R ² , a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 118.

Embodiment 123.

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is triazolyl, *** of X indicates the point of attachment to W, * of X indicates the point of attachment to R ² ;

A compound of formula A' or a compound of any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein * of L ₃ represents an attachment point to R ² , a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 83 to 118.

Embodiment 124.

L ₃ is a structure As a spacer moiety having,

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and ** of W represents the point of attachment to X;

X is ***-CH ₂ -triazolyl-*, the *** of X represents the point of attachment to W, and the * of X represents the point of attachment to R ² ;

A compound of formula A' or a compound of any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein * of L ₃ represents an attachment point to R ² , a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 118.

Embodiment 125. R ² is polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl (1 to 3 A compound of formula A' or a compound of any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 86 to 124, wherein the hydrophilic moiety is selected from the group substituted with a group.

Embodiment 126. R ² is a sugar, a compound of Formula A' or a compound of any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a conjugate linker of Formula C' or a conjugate linker of any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or an immunoconjugate of any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 125.

Embodiment 127. A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein R ² is an oligosaccharide, a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or any one of Embodiments 87 to 125.

Embodiment 128. A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein R ² is a polypeptide, a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or any one of Embodiments 87 to 125.

Embodiment 129. A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein R ² is a polyalkylene glycol, a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or any one of Embodiments 87 to 125.

Embodiment 130. A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein R ² is a polyalkylene glycol having the structure -(O(CH ₂ ) _m ) _t R', wherein R' is OH, OCH ₃ or OCH ₂ CH ₂ C(=O)OH, m is 1 to 10, and t is 4 to 40, a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 125.

Embodiment 131. A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein R ² is a polyalkylene glycol having the structure -((CH ₂ ) _m O) _t R''-, wherein R'' is H, CH ₃ or CH ₂ CH ₂ C(=O)OH, m is 1 to 10, and t is 4 to 40, a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 125.

Embodiment 132. A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein R ² is polyethylene glycol, a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or any one of Embodiments 87 to 125.

Embodiment 133. A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein R ² is polyethylene glycol having the structure -(OCH ₂ CH ₂ ) _t R', wherein R' is OH, OCH ₃ or OCH ₂ CH ₂ C(=O)OH, and t is 4 to 40, a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 125.

Embodiment 134. A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein R ² is polyethylene glycol having the structure -(CH ₂ CH ₂ O) _t R''-, wherein R'' is H, CH ₃ or CH ₂ CH ₂ C(=O)OH, and t is 4 to 40, a conjugate linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 125.

Embodiment 135.

R ² is A compound of formula A' or a compound of any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein * of R ² represents a point of attachment to X or L ₃ , a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 125.

Embodiment 136.

R ² is A compound of formula A' or a compound of any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein * of R ² represents a point of attachment to X or L ₃ , a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 125.

Embodiment 137.

R ² is A compound of formula A' or a compound of any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein * of R ² represents a point of attachment to X or L ₃ , a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 125.

Embodiment 138.

R ² is A compound of formula A' or a compound of any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein * of R ² represents a point of attachment to X or L ₃ , a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 125.

Embodiment 139.

X ₁ is A compound of formula A' or a compound of any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 138.

Embodiment 140.

X ₁ is A compound of formula A' or a compound of any one of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a conjugate linker of formula C' or a conjugate linker of any one of embodiments 33 to 47, and an immunoconjugate of formula E' or an immunoconjugate of any one of embodiments 62 to 72, or an immunoconjugate of any one of embodiments 87 to 138.

Embodiment 141.

A compound of Formula A' or a compound of any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein each m is independently selected from 1, 2, 3, 4, and 5, a conjugate linker of Formula C' or a conjugate linker of any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or an immunoconjugate of any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 140.

Embodiment 142.

A compound of Formula A' or a compound of any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein each m is independently selected from 1, 2, and 3, a conjugate linker of Formula C' or a conjugate linker of any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or an immunoconjugate of any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 140.

Embodiment 143.

A compound of Formula A' or a compound of any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein each n is independently selected from 1, 2, 3, 4, and 5, a conjugate linker of Formula C' or a conjugate linker of any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 142.

Embodiment 144.

A compound of Formula A' or a compound of any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein each n is independently selected from 1, 2, and 3, a conjugate linker of Formula C' or a conjugate linker of any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or an immunoconjugate of any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 142.

Embodiment 145.

A compound of Formula A' or any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 144, wherein each t is independently selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30, or a pharmaceutically acceptable salt thereof, a linker of Formula C' or any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 144.

Embodiment 146.

A compound of Formula A' or a compound of any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, a conjugate linker of Formula C' or a conjugate linker of any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 144, wherein each t is independently selected from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25.

Embodiment 147.

A compound of Formula A' or a compound of any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt thereof, wherein each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18, a conjugate linker of Formula C' or a conjugate linker of any one of Embodiments 33 to 47, and an immunoconjugate of Formula E' or an immunoconjugate of any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 144.

Embodiment 148. An immunoconjugate of formula E', wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, or an immunoconjugate of any one of Embodiments 62 to 72, or any one of Embodiments 87 to 147.

Embodiment 149. An immunoconjugate of formula E', wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, or an immunoconjugate of any one of embodiments 62 to 72, or any one of embodiments 87 to 147.

Embodiment 150. An immunoconjugate of formula E', wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or an immunoconjugate of any one of embodiments 62 to 72, or any one of embodiments 87 to 147.

Embodiment 151. An immunoconjugate of formula E', wherein y is 1, 2, 3, 4, 5, 6, 7 or 8, or an immunoconjugate of any one of embodiments 62 to 72, or any one of embodiments 87 to 147.

Embodiment 152. An immunoconjugate of formula E', wherein y is 1, 2, 3, 4, 5 or 6, or an immunoconjugate of any one of embodiments 62 to 72, or any one of embodiments 87 to 147.

Embodiment 153. An immunoconjugate of formula E', wherein y is 1, 2, 3 or 4, or an immunoconjugate of any one of embodiments 62 to 72, or any one of embodiments 87 to 147.

Embodiment 154. An immunoconjugate of formula E', wherein y is 1 or 2, or an immunoconjugate of any one of embodiments 62 to 72, or any one of embodiments 87 to 147.

Embodiment 155. An immunoconjugate of formula E', wherein y is 2, or an immunoconjugate of any one of embodiments 62 to 72, or any one of embodiments 87 to 147.

Embodiment 156. An immunoconjugate of formula E', wherein y is 4, or an immunoconjugate of any one of embodiments 62 to 72, or any one of embodiments 87 to 147.

Embodiment 157. An immunoconjugate of formula E', wherein y is 6, or an immunoconjugate of any one of embodiments 62 to 72, or any one of embodiments 87 to 147.

Embodiment 158. An immunoconjugate of formula E', wherein y is 8, or an immunoconjugate of any one of embodiments 62 to 72, or any one of embodiments 87 to 147.

Embodiment 159. A compound of Formula A' or a compound of any one of Embodiments 1 to 31, or a pharmaceutically acceptable salt thereof, an immunoconjugate of Formula E' or an immunoconjugate of any one of Embodiments 62 to 72, or an immunoconjugate of any one of Embodiments 87 to 158, wherein when released from the immunoconjugate, D is a panRAS inhibitor.

Other conjugate linker groups

Other examples of conjugate linker groups suitable for preparing ADCs or immunoconjugates of the panRAS inhibitors disclosed herein include those disclosed in international application publications such as WO 2018200812, WO 2017214456, WO 2017214458, WO 2017214462, WO 2017214233, WO 2017214282, WO 2017214301, WO 2017214322, WO 2017214335, WO 2017214339, WO 2016094509, WO 2016094517, and WO 2016094505, the contents of each of which are incorporated by reference in their entirety.

For example, immunoconjugates of panRAS inhibitors disclosed herein may have a linker-payload (“-L-D”) structure selected from:

[Here,

Lc is a conjugate linker component, and each Lc is independently selected from a conjugate linker component as disclosed herein;

x is an integer chosen from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20;

y is an integer chosen from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20;

p is an integer chosen from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

D is a panRAS inhibitor disclosed herein;

Each cleavage element (C _E ) is independently selected from a self-immolative spacer and a cleavage sensitive group selected from acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase-induced cleavage, phosphodiesterase-induced cleavage, phosphatase-induced cleavage, protease-induced cleavage, lipase-induced cleavage, or disulfide bond cleavage.

In some embodiments, L has a structure selected from, or L comprises a structural component selected from:

.

In some embodiments, Lc is a conjugate linker component and each Lc is

are independently selected from.

In some embodiments, the conjugate linker L comprises a conjugate linker component selected from:

-**C(=O)O(CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m -; -**C(=O)O(CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m O(CH ₂ ) _m -;

-**C( ₌ O)O ₍ CH ₂ ) _m NR ¹¹ C(= _O )X _1a

-**C( ₌ O)OC(R ¹² ) ₂ (CH ₂ ) _m NR ¹¹ C(= _O ) _{X 1a}

_- **C(= _O )O(CH ₂ ) _m NR ¹¹ C ₍ = _O ) _{X 1a}

_- **C( _{= O} )O(CH ₂ ) _{m NR} ¹¹ C( ₌ O)X _1a

-**C(=O)O(CH ₂ ) _m NR ¹¹ C(=O)X ₄ C(=O)NR ¹¹ (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m O(CH ₂ ) _m -;

-**C(=O)O(CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)(CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m -;

-**C(=O)X ₄ C(=O)NR ¹¹ (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m O(CH ₂ ) _m -;

-**C( ₌ O)(CH ₂ ) _m NR ¹¹ C( _{= O} )X _1a

-** _{C ( = O} )O( _{CH 2} ) _m

-**C(=O)(CH ₂ ) _m NR ¹¹ C(=O)((CH ₂ ) _m O) _n (CH ₂ ) _m -

-** _C ( ₌ O ₎ O(CH ₂ ) _m -** _C ( _{= O} )O _{( CH 2} ) _m

-** _{C ( = O} )O( _{CH 2} ) _m

_{- **} C _{( = O} )O( _{CH 2 ) m}

_{- ** C} ( _{= O )} O( _{CH 2} ) _m

^- _{** C (} ⁼ _{O )} O _{( CH 2 ) m}

-**C(=O)X ₄ C(=O)X ₆ (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m O(CH ₂ ) _m -;

-** _{C ( = O} ) ₍ CH ₂ ) _m

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)(CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)(CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m -;

-**C(=O)O(( _{CH 2} ) _m O _{) n (} CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)(CH ₂ ) _m

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)((CH ₂ ) _m O) _n (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _{m NR} ¹¹ C(=O)X ₅ C(=O)((CH _{2 ) m} O _{) n} (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)) _{X 5} C(= _O )((CH ₂ ) _m O _{) n} (CH ₂ ) _m

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)(CH ₂ ) _m NR ¹¹ C(=O)((CH ₂ ) _m O) _n (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _{m NR} ¹¹ C(=O)X ₅ C(=O)(CH ₂ ) _m NR ¹¹ C(= _O )(( _{CH 2} ) _m O) _n (CH ₂ ) _m

-**C( ₌ O)O(( _{CH 2} ) _m O _{) n} (CH ₂ ) _m NR ¹¹ C(=O)X ₅ (CH ₂ ) _m

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ ((CH ₂ ) _m O) _n (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ ((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O) _{X 5} (( _{CH 2} ) _m O) _n (CH ₂ ) _{m NR} ¹¹ C(=O)(CH ₂ ) _m

-**C(=O)O((CH ₂ ) _m O _{) n (} CH ₂ ) _{m NR} ¹¹ C(=O)X ₅ ((CH ₂ ) _m O) _n (CH ₂ ) _m

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ (CH ₂ ) _m NR ¹¹ ((CH ₂ ) _m O) _n (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O) _{X 5} C(=O)(CH ₂ ) _m NR ¹¹ (( _{CH 2} ) _{m O} ) _n (CH ₂ ) _m

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)((CH ₂ ) _m O) _n (CH ₂ ) _m -;

-**C( ₌ O)O(( _{CH 2} ) _m O _{) n} (CH ₂ ) _m NR ¹¹ C(=O)X ₅ (CH ₂ ) _m -**C(=O)O(CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m -; -**C(=O)O(CH ₂ ) _m NR ¹¹ (CH ₂ ) _m -;

-**C(=O)O(CH ₂ ) _m NR ¹¹ (CH ₂ ) _m C(=O) _{X 2a}

-**C( ₌ O)O _{( CH 2} ) _m -** _C (=O)O( _{( CH 2} ) _m O) _n (CH ₂ ) _m

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m -;

-** _C ( ₌ O)O(CH ₂ ) _m NR ¹¹ C(= _O (CH ₂ ) _m

-**C(=O)O( _{( CH 2} ) _m O _{) n} (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m

_- **C(=O) _O ((CH ₂ ) _{m O} ) _n -** _C (=O)O( _{( CH 2} ) _m O) _n (CH ₂ ) _m

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m C(=O)NR ¹¹ (CH ₂ ) _m -; -**C(=O)O(CH ₂ ) _m C(R ¹² ) ₂ -;

-**C(=O)OCH ₂ ) _m C(R ¹² ) ₂ SS(CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m -, and

-**C(=O)O(CH ₂ ) _m C(=O)NR ¹¹ (CH ₂ ) _m -(wherein ** represents the point of attachment to the drug moiety (D) and the other terminal can be linked to R ¹⁰⁰ , i.e. a coupling group as described herein;

X _1a is

As, * indicates the attachment point to X _2a ;

X _2a is

As selected from, * represents an attachment point to X _1a ;

X ₃ is and;

X ₄ is -O(CH ₂ ) _n SSC(R ¹² ) ₂ (CH ₂ ) _n - or -(CH ₂ ) _n C(R ¹² ) ₂ SS(CH ₂ ) _n O-;

X ₅ is _ As, ** indicates orientation with respect to the drug moiety;

X ₆ is As, ** indicates orientation with respect to the drug moiety;

Each R ¹¹ is independently selected from H and C ₁ -C ₆ alkyl;

Each R ¹² is independently selected from H and C ₁ -C ₆ alkyl;

Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18).

Conjugation method

The present invention provides various methods for producing antibody drug conjugates comprising a conjugate linker having one or more hydrophilic moieties by conjugating a linker-drug group of the present invention to an antibody or antibody fragment.

A general reaction scheme for the formation of an antibody drug conjugate of formula E' is illustrated in Scheme 2 below:

Reaction Scheme 2

Here, RG ₂ is a reactive group that reacts with a compatible R ¹ group to form the corresponding R ¹⁰⁰ group (these groups are exemplified in Tables 8 and 9). D, R ¹ , L ₁ , Lp, L ₂ , L ₃ , R ² , A, G, Ab, y and R ¹⁰⁰ are as defined herein.

Scheme 3 further illustrates this general approach to the formation of antibody-drug conjugates of formula E', wherein the antibody comprises a reactive group (RG ₂ ) that reacts with an R ¹ group (as defined herein) to covalently attach a linker-drug group to the antibody via an R ¹⁰⁰ group (as defined herein). For illustrative purposes only, Scheme 3 shows an antibody having four RG ₂ groups.

Reaction Scheme 3

In one embodiment, the linker-drug group is conjugated to the antibody via a modified cysteine residue of the antibody (see, e.g., WO2014/124316). Scheme 4 illustrates this approach for the formation of an antibody-drug conjugate of formula E', wherein a free thiol group generated from an engineered cysteine residue of the antibody reacts with an R ¹ group (wherein R ¹ is maleimide) to covalently attach the linker-drug group to the antibody via an R ¹⁰⁰ group (wherein R ¹⁰⁰ is a succinimide ring). For illustrative purposes only, Scheme 4 shows an antibody having four free thiol groups.

Reaction Scheme 4

In another embodiment, the linker-drug group is conjugated to the antibody via a lysine residue of the antibody. Scheme 5 illustrates this approach for the formation of an antibody-drug conjugate of formula E', wherein a free amine group from a lysine residue of the antibody reacts with an R ¹ group (wherein R ¹ is an NHS ester, pentafluorophenyl, or tetrafluorophenyl) to covalently attach the linker-drug group to the antibody via an R ¹⁰⁰ group (wherein R ¹⁰⁰ is an amide). For illustrative purposes only, Scheme 5 shows an antibody having four amine groups.

Reaction Scheme 5

In another embodiment, the linker-drug group is conjugated to the antibody via the formation of an oxime crosslinker from the antibody's naturally occurring disulfide crosslinker. The oxime crosslinker is initially formed by reducing the antibody's interchain disulfide crosslinker to form a ketone crosslinker, which is then recrosslinked using a 1,3-dihaloacetone (e.g., 1,3-dichloroacetone). Subsequent reaction with a linker-drug group comprising a hydroxylamine forms an oxime bond (oxime crosslinker) that attaches the linker-drug group to the antibody (see, e.g., WO2014/083505). Scheme 6 exemplifies this approach for the formation of an antibody-drug conjugate of formula E'.

Reaction Scheme 6

A general reaction scheme for the formation of an antibody drug conjugate of formula F' is illustrated in Scheme 7 below:

Reaction Scheme 7

Here, RG ₂ is a reactive group that reacts with a compatible R ¹ group to form the corresponding R ¹⁰⁰ group (these groups are exemplified in Tables 8 and 9). D, R ¹ , L ₁ , Lp, Ab, y and R ¹⁰⁰ are as defined herein.

Scheme 8 further illustrates this general approach to the formation of antibody-drug conjugates of formula F', wherein the antibody comprises a reactive group (RG ₂ ) that reacts with an R ¹ group (as defined herein) to covalently attach a linker-drug group to the antibody via an R ¹⁰⁰ group (as defined herein). For illustrative purposes only, Scheme 8 shows an antibody having four RG ₂ groups.

Reaction Scheme 8

In one embodiment, the linker-drug group is conjugated to the antibody via a modified cysteine residue of the antibody (see, e.g., WO2014/124316). Scheme 9 illustrates this approach for the formation of an antibody-drug conjugate of formula F', wherein a free thiol group generated from an engineered cysteine residue of the antibody reacts with an R ¹ group (wherein R ¹ is maleimide) to covalently attach the linker-drug group to the antibody via an R ¹⁰⁰ group (wherein R ¹⁰⁰ is a succinimide ring). For illustrative purposes only, Scheme 9 shows an antibody having four free thiol groups.

Reaction Scheme 9

In another embodiment, the linker-drug group is conjugated to the antibody via a lysine residue of the antibody. Scheme 10 illustrates this approach for the formation of an antibody-drug conjugate of formula F', wherein a free amine group from a lysine residue of the antibody reacts with an R ¹ group (wherein R ¹ is an NHS ester, pentafluorophenyl, or tetrafluorophenyl) to covalently attach the linker-drug group to the antibody via an R ¹⁰⁰ group (wherein R ¹⁰⁰ is an amide). For illustrative purposes only, Scheme 10 shows an antibody having four amine groups.

Reaction Scheme 10

In another embodiment, the linker-drug group is conjugated to the antibody via the formation of an oxime crosslinker from the antibody's naturally occurring disulfide crosslinker. The oxime crosslinker is initially formed by reducing the antibody's interchain disulfide crosslinker to form a ketone crosslinker, which is then recrosslinked using a 1,3-dihaloacetone (e.g., 1,3-dichloroacetone). Subsequent reaction with a linker-drug group comprising a hydroxylamine forms an oxime bond (oxime crosslinker) that attaches the linker-drug group to the antibody (see, e.g., WO2014/083505). Scheme 11 exemplifies this approach for the formation of an antibody-drug conjugate of formula F'.

Reaction Scheme 11

Protocols for certain aspects of analytical methodologies for evaluating antibody conjugates of the present invention are also provided. Such analytical methodologies and results may demonstrate that the conjugate possesses advantageous properties, such as properties that make it easier to manufacture, easier to administer to a patient, more effective, and/or potentially safer for a patient. One example is the determination of molecular size by size exclusion chromatography (SEC), wherein the amount of a desired antibody species in a sample is determined by comparing the amount of high molecular weight contaminants (e.g., dimers, multimers, or aggregated antibodies) or low molecular weight contaminants (e.g., antibody fragments, degradation products, or individual antibody chains) present in the sample. Generally, it is desirable to have a greater amount of monomer and a lower amount of, for example, aggregated antibodies, due to the impact of, for example, aggregates on other properties of the antibody sample, such as, but not limited to, clearance rate, immunogenicity, and toxicity. A further example is the determination of hydrophobicity by hydrophobic interaction chromatography (HIC), where the hydrophobicity of a sample is assessed by comparison with a set of standard antibodies of known properties. Generally, lower hydrophobicity is desirable due to the influence of hydrophobicity on other properties of the antibody sample, such as, but not limited to, aggregation, aggregation over time, adhesion to surfaces, hepatotoxicity, clearance, and pharmacokinetic exposure. See Damle, N.K., Nat Biotechnol. 2008; 26(8):884-885; Singh, S.K., Pharm Res. 2015; 32(11):3541-71. When measured by hydrophobic interaction chromatography, a higher hydrophobicity index score (i.e., faster elution from the HIC column) reflects a lower hydrophobicity of the conjugate. As shown in the examples below, most of the antibody conjugates tested exhibited hydrophobicity indices greater than 0.8. In some embodiments, an antibody conjugate is provided having a hydrophobicity index of greater than or equal to 0.8 as measured by hydrophobic interaction chromatography.

Example

The following examples provide illustrative embodiments of the present invention. Those skilled in the art will recognize numerous modifications and variations that can be made without altering the spirit or scope of the present invention. Such modifications and variations are within the scope of the present invention. The provided examples do not limit the present invention in any way.

Example 1. Synthesis and characterization of the PanRAS payload.

Exemplary payloads were synthesized using the exemplary methods described in WO2021/091956 or WO2022/060836, which are incorporated herein by reference in their entirety. All reagents were obtained from commercial sources and used without further purification. Anhydrous solvents were obtained from commercial sources and used without further drying.

Example 2. Synthesis and characterization of conjugate linkers

Exemplary conjugate linkers and precursors thereof were synthesized using the exemplary methods described in PCT Application No. PCT/US2021/060620, which is incorporated herein by reference in its entirety.

Example 3. Synthesis and characterization of linkers, linker-payloads, and precursors thereof.

Exemplary linkers, linker-payloads, and precursors thereof were synthesized using the exemplary methods described in this Example.

Materials, methods, and general procedures:

All reagents were obtained from commercial sources and used without further purification. Anhydrous solvents were obtained from commercial suppliers and used without further drying. Flash chromatography was performed on a CombiFlash Rf (Teledyne ISCO) using prepacked silica gel cartridges (Macherey-Nagel Chromabond Flash). Thin-layer chromatography was performed using 5 × 10 cm plates coated with Merck Type 60 F254 silica gel. Microwave heating was performed on a CEM Discover® instrument.

NMR data were collected at 298 K on a Bruker Avance NMR spectrometer equipped with a 5 mm BBFO CryoProbe with a z-gradient operating at frequencies of 400.13 MHz for ¹ H, 376.50 MHz for ¹⁹ F, and 100.61 MHz for ¹³ C. Chemical shifts for ¹ H and ¹³ C spectra were referenced to internal tetramethylsilane (TMS) set to 0 ppm.

Analysis method

LC/MS data were acquired using an instrument with the following parameters:

The method used to generate LC/MS data was as follows:

2-minute acid method :

2-minute alkaline method:

5-minute acid method

HRMS data were acquired using an instrument with the following parameters:

HRMS_QT01

HRMS_QT02

The methods used to generate HRMS data for linkers/payloads and synthetic intermediates were as follows:

Preparative RP-HPLC:

Preparative HPLC (“Prep-HPLC”) data were acquired using a Teledyne ISCO purification system using a C18 or C4 RP ISCO or ISCO-gold column.

The following four Prep-HPLC methods were used:

a. TFA method: Solvent: A: water + 0.05% TFA, B: acetonitrile + 0.05% TFA, gradient: 5–100% B in 15–30 CV

b. NH ₄ HCO ₃ Method: Solvent: A: Water + 0.02 M NH ₄ HCO ₃ , B: Acetonitrile/water (80/20) + 0.02 M NH ₄ HCO ₃ , Gradient: 5–100% B in 15–30 CV

c. Neutral method: Solvent: A: water, B: acetonitrile, 5–100% B in 15–30 CV

d. Formic acid method: Solvent: A: water + 0.05% formic acid, B: acetonitrile + 0.05% formic acid, gradient: 5–100% B in 15–30 CV

The gradient variation of method a.~d. was appropriately used. All fractions containing the pure compound were combined and directly freeze-dried to obtain the compound as an amorphous powder.

1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -Ethyl-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-2 ⁵ -((triisopropylsilyl)oxy)-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -Hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-1 ² Synthesis of -1)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate

(2R,3R)-N-((2S)-1-((6 ³ S,4S)-1 ¹ -ethyl-12-(2-((S)-1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo- ^{2 5} -((triisopropylsilyl)oxy)-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro- ^{1 1} To a mixture of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapan-4-yl)amino)-3-methyl-1-oxobutan-2-yl)-N-methyl-3-phenyltetrahydrofuran-2-carboxamide formate (200 mg, 154.4 μmol), tert-butyl ((S)-1-(((S)-1-((4-(chloromethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (100 mg, 201 μmol) and sodium iodide (23.1 mg, 154 μmol) was added DIEA (81 μL, 463 μmol). After stirring for 6 h, the solution was diluted with DMSO (3 mL) and purified directly by ISCO Gold RP-HPLC. After lyophilization, 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -ethyl-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-2 ⁵ -((triisopropylsilyl)oxy)-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro- ^{1 1} H-8-Oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapan-1 ² -yl)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate (217 mg, 79% yield) was obtained. HRMS: M+=1642.9900, Rt=3.11 min, 5 min acidic method.

1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -Ethyl-2 ⁵ -Hydroxy-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -Hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-1 ² Synthesis of -1)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate

1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -ethyl-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-2 ⁵ -((triisopropylsilyl)oxy)-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro- ^{1 1} To a solution of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapan-1 ² -yl)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate (217 mg, 123.4 μmol) was added 1.0 M TBAF in THF (248 μL, 248 μmol). After stirring for 30 min, the solution was diluted with DMSO (4 mL) and purified directly by ISCO Gold RP-HPLC. After lyophilization, 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -ethyl- ^{2 5} -hydroxy-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo- ^{6 1} ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-1 ² -yl)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate (198 mg, 90% yield) was obtained. HRMS: M+=1486.8500, Rt=2.25 min, 5 min acidic method. The material contained a small amount of Bu4N+ salt impurity, which was passed to the next step.

1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -Ethyl-2 ⁵ -Hydroxy-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -Hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-1 ² Synthesis of -1)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate (L102-D101)

1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -ethyl- ^{2 5} -hydroxy-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro- ^{1 1} H-8-Oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapan-1 ² -yl)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate (175 mg, 109 μmol) was treated with 8 mL of 25% TFA/CH2Cl2 containing 0.1% triethylsilane for 1 h, during which time the volatiles were removed in vacuo. The residue was diluted with diethyl ether. The residue was dissolved in NMP (2 mL), and DIEA (133 μL, 765 μmol) and 2,5-dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (51 mg, 164 μmol) were added. After stirring for 2 h, the solution was diluted with DMSO and purified by ISCO RP-HPLC. After lyophilization, the material was further purified by LH-20 Sephadex size exclusion chromatography (1:1 CH2Cl2/MeOH eluent) to remove the Bu4N+ salt. The volatiles were removed in vacuo, dissolved in MeCN/H2O, and lyophilized. 1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -ethyl- ^{2 5} -hydroxy-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo- ^{6 1} ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro- ^{1 1} H-8-Oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapan-1 ² -yl)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate (123 mg, 66% yield) was obtained. HRMS: M+=1581.8400, Rt=2.11 min, 5 min acidic method. ^1H NMR (400 MHz, DMSO) δ 10.21 (s, 1H), 9.34 - 9.18 (m, 0H), 9.05 (s, 1H), 8.51 (d, J = 2.6 Hz, 1H), 8.39 (d, J = 7.0 Hz, 0H), 8.15 (d, J = 7.2 Hz, 1H), 7.95 - 7.80 (m, 2H), 7.76 (d, J = 8.2 Hz, 3H), 7.65 - 7.47 (m, 4H), 7.47 - 7.34 (m, 1H), 7.34 - 7.10 (m, 5H), 7.01 (s, 3H), 6.91 - 6.63 (m, 1H), 6.58 (s, 1H), 5.99 (t, J = 5.9 Hz, 1H), 5.51 - 5.27 (m, 3H), 5.19 (dd, J = 15.7, 9.4 Hz, 1H), 5.09 (d, J = 7.7 Hz, 1H), 4.65 (s, 2H), 4.38 (q, J = 7.3 Hz, 1H), 4.34 - 3.99 (m, 7H), 3.84 (dtd, J = 31.9, 15.7, 7.5 Hz, 5H), 3.71 - 3.40 (m, 15H), 3.33 (s, 39H), 3.11 (s, 3H), 3.09 - 2.88 (m, 6H), 2.88 - 2.59 (m, 8H), 2.47 - 2.16 (m, 6H), 1.94 (dq, J = 10.7, 5.5 Hz, 2H), 1.68 (tdd, J = 31.6, 16.6, 7.5 Hz, 5H), 1.54 - 1.42 (m, 2H), 1.37 (dd, J = 10.2, 6.0 Hz, 4H), 1.09 - 0.92 (m, 4H), 0.92 - 0.78 (m, 9H), 0.73 (d, J = 6.7 Hz, 1H), 0.68 (d, J = 6.5 Hz, 3H), 0.50 (s, 3H), -0.06 (d, J = 6.5 Hz, 2H).

1-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -Ethyl-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-2 ⁵ -((triisopropylsilyl)oxy)-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -Hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-1 ² Synthesis of -1)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate

(2R,3R)-N-((2S)-1-((6 ³ S,4S)-1 ¹ -ethyl-12-(2-((S)-1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo- ^{2 5} -((triisopropylsilyl)oxy)-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro- ^{1 1} To a mixture of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapan-4-yl)amino)-3-methyl-1-oxobutan-2-yl)-N-methyl-3-phenyltetrahydrofuran-2-carboxamide formate (250 mg, 0.193 mmol), (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(methyl)carbamate (177 mg, 0.232 mmol) and sodium iodide (28.9 mg, 0.193 mmol) was added DIEA (67 μL, 0.386 mmol). was added. After stirring for 5 hours, the solution was diluted with DMSO (3 mL) and purified directly by ISCO Gold RP-HPLC . After lyophilization, 1-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -ethyl-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-2 ⁵ -((triisopropylsilyl)oxy)-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro- ^{1 1} H-8-Oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapan-1 ² -yl)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate was obtained (379 mg, 96% yield). HRMS: M+=1908.1000, Rt=3.38 min, 5 min acidic method.

1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -Ethyl-2 ⁵ -Hydroxy-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -Hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-1 ² Synthesis of -1)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate

1-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -ethyl-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-2 ⁵ -((triisopropylsilyl)oxy)-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-1 ² -yl)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate To a solution of (379 mg, 194 μmol) was added 1.0 TBAF (210 μL, 210 μmol) in THF. After stirring for 45 min, piperidine (230 μL, 2.33 mmol) was added. After stirring for 30 min, the solution was diluted with DMSO (9 ml) and purified directly by ISCO Gold RP-HPLC. After lyophilization, the material was further purified by LH-20 Sephadex size exclusion chromatography (1:1 CH2Cl2/MeOH eluent) to remove Bu4N+ salt. After removing the volatiles in vacuo, dissolving in MeCN/H2O and lyophilizing, 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -ethyl- ^{2 5} -hydroxy-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro- ^{1 1} H-8-Oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapan-1 ² -yl)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate (235 mg, 68% yield) was obtained. HRMS: M+=1529.9000, Rt=2.02 min, 5 min acidic method.

1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(78-carboxy-2-methyl-3-oxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4-diazaoctaheptacontyl)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -Ethyl-2 ⁵ -Hydroxy-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -Hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-1 ² Synthesis of -1)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate

To a solution of DIEA (105 μL, 601 μmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontane-75-oate (222 mg, 194 μmol) in NMP (1.5 mL) was added bis(4-nitrophenyl) carbonate (54.9 mg, 180 μmol). After stirring for 2 h, the activated amine solution was added to a solution of DIEA (140 μL, 802 μmol) and 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -ethyl- ^{2 5} -hydroxy-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro- ^{1 1} H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-1 ² -yl)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate (235 mg, 133.6 μmol) was added to the solution. The resulting solution was stirred for 30 minutes, diluted with DMSO, and purified by ISCO RP-HPLC. When lyophilized, 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(78-carboxy-2-methyl-3-oxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4-diazaoctaheptacontyl)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -ethyl- ^{2 5} -Hydroxy-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapan-1 ² -yl)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate (291 mg, 77% yield) was obtained. HRMS: M+=2701.5600, Rt=2.30 min (5 min acid method).

1-(2-(78-Carboxy-2-methyl-3-oxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4-diazaoctaheptacontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -Ethyl-2 ⁵ -Hydroxy-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -Hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-1 ² Synthesis of -1)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate (L101-D101)

1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(78-carboxy-2-methyl-3-oxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4-diazaoctaheptacontyl)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -ethyl- ^{2 5} -Hydroxy-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro-1 ¹ H -8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-1 ² -yl)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate (356 mg, 126.4 μmol) was dissolved in 25% TFA/CH2Cl2 containing 0.1% triethylsilane The mixture was treated with 15 mL for 1 h, during which time the volatiles were removed in vacuo. The residue was diluted with diethyl ether. The residue was dissolved in NMP (4 mL), and DIEA (352 μL, 2022 μmol) and 2,5-dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (55 mg, 177 μmol) were added. After stirring for 2 h, the solution was diluted with DMSO and purified by ISCO RP-HPLC. When lyophilized, 1-(2-(78-carboxy-2-methyl-3-oxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4-diazaoctaheptacontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(5-((6 ³ S,4S)-1 ¹ -ethyl-2 ⁵ -Hydroxy-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-1 ² -yl)-6-((S)-1-methoxyethyl)pyridin-3-yl)-1-methylpiperazin-1-ium trifluoroacetate (300 mg, 81% yield) was obtained. HRMS: M+= 2796.5701, Rt=2.20 min (5 min acidic method). ^1H NMR (400 MHz, DMSO) δ 12.15 (s, 1H), 10.25 (s, 1H), 9.21 (s, 0H), 9.04 (s, 1H), 8.50 (d, J = 2.6 Hz, 1H), 8.38 (d, J = 6.9 Hz, 0H), 8.13 (d, J = 7.1 Hz, 1H), 7.96 - 7.66 (m, 3H), 7.64 - 7.50 (m, 1H), 7.50 - 7.32 (m, 2H), 7.32 - 7.10 (m, 4H), 7.00 (s, 2H), 6.92 - 6.61 (m, 1H), 6.61 - 6.44 (m, 1H), 5.99 (t, J = 5.9 Hz, 1H), 5.57 - 5.14 (m, 3H), 5.09 (d, J = 7.7 Hz, 1H), 4.73 (s, 1H), 4.60 (s, 1H), 4.46 - 4.32 (m, 1H), 4.32 - 4.00 (m, 5H), 3.84 (dtd, J = 32.1, 15.8, 7.5 Hz, 4H), 3.71 - 3.63 (m, 2H), 3.59 (q, J = 6.4 Hz, 4H), 3.55 - 3.45 (m, 78H), 3.41 (d, J = 6.5 Hz, 21H), 3.22 (q, J = 6.1 Hz, 2H), 3.17 - 2.99 (m, 6H), 2.93 (q, J = 6.1 Hz, 1H), 2.76 (s, 4H), 2.66 (s, 3H), 2.43 (t, J = 6.3 Hz, 3H), 2.38 - 2.19 (m, 3H), 1.94 (dq, J = 13.6, 7.2 Hz, 2H), 1.84 - 1.43 (m, 5H), 1.36 (dd, J = 10.1, 6.1 Hz, 4H), 1.05 - 0.89 (m, 3H), 0.89 - 0.77 (m, 6H), 0.77 - 0.59 (m, 3H), 0.50 (s, 2H), -0.06 (d, J = 6.5 Hz, 2H).

(6 ³ S,4S)-1 ¹ -Ethyl-1 ² -(2-((S)-1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -Hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-2 ⁵ - Synthesis of (4-nitrophenyl) carbonate trifluoroacetate

(2R,3R)-N-((2S)-1-((6 ³ S,4S)-1 ¹ -ethyl-12-(2-((S)-1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-2 ⁵ -((triisopropylsilyl)oxy)-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro-1 ¹ H-8-Oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapan-4-yl)amino)-3-methyl-1-oxobutan-2-yl)-N-methyl-3-phenyltetrahydrofuran-2-carboxamide formate (37 mg, 35 μmol) was treated with DMF (1.0 mL), followed by DIPEA (12.0 μL, 69 μmol) and finally bis(4-nitrophenyl) carbonate (21 mg, 69 μmol). The reaction was stirred for a total of 2 h, at which point it was diluted with a total of 2 mL of DMSO and loaded onto a 50 g C18 column. The material was purified through a gradient of CH ₃ CN and water (containing 0.1% TFA modifier) (20% to 80% CH ₃ CN). The desired fractions were combined, frozen and lyophilized to afford (6 ³ S,4S)-1 ¹ -ethyl-1 ² -(2-((S)-1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo- ^{6 1} ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-2 ⁵ -yl (4-nitrophenyl) carbonate Trifluoroacetate (39 mg, 85% yield) was produced as a yellow powder. HRMS: M+= 1212.5699, Rt=2.43 min (5 min acidic method).

4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl ((6 ³ S,4S)-1 ¹ -Ethyl-1 ² -(2-((S)-1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -Hexahydro-11H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-2 ⁵ -Synthesis of ethane-1,2-diylbis(methylcarbamate)trifluoroacetate

(6 ³ S,4S)-1 ¹ -ethyl-1 ² -(2-((S)-1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane- ^{2 5} -yl (4-nitrophenyl) carbonate in a 4 mL vial exposed to air at 23°C Trifluoroacetate (39 mg, 30 μmol) was treated with 2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl methyl(2-(methylamino)ethyl)carbamate trifluoroacetate (34 mg, 35 μmol), followed by DMF (0.60 mL). Next, DIPEA (31 μL, 180 μmol) was added. The reaction was stirred at 23 °C for 1 h. To this mixture, dimethylamine (150 μL, 300 μmol) in THF was added. The reaction was stirred at 23°C for 1.75 h, at which point it was diluted with 2 mL of DMSO and loaded onto a 50 g C18 column. The material was purified through a gradient of CH ₃ CN and water (15% to 60% CH ₃ CN, 0.1% TFA). The desired fractions were combined, frozen and lyophilized to afford 4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl ((6 ³ S,4S)-1 ¹ -ethyl- ^{1 2} -(2-((S)-1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -Hexahydro-11H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-2 ⁵ -yl) ethane-1,2-diylbis(methylcarbamate) trifluoroacetate (37 mg, 70% yield) was produced as a yellow powder. HRMS: M+= 1687.9700, Rt=1.95 min (5 min acidic method).

1-(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((((2-((((6 ³ S,4S)-1 ¹ -Ethyl-1 ² -(2-((S)-1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -Hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-2 ⁵ - Synthesis of (methyl)amino)methyl)phenyl)-2-methyl-3-oxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4-diazanonaheptacontane-79-oic acid trifluoroacetate

1-Amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-Tetracosaoxapentaheptacontan-75-oic acid (35 mg, 30 μmol) in a 4 mL vial exposed to air at 23°C was treated with DMF (0.50 mL). The mixture was stirred for 1–2 minutes until all material was dissolved. Next, DIPEA (22 μL, 120 μmol) was added, followed by bis(4-nitrophenyl) carbonate (8.5 mg, 28 μmol). The reaction was stirred at 23°C for 30 min, at which point additional DIPEA (11 μL, 62 μmol) was added, and the reaction was transferred to a 4 mL vial exposed to air at 23°C to give 4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl((6 ³ S,4S)-1 ¹ -ethyl-1 ² -(2-((S)-1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo- ^{6 1} ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -hexahydro-11H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapan- ^{2 5} -yl) ethane-1,2-diylbis(methylcarbamate) trifluoroacetate (37 mg, 21 μmol) was added. The reaction was stirred at 23°C for 45 min, at which point it was diluted with DMSO to a total volume of 3 mL and loaded onto a 50 g C18 column. The material was purified through a gradient of CH ₃ CN and water (15% to 80% CH ₃ CN, 0.1% TFA). The desired fractions were combined, frozen and lyophilized to give 1-(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((((2-((((6 ³ S,4S)-1 ¹ -ethyl-1 ² -(2-((S)-1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo- ^{6 1} ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -Hexahydro- ^{1 1} H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-2 ⁵ -yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)methyl)phenyl)-2-methyl-3-oxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4-diazanonaheptacontane-79-oic acid trifluoroacetate (22 mg, 35% yield) was produced as a yellow powder. HRMS: M+2H+= 1430.8101, Rt=2.23 min (5 min acidic method). ^1H NMR (400 MHz, DMSO) δ 12.13 (s, 1H), 10.11 (s, 1H), 9.63 (s, 1H), 8.51 (d, J = 2.9 Hz, 1H), 7.96 (d, J = 7.8 Hz, 2H), 7.83 - 6.88 (m, 14H), 6.88 - 6.58 (m, 3H), 6.43 (s, 1H), 5.96 (s, 1H), 5.66 - 4.84 (m, 7H), 4.47 (d, J = 24.7 Hz, 3H), 4.33 - 4.08 (m, 5H), 4.01 (t, J = 9.8 Hz, 3H), 3.90 (s, 1H), 3.81 (q, J = 8.9 Hz, 2H), 3.71 (d, J = 6.7 Hz, 1H), 3.65 (d, J = 13.0 Hz, 2H), 3.59 (t, J = 6.4 Hz, 3H), 3.52 - 3.48 (m, 85H), 3.21 - 3.12 (m, 5H), 3.06 (d, J = 4.6 Hz, 9H), 2.96 - 2.80 (m, 10H), 2.80 - 2.64 (m, 8H), 2.58 (d, J = 6.2 Hz, 3H), 2.43 - 2.31 (m, 9H), 1.95 (d, J = 15.7 Hz, 3H), 1.79 (s, 1H), 1.60 (d, J = 44.3 Hz, 5H), 1.41 - 1.29 (m, 14H), 0.99 (d, J = 6.8 Hz, 4H), 0.88 - 0.46 (m, 16H), -0.07 (dd, J = 6.6, 3.0 Hz, 3H).

1-(5-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((((2-((((6 ³ S,4S)-1 ¹ -Ethyl-1 ² -(2-((S)-1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -Hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-2 ⁵ - Synthesis of (methyl)amino)methyl)phenyl)-2-methyl-3-oxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4-diazanonaheptacontane-79-oic acid trifluoroacetate (L103-D101)

1-(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((((2-((((6 ³ S,4S)-1 ¹ -ethyl-1 ² -(2-((S)-1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo- ^{6 1} ,6 ² ,6 ³ ,6 ^{4 ,6 5 ,} 6 ⁶ -Hexahydro- ^{1 1} H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane-2 ⁵ -yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)methyl)phenyl)-2-methyl-3-oxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4-diazanonaheptacontane-79-oic acid trifluoroacetate (22 mg, 7.4 μmol) was treated with 25% TFA/DCM, 0.1% Et ₃ SiH (1.0 mL). The reaction was stirred at 23°C for 1 h, at which point it was transferred to a separate 4 mL vial, washed with 2 mL DCM, and the mixture concentrated under reduced pressure (room temperature water bath). The resulting oil was diluted twice with 3 mL _Et2O , and the _Et2O was decanted. The vial was placed under reduced pressure, which gave a yellow solid. The solid was dissolved in NMP (0.50 mL), and DIPEA (19 μL, 110 μmol) was added. Next, 2,5-dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (3.2 mg, 10 μmol) was added in one portion, and the reaction was stirred at 23 °C for 2 h, at which point it was diluted with DMSO (2.5 mL) and loaded onto a 50 g C18 column. The material was purified through a gradient of CH ₃ CN and water (20% to 80% CH ₃ CN, 0.1% TFA). The desired fractions were combined, frozen and lyophilized to give 1-(5-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((((2-((((6 ³ S,4S)-1 ¹ -ethyl- ^{1 2} -(2-((S)-1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-4-((S)-3-methyl-2-((2R,3R)-N-methyl-3-phenyltetrahydrofuran-2-carboxamido)butanamido)-5,7-dioxo-6 ¹ ,6 ² ,6 ³ ,6 ⁴ ,6 ⁵ ,6 ⁶ -Hexahydro-1 ¹ H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecapane- ^{2 5} -yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)methyl)phenyl)-2-methyl-3-oxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4-diazanonaheptacontane-79-oic acid trifluoroacetate (10 mg, 45% yield) as a yellowish-white solid Generated. HRMS: M+2H+= 1478.3101, Rt=2.12 min (5 min acidic method). ^1H NMR (400 MHz, DMSO) δ 12.10 (s, 0H), 10.03 (s, 1H), 9.66 (s, 1H), 8.51 (d, J = 3.0 Hz, 1H), 8.16 - 7.94 (m, 2H), 7.83 (d, J = 8.6 Hz, 1H), 7.79 - 7.33 (m, 7H), 7.33 - 7.09 (m, 7H), 7.00 (s, 2H), 6.86 - 6.66 (m, 2H), 6.43 (s, 1H), 5.99 (s, 1H), 5.72 - 4.88 (m, 6H), 4.70 - 3.98 (m, 12H), 3.96 - 3.57 (m, 19H), 3.50 (s, 76H), 3.27 - 2.64 (m, 36H), 2.58 (d, J = 6.4 Hz, 5H), 2.43 (q, J = 5.6 Hz, 6H), 2.34 - 2.18 (m, 4H), 2.09 - 1.86 (m, 3H), 1.81 (s, 1H), 1.75 - 1.49 (m, 5H), 1.37 (dd, J = 9.9, 6.1 Hz, 6H), 0.99 (d, J = 6.9 Hz, 4H), 0.92 - 0.51 (m, 16H), -0.07 (dd, J = 6.6, 3.0 Hz,

Example 4. Conjugation and characterization of ADCs

In vitro manufacturing of Cysmab ADC

Antibody (typically 5–10 mg) was mixed with rProtein A Sepharose resin (GE) in an appropriately sized disposable column and incubated for 15 minutes at a ratio of 10 mg Ab to 1 ml resin in PBS. Cysteine HCl was added to a final concentration of 20 mM and incubated at room temperature for 30 minutes with stirring to allow deblocking of reactive cysteines. The resin was rapidly washed (multiple additions) with 50 column volumes of PBS on a vacuum manifold. The resin was then resuspended in an equal volume of PBS containing 250 nM CuCl ₂ . Time points were taken to monitor the reformation of antibody interchain disulfides. At each time point, 25 μL of resin slurry was removed, 1 μL of 20 mM MC-valcit-MMAE was added, and the tube was flicked several times. The resin was spun down, the supernatant was removed, and eluted with 50 μL of antibody elution buffer (Thermo). The resin was pelleted, and the supernatant was analyzed by reversed-phase chromatography using an Agilent PLRP-S 4000A 5 μm, 4.6x50 mm column (Buffer A is water, 0.1% TFA; Buffer B is acetonitrile, 0.1% TFA; the column was maintained at 80°C; flow rate: 1.5 ml/min; gradient: 0 min - 30% B, 5 min - 45% B, 6.5 min - 100% B, 8 min - 100% B, 10 min - 30%).

Once the antibody was confirmed to have reformed the interchain disulfide bonds, the resin was washed with 10 column volumes of PBS, resuspended in an equal volume of PBS, and 12 equivalents of the appropriate linker-payload (20 mM) in DMSO were added, followed by incubation at room temperature for 2 hours. The resin was then washed with 50 column volumes of PBS to remove excess linker-payload. The ADC was eluted from the protein A resin with antibody elution buffer. The ADC was then dialyzed into PBS. The material was then concentrated to 4.5 mg/ml using a centrifugal concentrator using Amicon Ultra-15, 50 KDa, regenerated cellulose (Millipore, UFC0905024), sterile filtered through a 0.22 μm sterile PVDF filter, 25 mm (Millapore, SLGV013SL), and stored at 4°C. The following analyses were performed: analytical SEC to determine % monomer, reducing mass spectrometry to determine DAR, LAL test to determine endotoxin load, and protein concentration was determined by A280 utilizing the extinction coefficient and molecular weight of the antibody. All in vitro material was >90% monomeric (% aggregation determined by comparing the high molecular weight peak absorbance areas at 210 and 280 nm with the peak absorbance areas of monomeric ADC). HRMS data (protein method) showed a predominant mass of the heavy chain + 2 species, and compared the MS intensities of the peaks for the DAR1, DAR2, and DAR3 species to give a DAR of approximately 4.0.

General Methodology : The drug-to-antibody ratio (DAR) of exemplary ADCs was determined by liquid chromatography-mass spectrometry (LC/MS) according to the following method. For all LC methods, mobile phase A was purified MS-grade water (Honeywell, LC015-1), and mobile phase B was MS-grade 80% isopropanol (Honeywell LC323-1):20% acetonitrile (Honeywell, LC015-1), LC323-1) supplemented with 1% formic acid (FA) (Thermo Scientific, 85178). The column temperature was set to 80°C. The general MS method was optimized for all synthesized ADCs. The column used for analysis was an Agilent PLRP-S 4000 A; 2.1x150 mm, 8 μm (Agilent, PL1912-3803 ). The flow rate used was 0.3 ml/min. The gradient used was as follows: 0–0.75 min 95% A, 0.76–1.9 min 75% A, 1.91–11.0 min 50% A, 11.01–11.50 min 10% A, 11.51–13.50 min 95% A, 13.51–18 min 95% A on an Acuity Bio H-Class Quaternary UPLC (Waters). The MS system was a Xevo G2-XS QToF ESI mass spectrometer (Waters), and data were acquired from 1.5 to 11 min, with masses analyzed between 15,000 and 80,000 daltons. DAR was determined from deconvoluted spectra or UV chromatograms by summing the integrated MS (total ion current) or UV (280 nm) peak areas of the unconjugated and conjugated species (mAb or related fragments), and weighting each area by the number of drugs attached. The summed weighted area was divided by the sum of the total areas, resulting in a final average DAR value for the entire ADC.

Size exclusion chromatography (SEC) : The quality and percent aggregate (%) of the purified ADC were determined by SEC. The analysis was performed on an analytical column Superdex 200 Increase 5/150 GL (GE Healthcare, 28990945) under isocratic conditions of 100% PBS pH 7.2 ((Hyclone SH30028.03)) at a flow rate of 0.45 ml/min for 8 minutes. The percent aggregate fraction of the ADC samples was quantified based on the peak area absorbance at 280 nm. The calculation was based on the ratio of the high molecular weight eluate at 280 nm divided by the sum of the peak area absorbances of the high molecular weight and monomer eluates at that wavelength, multiplied by 100%. Data were acquired on an Agilent Bio-Inert 1260 HPLC (Wyatt Technologies, Santa Barbara, CA, USA) equipped with a Wyatt miniDAWN light scattering and Treos refractive index detector.

Example 5. In vitro evaluation of panRAS antibody-drug conjugates in various cancer cell lines.

PanRAS antibody-drug conjugates were tested against the following cancer cell lines.

LU65 : JCRB number JCRB0079 (cultured in RPMI-1640 + 10% FBS)

HPAC : ATCC No. CRL-2119 (cultured in DMEM:HAM's F12 + 5% FBS)

NCI-H727 : ATCC No. CRL-5815 (cultured in RPMI-1640 + 10% FBS)

SW1271 : ATCC No. CRL-2177 (cultured in DMEM + 10% FBS)

Inhibition of cell proliferation and survival

The ability of panRAS antibody-drug conjugates to inhibit cell proliferation and survival was assessed using the Promega CellTiter-Glo ^® proliferation assay.

Cell lines were cultured in optimal growth medium at 37°C in a tissue culture incubator _with 5% CO2. Prior to seeding for proliferation assays, cells were split at least 2 days prior to analysis to ensure optimal growth density. On the day of seeding, cell viability and cell density were determined using a cell counter (Vi-Cell XR Cell Viability Analyzer, Beckman Coulter). Cells with greater than 85% viability were seeded into white, clear-bottom, 384-well TC-treated plates (Corning catalog no. 3765). Cells were seeded at a density of 1,000 cells per well in 45 μL of standard growth medium. Plates were incubated overnight at 37°C in a tissue culture incubator _with 5% CO2. The following day, the indicated compounds and ADCs were prepared at 10X in standard growth medium. The prepared compounds and ADCs were then added to the cells to yield a final volume of 50 μL per well and a final concentration of 0.005–100 nM. Each drug concentration was tested in quadruplicate. The plates were incubated in a tissue culture incubator at 5% CO ₂ and 37°C for 5 days, after which 25 μL of CellTiter Glo® (Promega, catalog no. G7573), a reagent that lyses cells and measures total adenosine triphosphate (ATP) content, was added to assess cell viability. The plates were incubated at room temperature for 10 minutes to stabilize the luminescence signal before reading using a luminescence reader (EnVision Multilabel Plate Reader, PerkinElmer). To assess the effectiveness of the drug treatments, the luminescence counts of wells containing untreated cells (100% viability) were used to normalize the treated samples. A nonlinear regression curve was fitted to the data using GraphPad PRISM version 7.02 software using a variable slope model. IC50 and Amax values were extrapolated from the generated curve.

Dose-response curves for representative cancer cell lines are illustrated in Figure 1. The concentration of treatment required to inhibit cell growth or survival by 50% (IC50) was calculated using representative IC50 values for the tested cell lines summarized in Table I. The panRAS ADC tested in the representative cell lines (in the table below, “panRAS ADC” is Ab-L101-D101) demonstrated in vitro efficacy compared to an isotype-matched non-targeting control ADC.

These studies demonstrate that panRAS ADCs can inhibit cell proliferation in a variety of cancer cell lines expressing the antigen of interest. No cytotoxic activity was observed in the tested cancer cell lines compared to the isotype-matched non-targeting control.

Table I. PanRAS ADC cytotoxicity

Attach an electronic file of the sequence list

Claims (115)

Antibody-drug conjugate of chemical formula 1:
[Chemical Formula 1]

(wherein Ab is an antibody or an antigen-binding fragment thereof;
L is a conjugate linker that covalently attaches Ab to D;
p is an integer from 1 to 16;
D is a panRAS inhibitor). An antibody-drug conjugate in claim 1, wherein p is an integer from 1 to 6 or from 2 to 4, or p is 2 or 4; or p is determined by liquid chromatography-mass spectrometry (LC-MS). In claim 1 or 2, L is an antibody-drug conjugate comprising:
Attachment;
one or more crosslinking spacer groups; and
One or more cleavable groups, optionally one or more cleavable groups comprising a pyrophosphate group and/or a self-immolative group. In the third paragraph, -(LD) is an antibody-drug conjugate having the chemical formula A:
[Chemical Formula A]

(Here,
R ¹ is an attachment group;
L ₁ is a crosslinking spacer group;
E is a cuttable device). In claim 3 or 4, the cleavable group comprises a pyrophosphate group or the cleavable group comprises a pyrophosphate group. An antibody-drug conjugate comprising: In claim 3 or 4, the crosslinking spacer group comprises an antibody-drug conjugate:
(i) polyoxyethylene (PEG) group;
(ii) a PEG group selected from PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, and PEG15;
(iii) -CO-CH ₂ -CH ₂ -PEG12- group;
(iv) a butanoyl, pentanoyl, hexanoyl, heptanoyl, or octanoyl group; or
(v) Hexanoyl group. In the sixth paragraph, (i) the attachment group is formed from one or more reactive groups selected from a maleimide group, a thiol group, a cyclooctyne group, and an azido group; optionally,
a) Maleimide group structure: have or;
b) The structure of the azido is: -N=N ⁺ =N ^- or;
c) Cyclooctene group structure: As having, is binding to an antibody or an antigen-binding fragment thereof;
d) Cyclooctene group structure: As having, is binding to an antibody or an antigen-binding fragment thereof; or
(ii) Attachment
As having a chemical formula including,
An antibody-drug conjugate, which binds to an antibody or an antigen-binding fragment thereof. In claim 7, the antibody or antigen-binding fragment thereof is linked to a conjugate linker (L) by an attachment group selected from the following:

(Here, is binding to an antibody or an antigen-binding fragment thereof, is a bond to the cross-linking spacer group). An antibody-drug conjugate in claim 8, wherein the crosslinking spacer group is -CH ₂ CH ₂ -O-CH ₂ CH ₂ -CO-. An antibody-drug conjugate according to claim 8 or 9, wherein the crosslinking spacer group is bonded to a cleavable group; optionally, the cleavable group is -pyrophosphate-CH ₂ -CH ₂ -NH ₂ -. An antibody-drug conjugate according to any one of claims 8 to 10, wherein the cleavable group is bound to a panRAS inhibitor (D). An antibody-drug conjugate according to any one of claims 1 to 3, wherein the conjugate linker comprises:
Attachment,
One or more crosslinking spacer groups,
Peptide groups, and
One or more cuttable elements. In claim 12, -(LD) is an antibody-drug conjugate having the chemical formula B:
[Chemical Formula B]

(Here,
R ¹ is an attachment group;
L ₁ is a crosslinking spacer;
Lp is a peptide group containing 1 to 6 amino acid residues or Lp is a group Includes;
E is a cuttable element;
L ₂ is a crosslinking spacer;
m is 0 or 1;
D is a panRAS inhibitor). In claim 12 or 13, (i) the attachment group is formed from one or more reactive groups including a maleimide group, a thiol group, a cyclooctyne group, and/or an azido group, and optionally,
a) Maleimide group structure: have or;
b) The structure of the azido is: -N=N ⁺ =N ^- or;
c) Cyclooctene group structure: As having, is binding to an antibody or an antigen-binding fragment thereof;
(ii) Attachment
As having a chemical formula including,
An antibody-drug conjugate, which binds to an antibody or an antigen-binding fragment thereof. In any one of the 12th to 14th clauses,
(i) one or more crosslinking spacers comprise a PEG group, optionally the PEG group is selected from PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, and PEG15; or
(ii) an antibody-drug conjugate, wherein at least one cross-linking spacer is selected from *-C(O)-CH ₂ -CH ₂ -PEG1-**, *-C(O)-CH ₂ -PEG3-**, *-C(O)-CH ₂ -CH ₂ -PEG12**, *-NH-CH ₂ -CH ₂ -PEG1-**, a polyhydroxyalkyl group, *-C(O)-N(CH ₃ )-CH ₂ -CH ₂ -N(CH ₃ )-C(O)-**, and *-C(O)-CH ₂ -CH ₂ -PEG12-NH-C(O)CH ₂ -CH ₂ -**, wherein ** represents a direct or indirect attachment point of the at least one cross-linking spacer to an attachment group, and * represents a direct or indirect attachment point of the at least one cross-linking spacer to a peptide group. An antibody-drug conjugate according to any one of claims 12 to 15, wherein L ₁ is selected from *-C(O)-CH ₂ -CH ₂ -PEG1-**, *-C(O)-CH ₂ -PEG3-**, *-C(O)-CH ₂ -CH ₂ -PEG12**, *-NH-CH ₂ -CH ₂ -PEG1-**, and a polyhydroxyalkyl group, wherein ** represents a direct or indirect attachment point of L ₁ to R ¹ , and * represents a direct or indirect attachment point of L ₁ to Lp. An antibody-drug conjugate according to any one of claims 12 to 16, wherein m is 1 and L ₂ is -C(O)-N(CH ₃ )-CH ₂ -CH ₂ -N(CH ₃ )-C(O)-. In any one of Articles 12 to 17,
(i) the peptide group comprises 1 to 6, 1 to 4, 1 to 3 or 1 to 2 amino acid residues, optionally wherein the amino acid residues are selected from L-glycine (Gly), L-valine (Val), L-citrulline (Cit), L-cysteic acid (sulfo-Ala), L-lysine (Lys), L-isoleucine (Ile), L-phenylalanine (Phe), L-methionine (Met), L-asparagine (Asn), L-proline (Pro), L-alanine (Ala), L-leucine (Leu), L-tryptophan (Trp), and L-tyrosine (Tyr);
(ii) the peptide group comprises Val-Cit, Val-Ala, Val-Lys, and/or sulfo-Ala-Val-Ala; or
(iii) Peptide group
An antibody-drug conjugate selected from. An antibody-drug conjugate according to any one of claims 12 to 18, wherein (i) the cleavable group comprises a pyrophosphate and/or a self-immolative group; (ii) the cleavable group comprises a self-immolative group; or (iii) the cleavable group comprises a self-immolative group comprising para-aminobenzyl-carbamate, para-aminobenzyl-ammonium, para-amino-(sulfo)benzyl-ammonium, para-amino-(sulfo)benzyl-carbamate, para-amino-(alkoxy-PEG-alkyl)benzyl-carbamate, para-amino-(polyhydroxycarboxytetrahydropyranyl)alkyl-benzyl-carbamate, or para-amino-(polyhydroxycarboxytetrahydropyranyl)alkyl-benzyl-ammonium. In any one of claims 13 to 19, m is 0 or 1, or m is 1 and the crosslinking spacer is An antibody-drug conjugate comprising: In any one of claims 13 to 20, -(LD) is an antibody-drug conjugate formed from a compound selected from:




An antibody-drug conjugate according to any one of claims 13 to 21, wherein -(LD) comprises a chemical formula selected from:








(Here, is binding to an antibody or an antigen-binding fragment thereof). In claim 1 or 2, -(LD) is an antibody-drug conjugate having the chemical formula C:
[Chemical Formula C]

(Here,
R ¹ is an attachment group;
L ₁ is a crosslinking spacer;
L _p is a peptide group containing 1 to 6 amino acids;
D is a panRAS inhibitor;
G ₁ -L ₂ -A is a self-immolative spacer;
L ₂ is a bond, methylene, neopentylene or C ₂ -C ₃ alkenylene;
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
L ₃ is a spacer moiety;
R ² is a hydrophilic moiety). In claim 23, -(LD) is an antibody-drug conjugate having the formula D or a pharmaceutically acceptable salt thereof:
[Chemical Formula D]

(Here,
R ¹ is an attachment group;
L ₁ is a crosslinking spacer;
Lp is a peptide group containing 1 to 6 amino acids;
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
L ₃ is a spacer moiety;
R ² is a hydrophilic moiety). In paragraph 23 or 24,
(1) L ₁ is , or
As containing *-CH(OH)CH(OH)CH(OH)CH(OH)CH(OH)-**,
Each n is an integer from 1 to 12, * of L ₁ represents a direct or indirect attachment point to Lp, ** of L ₁ represents a direct or indirect attachment point to R ¹ ; or;
(2) L ₁ is , n is an integer from 1 to 12, or n is 1 or n is 12, * of L ₁ represents a direct or indirect attachment point to Lp, ** of L ₁ represents a direct or indirect attachment point to R ¹ ; or
(3) L ₁ is , n is an integer from 1 to 12, * of L ₁ represents a direct or indirect attachment point to Lp, ** of L ₁ represents a direct or indirect attachment point to R ¹ ; or
(4) L ₁ is , and * of L ₁ represents a direct or indirect attachment point to Lp, and ** of L ₁ represents a direct or indirect attachment point to R ¹ ; or
(5) L ₁ is
*-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**;
*-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**;
*-C(=O)O(CH ₂ ) _m SSC(R ³ ) ₂ (CH ₂ ) _m C(=O)NR ³ (CH ₂ ) _m NR ³ C(=O)(CH ₂ ) _m -**;
*-C(=O)O(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**;
*-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**; _{* -C} ( ₌ O)(CH ₂ ) _m
* -C(=O)(( _{CH 2 ) m} O) _t (CH ₂ ) _n *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**;
*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n -**;
* _-C (=O) _{( CH 2} ) _m NHC(=O)(CH ₂ ) _n
*-C(=O)(( _{CH 2} ) _m O _{) t (} CH ₂ ) _n NHC(=O)(CH ₂ ) _n
*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m C(R ³ ) ₂ -** or
*-C(=O)(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -** as a bridge spacer, wherein * of L ₁ represents a direct or indirect attachment point to Lp, and ** of L ₁ represents a direct or indirect attachment point to R ¹ ;
X ₁ is and;
Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
Each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
Each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;
An antibody-drug conjugate, wherein each R ³ is independently selected from H and C ₁ -C ₆ alkyl. In any one of claims 23 to 25, R ² is polyethylene glycol, polyalkylene glycol, polyol, polysarcosine, sugar, oligosaccharide, polypeptide, 1 to 3 An antibody-drug conjugate, wherein the antibody-drug conjugate is a hydrophilic moiety comprising a C ₂ -C ₆ alkyl substituted with one or two substituents _{independently} selected from -OC(=O)NHS(O) ₂ NHCH ₂ CH ₂ OCH ₃ , -NHC(=O)C _{1-4 alkylene} -P(O)(OCH ₂ CH ₃ ) ₂ and -COOH groups. In any one of claims 23 to 26, R ² is

(where n is an integer from 1 to 6), ,

In, antibody-drug conjugate. In claim 23 or 24, the hydrophilic moiety comprises an antibody-drug conjugate:
(i) Polysarcosine having the following moieties:
(wherein, n is an integer from 3 to 25; R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH); or
(ii) Polyethylene glycol of the following chemical formula:
(wherein, R is H, -CH ₃ , CH ₂ CH ₂ NHC(=O)OR _a , -CH ₂ CH ₂ NHC(=O)R _a , or -CH ₂ CH ₂ C(=O)OR _a , R' is OH, -OCH ₃ , -CH ₂ CH ₂ NHC(=O)OR _a , -CH ₂ CH ₂ NHC(=O)R _a , or -OCH ₂ CH ₂ C(=O)OR _a , R _a is H, or C _1-4 alkyl optionally substituted with any one of OH or C _1-4 alkoxyl, and each m and n is independently an integer from 2 to 25.) In any one of claims 23 to 27, the hydrophilic moiety An antibody-drug conjugate comprising: In any one of Articles 23 to 29,
(i) L ₃ is a structure
[Here,
W is -CH ₂ -, -CH ₂ O-, -CH ₂ N(R ^b )C(=O)O-, -NHC(=O)C(R ^b ) ₂ NHC(=O)O-,
-NHC(=O)C(R ^b ) ₂ NH-, -NHC(=O)C(R ^b ) ₂ NHC(=O)-, -CH ₂ N(XR ² )C(=O)O-, -C(=O)N(XR ² )-, -CH ₂ N(XR ² )C(=O)-, -C(=O)NR ^b -, -C(=O)NH-, -CH ₂ NR ^b C(=O)-, -CH ₂ NR ^b C(=O)NH-, -CH ₂ NR ^b C(=O)NR ^b -, -NHC(=O)-, -NHC(=O)O-, -NHC(=O)NH-, -OC(=O)NH-, -S(O) ₂ NH-, -NHS(O) ₂ -, -C(=O)-, -C(=O)O- or -NH-(wherein each R ^b is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl);
X is a bond, triazolyl, or -CH ₂ -triazolyl-,
X is a spacer moiety having [linked to R ² ]; or
(ii) L ₃ is a structure
[Here,
W is -CH ₂ -, -CH ₂ O-, -CH ₂ N(R ^b )C(=O)O-, -NHC(=O)C(R ^b ) ₂ NHC(=O)O-, -NHC(=O)C(R ^b ) ₂ NH-, -NHC(=O)C(R ^b ) ₂ NHC(=O)-, -CH ₂ N(XR ² )C(=O)O-, -C(=O)N(XR ² )-, -CH ₂ N(XR ² )C(=O)-, -C(=O)NR ^b -, -C(=O)NH-, -CH ₂ NR ^b C(=O)-, -CH ₂ NR ^b C(=O)NH-, -CH ₂ NR ^b C(=O)NR ^b -, -NHC(=O)-, -NHC(=O)O-, -NHC(=O)NH-, -OC(=O)NH-, -S(O) ₂ NH-, -NHS(O) ₂ -, -C(=O)-, -C(=O)O- or -NH- (wherein each R ^b is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl);
X is -CH ₂ -triazolyl-C _1-4 alkylene-OC(O)NHS(O) ₂ NH-, -C _4-6 cycloalkylene-OC(O)NHS(O) ₂ NH-, -(CH ₂ CH ₂ O) _n -C(O)NHS(O) ₂ NH-, -(CH ₂ CH ₂ O) _n -C(O)NHS(O) ₂ NH-(CH ₂ CH ₂ O) _n -, -CH ₂ -triazolyl-C _1-4 alkylene-OC(O)NHS(O) ₂ NH-(CH ₂ CH ₂ O) _n -, -C _4-6 cycloalkylene-OC(O)NHS(O) ₂ NH-(CH ₂ CH ₂ O) _n - (wherein each n is independently 1, 2, or 3),
An antibody-drug conjugate having a spacer moiety having [X is linked to R ² ]. An antibody-drug conjugate according to any one of claims 3 to 30, wherein the attachment group is formed by a reaction comprising one or more reactive groups. In any one of claims 3 to 31, the attachment group
a first reactive group attached to a conjugate linker, and
A second reactive group attached to an antibody or an antigen-binding fragment thereof or which is an amino acid residue of an antibody or an antigen-binding fragment thereof;
is formed by reacting, and optionally,
(i) at least one of the reactors;
thiol,
maleimide,
Haloacetamide,
Azid,
Alkyne,
cyclooctene,
Triaryl phosphine,
Oxanobornadiene,
cyclooctyne,
Diaryl tetrazine,
Monoaryl tetrazine,
Norbornene,
aldehyde,
Hydroxylamine,
Hydrazine,
NH ₂ -NH-C(=O)-,
ketones,
vinyl sulfone,
Aziridine,
amino acid residue,



(Here,
Each R ³ is independently selected from H and C ₁ -C ₆ alkyl;
Each R ⁴ is 2-pyridyl or 4-pyridyl;
Each R ⁵ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl, and -OH;
Each R ⁶ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl, -NH ₂ , -OCH ₃ , -OCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -CN, -NO ₂ and -OH;
Each R ⁷ is independently selected from H, C _1-6 alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C _1-4 alkoxy substituted with -C(=O)OH, and C _1-4 alkyl substituted with -C(=O)OH.
including and/or;
(ii) The first reactor and the second reactor
thiols and maleimides,
thiols and haloacetamides,
thiols and vinyl sulfones,
thiols and aziridines,
Azides and alkynes,
Azide and cyclooctyne,
Azide and cyclooctene,
Azide and triaryl phosphines,
Azides and oxanobornadienes,
Diaryl tetrazine and cyclooctene,
Monoaryl tetrazine and norbornene,
Aldehydes and hydroxylamine,
Aldehydes and hydrazines,
Aldehyde and NH ₂ -NH-C(=O)-,
Ketones and hydroxylamines,
Ketones and hydrazines,
Ketones and NH ₂ -NH-C(=O)-,
Hydroxylamine and
Amine and
, or
CoA or CoA analogue and serine residue
An antibody-drug conjugate comprising: An antibody-drug conjugate according to any one of claims 3 to 32, wherein the attachment group comprises a group selected from:




; and
Disulfide
(Here,
R ³² is H, C _1-4 alkyl, phenyl, pyrimidine or pyridine;
R ³⁵ is H, C _1-6 alkyl, phenyl or C _1-4 alkyl substituted with 1 to 3 -OH groups;
Each R ⁷ is independently selected from H, C _1-6 alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C _1-4 alkoxy substituted with -C(=O)OH, and C _1-4 alkyl substituted with -C(=O)OH;
R ³⁷ is independently selected from H, phenyl and pyridine;
q is 0, 1, 2, or 3;
R ⁸ is H or methyl;
R ⁹ is H, -CH ₃ or phenyl). An antibody-drug conjugate according to any one of claims 23 to 33, wherein the peptide group comprises 1 to 4 or 1 to 3 or 1 or 2 amino acid residues, optionally wherein the amino acid residues are selected from L-glycine (Gly), L-valine (Val), L-citrulline (Cit), L-cysteic acid (sulfo-Ala), L-lysine (Lys), L-isoleucine (Ile), L-phenylalanine (Phe), L-methionine (Met), L-asparagine (Asn), L-proline (Pro), L-alanine (Ala), L-leucine (Leu), L-tryptophan (Trp), and L-tyrosine (Tyr). An antibody-drug conjugate according to any one of claims 23 to 33, wherein the peptide group comprises Val-Cit, Phe-Lys, Val-Ala, Val-Lys, Leu-Cit, sulfo-Ala-Val, and/or sulfo-Ala-Val-Ala. An antibody-drug conjugate according to any one of claims 23 to 35, wherein Lp is selected from:

. In any one of claims 23 to 36, -(LD) is an antibody-drug conjugate comprising or formed from a compound of the following chemical formula:
(1)
(Here,
R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor);
(2)
(Here,
R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor);
(3)
(Here,
R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor);
(4)
(Here,
Each R is independently selected from H, -CH ₃ , and -CH ₂ CH ₂ C(=O)OH;
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor);
(5)
(Here,
Each R is independently selected from H, -CH ₃ , and -CH ₂ CH ₂ C(=O)OH;
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor);
(6)
(Here,
Xa is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ -, and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH;
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor);
(7)
(Here,
R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor);
(8)
(Here,
Xb is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ -, and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH;
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor);
(9)
(Here,
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor);
(10)
(Here,
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor);
(11)
(Here,
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor);
(12)
(Here,
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor);
(13)
(Here,
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor);
(14)
(Here,
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor);
(15)
(Here,
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor); or
(16)
(Here,
Each R is independently H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor), or
(17)
(Here,
Each R is independently H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
n is an integer from 2 to 24;
D is a PanRAS inhibitor), or
(18)
(Here,
A is a bond, -OC(=O)-*,
, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or
As -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,
Each R ^a is independently selected from H, C ₁ -C ₆ alkyl, and C ₃ -C ₈ cycloalkyl, and the * in A indicates the point of attachment to D;
D is a panRAS inhibitor). An antibody-drug conjugate according to any one of claims 23 to 37, wherein A is a bond and/or R is -CH ₃ or -CH ₂ CH ₂ COOH. An antibody-drug conjugate according to any one of claims 23 to 37, wherein A is -OC(=O)-* and/or R is -CH ₃ or -CH ₂ CH ₂ COOH. In any one of claims 23 to 39, -(LD) is an antibody-drug conjugate formed from a compound selected from:














. An antibody-drug conjugate according to any one of claims 1 to 40, wherein D comprises a compound of formula Ia or a pharmaceutically acceptable salt thereof:
[Chemical Formula Ia]

[Here,
Dashed lines represent zero, one, two, three, or four non-adjacent double bonds;
A ^D is -N(H or CH ₃ )C(O)-(CH ₂ )- (wherein the amino nitrogen is bonded to a carbon atom of -C(R ^D10a )(R ^D10 )-), an optionally substituted 3 to 6 membered cycloalkylene, an optionally substituted 3 to 6 membered heterocycloalkylene, an optionally substituted 6 membered arylene, or an optionally substituted 5 to 6 membered heteroarylene;
Y ^X is

(Here, represents the point where X connects to ^D3 ; represents the point where W connects to ^X ); or
Y ^X is -N(R ^D11 )-CO-B ^D -L ^D -;
B ^D is -CH(R ^D9 )- or >C=CR ^D9 R ^D9' (wherein the carbon is bonded to the carbonyl carbon of -N(R ^D11 )C(O)-), an optionally substituted 3 to 6 membered cycloalkylene, an optionally substituted 3 to 6 membered heterocycloalkylene, an optionally substituted 6 membered arylene, or a 5 to 6 membered heteroarylene;
L ^D is absent or is a linker;
G ^D is optionally substituted C ₁ -C ₄ alkylene, optionally substituted C ₁ -C ₄ alkenylene, optionally substituted C ₁ -C ₄ heteroalkylene, -C(O)O-CH(R ^D6 )- (wherein -CH(R ^D6 )- is bonded to -C(R ^D7 R ^D8 )-), -C(O)NH-CH(R ^D6 )- (wherein -CH(R ^D6 )- is bonded to -C(R ^D7 R ^D8 )-), optionally substituted C ₁ -C ₄ heteroalkylene, or 3 to 8 membered heteroarylene;
W ^X is hydrogen, cyano, optionally substituted C ₁ -C ₃ heteroalkyl, optionally substituted amino, optionally substituted C ₁ -C ₄ alkoxy, optionally substituted C ₁ -C ₄ hydroxyalkyl, optionally substituted C ₁ -C ₄ aminoalkyl, optionally substituted C ₁ -C ₄ haloalkyl, optionally substituted C ₁ -C ₄ alkyl, optionally substituted C ₁ -C ₄ guanidinoalkyl, C ₀ -C ₄ alkyl, optionally substituted 3 to 11 membered heterocycloalkyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 3 to 8 membered heteroaryl;
X ^D1 is optionally substituted C ₁ -C ₂ alkylene, NR ^D , O, or S(O) _nD ;
X ^D2 is O or NH;
X ^D3 is N or CH;
nD is 0, 1, or 2;
R ^D is hydrogen, cyano, optionally substituted C ₁ -C ₄ alkyl, optionally substituted C ₂ -C ₄ alkenyl, optionally substituted C ₂ -C ₄ alkynyl, C(O)R ^D ', C(O)OR ^D ', C(O)N(R ^D ') ₂ , S(O)R ^D ', S(O) ₂ R ^D ', or S(O) ₂ N(R ^D ') ₂ ; each R ^D' is independently H or optionally substituted C ₁ -C ₄ alkyl;
Y ^D1 is C, CH, or N;
Y ^D2 , Y ^D3 , Y ^D4 , and Y ^D7 are independently C or N;
Y ^D5 is CH, CH ₂ , or N;
Y ^D6 is C(O), CH, CH ₂ , or N;
R ^D1 is cyano, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 6 membered cycloalkenyl, optionally substituted 3 to 6 membered heterocycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 5 to 10 membered heteroaryl, or
R ^D1 and R ^D2 are combined with the atoms to which they are attached to form an optionally substituted 3 to 14 membered heterocycloalkyl;
R ^D2 is absent, hydrogen, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 7 membered heterocycloalkyl, optionally substituted 6 membered aryl, optionally substituted 5 or 6 membered heteroaryl;
R ^D3 is absent or R ^D2 and R ^D3 are combined with the atoms to which they are attached to form an optionally substituted 3 to 8 membered cycloalkyl or an optionally substituted 3 to 14 membered heterocycloalkyl;
R ^D4 is absent, hydrogen, halogen, cyano, or methyl (optionally substituted with 1 to 3 halogens);
R ^D5 is hydrogen, C ₁ -C ₄ alkyl (optionally substituted with halogen, cyano, hydroxy, or C ₁ -C ₄ alkoxy), cyclopropyl, or cyclobutyl;
R ^D6 is hydrogen or methyl;
R ^D7 is hydrogen, halogen, or optionally substituted C ₁ -C ₃ alkyl, or
R ^D6 and R ^D7 are combined with the carbon atom to which they are attached to form an optionally substituted 3 to 6 membered cycloalkyl or an optionally substituted 3 to 7 membered heterocycloalkyl;
R ^D8 is hydrogen, halogen, hydroxy, cyano, optionally substituted C ₁ -C ₃ alkoxy, optionally substituted C ₁ -C ₃ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or
R ^D7 and R ^D8 are combined with the carbon atom to which they are attached to form C=CR ^D7' R ^D8' ; C=N(OH), C=N(OC ₁ -C ₃ alkyl), C=O, C=S, C=NH, an optionally substituted 3 to 6 membered cycloalkyl, or an optionally substituted 3 to 7 membered heterocycloalkyl;
R ^D7a and R ^D8a are, independently, hydrogen, halo, optionally substituted C ₁ -C ₃ alkyl, or combined with the carbon to which they are attached, form carbonyl;
R ^D7' is hydrogen, halogen, or optionally substituted C ₁ -C ₃ alkyl;
R ^D8' is hydrogen, halogen, hydroxyl, cyano, optionally substituted C ₁ -C ₃ alkoxyl, optionally substituted C ₁ -C ₃ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or
R ^D7' and R ^D8' are combined with the carbon atom to which they are attached to form an optionally substituted 3 to 6 membered cycloalkyl or an optionally substituted 3 to 7 membered heterocycloalkyl;
R ^D9 is hydrogen, F, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, or optionally substituted 3 to 7 membered heterocycloalkyl;
R ^D9 and L ^D are combined with the atoms to which they are attached to form an optionally substituted 3 to 14 membered heterocycloalkyl;
R ^D9' is hydrogen or optionally substituted C ₁ -C ₆ alkyl;
R ^D10 is hydrogen, halo, hydroxyl, C ₁ -C ₃ alkoxyl, or C ₁ -C ₃ alkyl;
R ^D10a is hydrogen or halogen;
R ^D11 is hydrogen or C ₁ -C ₃ alkyl;
R ^D16 is hydrogen or C ₁ -C ₃ alkyl]. In claim 41, D is an antibody-drug conjugate comprising a compound of formula I or a pharmaceutically acceptable salt thereof:
[Chemical Formula I]

[Here,
Dashed lines represent zero, one, two, three, or four non-adjacent double bonds;
A ^D is -N(H or CH ₃ )C(O)-(CH ₂ )- (wherein the amino nitrogen is bonded to a carbon atom of -C(R ^D10a )(R ^D10 )-), an optionally substituted 3 to 6 membered cycloalkylene, an optionally substituted 3 to 6 membered heterocycloalkylene, an optionally substituted 6 membered arylene, or an optionally substituted 5 to 6 membered heteroarylene;
B ^D is -CH(R ^D9 )- or >C=CR ^D9 R ^D9' (wherein the carbon is bonded to the carbonyl carbon of -N(R ^D11 )C(O)-), an optionally substituted 3 to 6 membered cycloalkylene, an optionally substituted 3 to 6 membered heterocycloalkylene, an optionally substituted 6 membered arylene, or a 5 to 6 membered heteroarylene;
G ^D is optionally substituted C ₁ -C ₄ alkylene, optionally substituted C ₁ -C ₄ alkenylene, optionally substituted C ₁ -C ₄ heteroalkylene, -C(O)O-CH(R ^D6 )- (wherein -CH(R ^D6 )- is bonded to -C(R ^D7 R ^D8 )-), -C(O)NH-CH(R ^D6 )- (wherein -CH(R ^D6 )- is bonded to -C(R ^D7 R ^D8 )-), optionally substituted C ₁ -C ₄ heteroalkylene, or 3 to 8 membered heteroarylene;
L ^D is absent or is a drug linker;
W ^D is hydrogen, cyano, optionally substituted amino, optionally substituted C ₁ -C ₄ alkoxy, optionally substituted C ₁ -C ₄ hydroxyalkyl, optionally substituted C ₁ -C ₄ aminoalkyl, optionally substituted C _{1 -C 4 haloalkyl, optionally substituted C 1 -C 4 alkyl ,} optionally substituted C ₁ -C ₄ guanidinoalkyl, C ₀ -C ₄ alkyl optionally substituted 3 to 11 membered heterocycloalkyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 3 to 8 membered heteroaryl;
X ^D1 is optionally substituted C ₁ -C ₂ alkylene, NR ^D , O, or S(O) _nD ;
X ^D2 is O or NH;
X ^D3 is N or CH;
nD is 0, 1, or 2;
R ^D is hydrogen, cyano, optionally substituted C ₁ -C ₄ alkyl, optionally substituted C ₂ -C ₄ alkenyl, optionally substituted C ₂ -C ₄ alkynyl, C(O)R ^D ', C(O)OR ^D ', C(O)N(R ^D ') ₂ , S(O)R ^D ', S(O) ₂ R ^D ', or S(O) ₂ N(R ^D ') ₂ ; each R ^D' is independently H or optionally substituted C ₁ -C ₄ alkyl;
Y ^D1 is C, CH, or N;
Y ^D2 , Y ^D3 , Y ^D4 , and Y ^D7 are independently C or N;
Y ^D5 is CH, CH ₂ , or N;
Y ^D6 is C(O), CH, CH ₂ , or N;
R ^D1 is cyano, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 6 membered cycloalkenyl, optionally substituted 3 to 6 membered heterocycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 5 to 10 membered heteroaryl, or
R ^D1 and R ^D2 are combined with the atoms to which they are attached to form an optionally substituted 3 to 14 membered heterocycloalkyl;
R ^D2 is absent, hydrogen, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 7 membered heterocycloalkyl, optionally substituted 6 membered aryl, optionally substituted 5 or 6 membered heteroaryl;
R ^D3 is absent or R ^D2 and R ^D3 are combined with the atoms to which they are attached to form an optionally substituted 3 to 8 membered cycloalkyl or an optionally substituted 3 to 14 membered heterocycloalkyl;
R ^D4 is absent, hydrogen, halogen, cyano, or methyl (optionally substituted with 1 to 3 halogens);
R ^D5 is hydrogen, C ₁ -C ₄ alkyl (optionally substituted with halogen, cyano, hydroxy, or C ₁ -C ₄ alkoxy), cyclopropyl, or cyclobutyl;
R ^D6 is hydrogen or methyl;
R ^D7 is hydrogen, halogen, or optionally substituted C ₁ -C ₃ alkyl, or
R ^D6 and R ^D7 are combined with the carbon atom to which they are attached to form an optionally substituted 3 to 6 membered cycloalkyl or an optionally substituted 3 to 7 membered heterocycloalkyl;
R ^D8 is hydrogen, halogen, hydroxy, cyano, optionally substituted C ₁ -C ₃ alkoxy, optionally substituted C ₁ -C ₃ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or
R ^D7 and R ^D8 are combined with the carbon atom to which they are attached to form C=CR ^D7' R ^D8' ; C=N(OH), C=N(OC ₁ -C ₃ alkyl), C=O, C=S, C=NH, an optionally substituted 3 to 6 membered cycloalkyl, or an optionally substituted 3 to 7 membered heterocycloalkyl;
R ^D7a and R ^D8a are, independently, hydrogen, halo, optionally substituted C ₁ -C ₃ alkyl, or combined with the carbon to which they are attached, form carbonyl;
R ^D7' is hydrogen, halogen, or optionally substituted C ₁ -C ₃ alkyl;
R ^D8' is hydrogen, halogen, hydroxyl, cyano, optionally substituted C ₁ -C ₃ alkoxyl, optionally substituted C ₁ -C ₃ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or
R ^D7' and R ^D8' are combined with the carbon atom to which they are attached to form an optionally substituted 3 to 6 membered cycloalkyl or an optionally substituted 3 to 7 membered heterocycloalkyl;
R ^D9 is hydrogen, F, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, or optionally substituted 3 to 7 membered heterocycloalkyl;
R ^D9 and L ^D are combined with the atoms to which they are attached to form an optionally substituted 3 to 14 membered heterocycloalkyl;
R ^D9' is hydrogen or optionally substituted C ₁ -C ₆ alkyl;
R ^D10 is hydrogen, halo, hydroxyl, C ₁ -C ₃ alkoxyl, or C ₁ -C ₃ alkyl;
R ^D10a is hydrogen or halogen;
R ^D11 is hydrogen or C ₁ -C ₃ alkyl;
R ^D16 is hydrogen or C ₁ -C ₃ alkyl]. In claim 41 or 42, an antibody-drug conjugate comprising a compound of formula Ic or a pharmaceutically acceptable salt thereof:
[Chemical formula Ic]

[Here,
Dashed lines represent zero, one, two, three, or four non-adjacent double bonds;
A ^D is -N(H or CH ₃ )C(O)-(CH ₂ )- (wherein the amino nitrogen is bonded to a carbon atom of -CH(R ^D10 )-), an optionally substituted 3 to 6 membered cycloalkylene, an optionally substituted 3 to 6 membered heterocycloalkylene, an optionally substituted 6 membered arylene, or an optionally substituted 5 to 6 membered heteroarylene;
B ^D is -CH(R ^D9 )- (wherein the carbon is bonded to the carbonyl carbon of -N(R ^D11 )C(O)-), an optionally substituted 3 to 6 membered cycloalkylene, an optionally substituted 3 to 6 membered heterocycloalkylene, an optionally substituted 6 membered arylene, or a 5 to 6 membered heteroarylene;
L ^D is absent or is a drug linker;
W ^D is hydrogen, optionally substituted amino, optionally substituted C ₁ -C ₄ alkoxy, optionally substituted C ₁ -C ₄ hydroxyalkyl, optionally substituted C ₁ -C ₄ aminoalkyl, optionally substituted C ₁ -C ₄ haloalkyl, optionally substituted C ₁ -C ₄ alkyl, optionally substituted C ₁ -C ₄ guanidinoalkyl, C ₀ -C ₄ alkyl, optionally substituted 3 to 11 membered heterocycloalkyl, optionally substituted 3 to 8 membered cycloalkyl, or optionally substituted 3 to 8 membered heteroaryl;
X ^D2 is O or NH;
X ^D3 is N or CH;
R ^D is hydrogen, cyano, optionally substituted C ₁ -C ₄ alkyl, optionally substituted C ₂ -C ₄ alkenyl, optionally substituted C ₂ -C ₄ alkynyl, C(O)R ^D ', C(O)OR ^D ', C(O)N(R ^D ') ₂ , S(O)R ^D ', S(O) ₂ R ^D ', or S(O) ₂ N(R ^D ') ₂ ;
Each R ^D' is independently H or optionally substituted C ₁ -C ₄ alkyl;
Y ^D1 is C, CH, or N;
Y ^D2 , Y ^D3 , Y ^D4 , and Y ^D7 are independently C or N;
Y ^D5 and Y ^D6 are independently CH or N;
R ^D1 is cyano, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 6 membered cycloalkenyl, optionally substituted 3 to 6 membered heterocycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 5 to 10 membered heteroaryl;
R ^D2 is hydrogen, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 7 membered heterocycloalkyl, optionally substituted 6 membered aryl, optionally substituted 5 or 6 membered heteroaryl;
R ^D3 is absent; or
R ^D2 and R ^D3 are combined with the atoms to which they are attached to form an optionally substituted 3 to 8 membered cycloalkyl or an optionally substituted 3 to 14 membered heterocycloalkyl;
R ^D4 is absent, hydrogen, halogen, cyano, or methyl (optionally substituted with 1 to 3 halogens);
R ^D5 is hydrogen, C ₁ -C ₄ alkyl (optionally substituted with halogen, cyano, hydroxy, or C ₁ -C ₄ alkoxy), cyclopropyl, or cyclobutyl;
R ^D6 is hydrogen or methyl;
R ^D7 is hydrogen, halogen, or optionally substituted C ₁ -C ₃ alkyl, or
R ^D6 and R ^D7 are combined with the carbon atom to which they are attached to form an optionally substituted 3 to 6 membered cycloalkyl or an optionally substituted 3 to 7 membered heterocycloalkyl;
R ^D8 is hydrogen, halogen, hydroxy, cyano, optionally substituted C ₁ -C ₃ alkoxy, optionally substituted C ₁ -C ₃ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or
R ^D7 and R ^D8 are combined with the carbon atom to which they are attached to form C=CR ^7' R ^8' ; C=N(OH), C=N(OC ₁ -C ₃ alkyl), C=O, C=S, C=NH, an optionally substituted 3 to 6 membered cycloalkyl, or an optionally substituted 3 to 7 membered heterocycloalkyl;
R ^D7' is hydrogen, halogen, or optionally substituted C ₁ -C ₃ alkyl;
R ^D8' is hydrogen, halogen, hydroxy, cyano, optionally substituted C ₁ -C ₃ alkoxy, optionally substituted C ₁ -C ₃ alkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or
R ^D7' and R ^D8' are combined with the carbon atom to which they are attached to form an optionally substituted 3 to 6 membered cycloalkyl or an optionally substituted 3 to 7 membered heterocycloalkyl;
R ^D9 is optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, or optionally substituted 3 to 7 membered heterocycloalkyl;
R ^D10 is hydrogen, hydroxy, C ₁ -C ₃ alkoxy or C ₁ -C ₃ alkyl;
R ^D11 is hydrogen or C ₁ -C ₃ alkyl]. An antibody-drug conjugate according to any one of claims 41 to 43, wherein D comprises a compound of formula If or a pharmaceutically acceptable salt thereof:
[Chemical formula If]

[Here,
A ^D is -N(H or CH ₃ )C(O)-(CH ₂ )- (wherein the amino nitrogen is bonded to the carbon atom of -CH ₂ -), optionally substituted 3 to 6 membered cycloalkylene, optionally substituted 3 to 6 membered heterocycloalkylene, optionally substituted 6 membered arylene, or optionally substituted 5 to 6 membered heteroarylene;
B ^D is -CH(R ^D9 )- (wherein the carbon is bonded to the carbonyl carbon of -NHC(O)-), an optionally substituted 3-6 membered cycloalkylene, an optionally substituted 3-6 membered heterocycloalkylene, an optionally substituted 6-membered arylene, or a 5-6 membered heteroarylene;
L ^D is absent or is a drug linker;
W ^D is hydrogen, optionally substituted amino, optionally substituted C ₁ -C ₄ alkoxy, optionally substituted C ₁ -C ₄ hydroxyalkyl, optionally substituted C ₁ -C ₄ aminoalkyl, optionally substituted C ₁ -C ₄ haloalkyl, optionally substituted C ₁ -C ₄ alkyl, optionally substituted C ₁ -C ₄ guanidinoalkyl, C ₀ -C ₄ alkyl, optionally substituted 3 to 11 membered heterocycloalkyl, optionally substituted 3 to 8 membered cycloalkyl, or optionally substituted 3 to 8 membered heteroaryl;
R ^D1 is cyano, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 6 membered cycloalkenyl, optionally substituted 3 to 6 membered heterocycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 5 to 10 membered heteroaryl;
R ^D2 is C ₁ -C ₆ alkyl or 3 to 6 membered cycloalkyl;
R ^D7 is C ₁ -C ₃ alkyl;
R ^D8 is C ₁ -C ₃ alkyl;
R ^D9 is optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, or optionally substituted 3 to 7 membered heterocycloalkyl. An antibody-drug conjugate according to any one of claims 41 to 44, wherein R ^D1 is a 5- to 10-membered heteroaryl. An antibody-drug conjugate according to any one of claims 41 to 45, wherein R ^D1 is an optionally substituted 6-membered aryl or an optionally substituted 6-membered heteroaryl. An antibody-drug conjugate according to any one of claims 41 to 46, wherein D is attached to a conjugate linker represented by L at position A ^D or R ^D1 . An antibody-drug conjugate according to any one of claims 41 to 47, wherein D comprises a compound of formula Ig or a pharmaceutically acceptable salt thereof:
[Chemical formula Ig]

[Here,
A ^D is an optionally substituted 3 to 6 membered cycloalkylene, an optionally substituted 3 to 6 membered heterocycloalkylene, an optionally substituted 6 membered arylene, or an optionally substituted 5 to 6 membered heteroarylene;
B ^D is -CH(R ^D9 )- (wherein the carbon is bonded to the carbonyl carbon of -NHC(O)-), an optionally substituted 3-6 membered cycloalkylene, an optionally substituted 3-6 membered heterocycloalkylene, an optionally substituted 6-membered arylene, or a 5-6 membered heteroarylene;
L ^D is absent or is a drug linker;
W ^D is hydrogen, optionally substituted amino, optionally substituted C ₁ -C ₄ alkoxy, optionally substituted C ₁ -C ₄ hydroxyalkyl, optionally substituted C ₁ -C ₄ aminoalkyl, optionally substituted C ₁ -C ₄ haloalkyl, optionally substituted C ₁ -C ₄ alkyl, optionally substituted C ₁ -C ₄ guanidinoalkyl, C ₀ -C ₄ alkyl, optionally substituted 3 to 11 membered heterocycloalkyl, optionally substituted 3 to 8 membered cycloalkyl, or optionally substituted 3 to 8 membered heteroaryl;
R ^D2 is C ₁ -C ₆ alkyl or 3 to 6 membered cycloalkyl;
R ^D7 is C ₁ -C ₃ alkyl;
R ^D8 is C ₁ -C ₃ alkyl;
R ^D9 is optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, or optionally substituted 3 to 7 membered heterocycloalkyl;
X ^De is N, CH, or CR ^D17 ;
X ^Df is N or CH;
R ^D12 is optionally substituted C ₁ -C ₆ alkyl or optionally substituted C ₁ -C ₆ heteroalkyl;
R ^D17 is optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 6 membered cycloalkenyl, optionally substituted 3 to 6 membered heterocycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 5 to 10 membered heteroaryl. An antibody-drug conjugate according to any one of claims 41 to 48, wherein A ^D is an optionally substituted 6-membered arylene. An antibody-drug conjugate according to any one of claims 41 to 49, wherein A ^D is an optionally substituted 5- to 6-membered heteroarylene. An antibody-drug conjugate according to any one of claims 41 to 50, wherein B ^D is -CHR ^D9 -. An antibody-drug conjugate according to any one of claims 41 to 51, wherein R ^D9 is an optionally substituted C ₁ -C ₆ alkyl or an optionally substituted 3 to 6 membered cycloalkyl. An antibody-drug conjugate according to any one of claims 41 to 52, wherein the drug linker has a structure of chemical formula II:
[Chemical Formula II]

(Here,
A ^D1 is a bond between the drug linker and B; A ^D2 is a bond between W and the drug linker;
B ^D1 , B ^D2 , B ^D3 , and B ^D4 are each independently selected from optionally substituted C ₁ -C ₂ alkylene, optionally substituted C ₁ -C ₃ heteroalkylene, O, S, and NR ^DN ; R ^DN is hydrogen, optionally substituted C ₁ -C ₄ alkyl, optionally substituted C ₁ -C ₃ cycloalkyl, optionally substituted C ₂ -C ₄ alkenyl, optionally substituted C ₂ -C ₄ alkynyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted C ₁ -C ₇ heteroalkyl;
C ^D1 and C ^D2 are each independently selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl;
fD, gD, hD, iD, jD, and kD are each independently 0 or 1;
D ^D1 is an optionally substituted C ₁ -C ₁₀ alkylene, an optionally substituted C ₂ -C ₁₀ alkenylene, an optionally substituted C ₂ -C ₁₀ alkynylene, an optionally substituted 3 to 14 membered heterocycloalkylene, an optionally substituted 5 to 10 membered heteroarylene, an optionally substituted 3 to 8 membered cycloalkylene, an optionally substituted 6 to 10 membered arylene, an optionally substituted C ₂ -C ₁₀ polyethylene glycolene, or an optionally substituted C ₁ -C ₁₀ heteroalkylene, or a chemical bond connecting A ^D1 -(B ^D1 ) _fD -(C ^D1 ) _gD -(B ^D2 ) _hD - to -(B ^D3 ) _iD -(C ^D2 ) _Dj -(B ^D4 ) _Dk -A ^D2 . An antibody-drug conjugate according to any one of claims 41 to 53, wherein the drug linker has a structure of chemical formula IIa:
[Chemical Formula IIa]

(Here,
X ^Da is absent or N;
R ^D14 is absent, hydrogen, optionally substituted C ₁ -C ₆ alkyl, or optionally substituted C ₁ -C ₃ cycloalkyl;
L ^D2 is absent, -C(O)-, -SO ₂ -, optionally substituted C ₁ -C ₄ alkylene or optionally substituted C ₁ -C ₄ heteroalkylene, wherein at least one of X ^Da , R ^D14 , or L ^D2 is present. An antibody-drug conjugate according to any one of claims 41 to 54, wherein W ^D is hydrogen. An antibody-drug conjugate according to any one of claims 41 to 54, wherein W ^D is a C ₀ -C ₄ alkyl optionally substituted 3 to 11 membered heterocycloalkyl. An antibody-drug conjugate according to any one of claims 46 to 56, wherein D is attached to a conjugate linker represented by L at position A ^D or R ^D17 . An antibody-drug conjugate according to any one of claims 41 to 47, wherein D comprises a compound of formula Ih or a pharmaceutically acceptable salt thereof:
[chemical formula Ih]

(Here,
R ^D2 is C ₁ -C ₃ alkyl;
R ^D7 is C ₁ -C ₃ alkyl;
R ^D8 is C ₁ -C ₃ alkyl;
R ^D9 is C ₁ -C ₆ alkyl;
R ^D14 is hydrogen, or C ₁ -C ₆ alkyl,
R ^D17 is an optionally substituted 3 to 6 membered cycloalkyl or an optionally substituted 3 to 6 membered heterocycloalkyl;
W ^D is an optionally substituted 3 to 11 membered heterocycloalkyl). In Article 58,
R ^D9 is C ₁ -C ₃ alkyl;
R ^D14 is C ₁ -C ₃ alkyl;
R ^D17 is an optionally substituted 3 to 6 membered heterocycloalkyl;
An antibody-drug conjugate wherein W ^D is an optionally substituted 5- to 6-membered heterocycloalkyl. In paragraph 58 or 59, D is
Compounds represented by or
An antibody-drug conjugate comprising a salt acceptable to the pharmaceutical industry. In claim 41, D is an antibody-drug conjugate comprising a compound of formula In or a pharmaceutically acceptable salt thereof:
[Chemical formula In]

(where aD is 0 or 1). In claim 41, D is an antibody-drug conjugate comprising a compound of formula Ij or a pharmaceutically acceptable salt thereof:
[Chemical formula Ij]

(where aD is 0 or 1). In claim 41, D is an antibody-drug conjugate comprising a compound of formula Ik or a pharmaceutically acceptable salt thereof:
[chemical formula Ik]

(where aD is 0 or 1). In claim 41, D is an antibody-drug conjugate comprising a compound of formula Im or a pharmaceutically acceptable salt thereof:
[Chemical formula Im]

(where aD is 0 or 1). An antibody-drug conjugate according to any one of claims 1 to 40, wherein D comprises a group represented by a chemical formula selected from the chemical formulas of Table A2. An antibody-drug conjugate according to any one of claims 1 to 65, wherein the antibody or antigen-binding fragment thereof binds to a target antigen on a cancer cell. An antibody-drug conjugate in claim 66, wherein the target antigen is EphA2 or B7-H3 (CD276). An antibody-drug conjugate according to any one of claims 1 to 67, wherein the antibody or antigen-binding fragment thereof is an anti-EphA2 antibody or antigen-binding fragment thereof. In claim 68, the anti-EphA2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs selected from the group consisting of:
1) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 17, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 18, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 19; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 26, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 27, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 28;
2) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 20, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 21, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 19; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 29, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 30, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 31;
3) a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 22, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 23, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 24; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 32, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 27, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 31; and
4) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 25, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 21, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 19; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 29, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 30, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 31. In claim 68 or 69, the anti-EphA2 antibody or antigen-binding fragment thereof comprises an antibody-drug conjugate comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12. The antibody-drug conjugate of any one of claims 68 to 70, wherein the anti-EphA2 antibody or antigen-binding fragment thereof comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 3, and a light chain comprising the amino acid sequence of SEQ ID NO: 5. An antibody-drug conjugate according to any one of claims 1 to 67, wherein the antibody or antigen-binding fragment thereof is an anti-B7-H3 (CD276) antibody or antigen-binding fragment thereof. In claim 72, the anti-B7-H3 (CD276) antibody comprises three heavy chain CDRs and three light chain CDRs selected from the group consisting of:
1) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 33, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 34, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 35; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 42, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 43, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 44;
2) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 36, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 37, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 35; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 45, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 46, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 47;
3) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 38, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 39, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 40; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 48, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 43, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 47;
4) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 41, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 37, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 35; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 45, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 46, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 47. In claim 72 or 73, the anti-B7-H3 (CD276) antibody is an antibody-drug conjugate comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In any one of claims 72 to 74, the anti-B7-H3 (CD276) antibody comprises an antibody-drug conjugate: a heavy chain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain comprising the amino acid sequence of SEQ ID NO: 8. In claim 72, the anti-B7-H3 (CD276) antibody comprises three heavy chain CDRs and three light chain CDRs selected from the group consisting of:
1) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 49, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 50, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 51; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 58, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 59, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 60;
2) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 52, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 53, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 51; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 61, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 62, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 63;
3) a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 54, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 55, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 56; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 58, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 59, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 63; and
4) A heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 57, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 53, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 51; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 61, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 62, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 63. In claim 72 or 76, the anti-B7-H3 (CD276) antibody is an antibody-drug conjugate comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16. An antibody-drug conjugate according to any one of claims 72, 76 or 77, wherein the anti-B7-H3 (CD276) antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10. In any one of Articles 1 to 78,
(a) the antibody or antigen-binding fragment thereof comprises an IgG1 heavy chain constant domain or a modified IgG1 heavy chain constant domain, and optionally the IgG1 heavy chain constant domain comprises cysteine residues (C) at positions 152 and 375 (wherein the positions are numbered according to the EU system); and/or;
(b) An antibody-drug conjugate, wherein the antibody or antigen-binding fragment thereof comprises an Ig kappa light chain constant domain. A composition comprising multiple copies of an antibody-drug conjugate of any one of claims 1 to 79, wherein the average p of the antibody-drug conjugate in the composition is from about 2 to about 16, for example, from about 2 to about 8, for example, from about 2 to about 4. A pharmaceutical composition comprising an antibody-drug conjugate of any one of claims 1 to 79 or a composition of claim 80, and a pharmaceutically acceptable carrier. A method of treating a subject having or suspected of having cancer, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate of any one of claims 1 to 79, a composition of claim 80, or a pharmaceutical composition of claim 81. A method according to claim 82, wherein the cancer expresses a target antigen, and optionally, the target antigen is EphA2 or B7-H3 (CD276). The method of claim 82 or 83, wherein the cancer is a tumor or a hematological cancer, and optionally, the cancer is breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, gastric cancer or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer. A method of reducing or inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate of any one of claims 1 to 79, a composition of claim 80, or a pharmaceutical composition of claim 81. A method according to claim 85, wherein the tumor expresses a target antigen, and optionally, the target antigen is EphA2 or B7-H3 (CD276). The method of claim 85 or 86, wherein the tumor is breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, gastric or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer. A method for reducing or suppressing a blood cancer in a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate of any one of claims 1 to 79, a composition of claim 80, or a pharmaceutical composition of claim 81. A method according to claim 88, wherein the blood cancer expresses a target antigen, and optionally, the target antigen is EphA2 or B7-H3 (CD276). The method of claim 88 or 89, wherein the blood cancer is chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelomonocytic leukemia (CML), chronic myelomonocytic leukemia (CMML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, non-Hodgkin's lymphoma, or myelodysplastic syndrome (MDS). A method according to any one of claims 85 to 90, wherein administration of the antibody-drug conjugate, composition, or pharmaceutical composition reduces or inhibits tumor growth or hematological malignancy by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. A method of reducing or slowing the expansion of a cancer cell population in a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate of any one of claims 1 to 79, a composition of claim 80, or a pharmaceutical composition of claim 81. A method according to claim 92, wherein the cancer cell population expresses a target antigen, and optionally, the target antigen is EphA2 or B7-H3 (CD276). The method of claim 92 or 93, wherein the cancer cell population is derived from a tumor or a hematological cancer, and optionally, the cancer cell population is derived from breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, gastric or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer. A method according to any one of claims 92 to 94, wherein administration of the antibody-drug conjugate, composition, or pharmaceutical composition reduces the cancer cell population or slows the expansion of the cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. A method according to any one of claims 82 to 95, wherein the antibody-drug conjugate is administered as a monotherapy. A method according to any one of claims 82 to 95, wherein the antibody-drug conjugate is administered adjuvantly to another therapeutic agent or radiotherapy. In claim 97, the antibody-drug conjugate is administered in an amount effective to sensitize tumor cells to one or more additional therapeutic agents and/or radiation therapy. A method according to any one of claims 82 to 95, further comprising administering to a subject in need thereof at least one additional therapeutic agent. A method of inhibiting panRAS activity in a cell expressing panRAS, comprising the step of contacting the cell with an antibody-drug conjugate of any one of claims 1 to 79 capable of binding to the cell, under conditions in which the antibody-drug conjugate binds to the cell. A method of determining whether a subject having or suspected of having cancer is responsive to treatment with an antibody-drug conjugate of any one of claims 1 to 79, a composition of claim 80, or a pharmaceutical composition of claim 81, comprising: providing a biological sample from the subject; contacting the sample with an antibody-drug conjugate; and detecting binding of the antibody-drug conjugate to cancer cells in the sample. A method according to claim 101, wherein the cancer cells in the sample express a target antigen, and optionally, the target antigen is EphA2 or B7-H3 (CD276). A method according to claim 101 or 102, wherein the cancer expresses a target antigen, and optionally, the target antigen is EphA2 or B7-H3 (CD276). The method of any one of claims 101 to 103, wherein the cancer is a tumor or a hematological cancer, and optionally, the cancer is breast cancer (including ER-positive breast cancer), multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, gastric or gastrointestinal cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, pancreatic cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancy of T-cell or B-cell origin, metastatic castration-resistant prostate cancer, bladder urothelial carcinoma, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, or head and neck cancer. A method according to any one of claims 101 to 104, wherein the sample is a tissue biopsy sample, a blood sample, or a bone marrow sample. A method according to any one of claims 83 to 105, wherein the target antigen is EphA2. A method according to any one of claims 83 to 105, wherein the target antigen is B7-H3 (CD276). A method for preparing an antibody-drug conjugate of any one of claims 1 to 80, comprising reacting an antibody or antigen-binding fragment with a cleavable linker linked to a panRAS inhibitor under conditions permitting conjugation. In claim 108, the method wherein the antibody or antigen-binding fragment is an anti-EphA2 antibody or antigen-binding fragment or a B7-H3 (CD276) antibody or antigen-binding fragment. In claim 109, the method wherein the antibody or antigen-binding fragment is an anti-EphA2 antibody or antigen-binding fragment. In claim 109, the method wherein the antibody or antigen-binding fragment is a B7-H3 (CD276) antibody or antigen-binding fragment. Use of an antibody-drug conjugate of any one of claims 1 to 79, a composition of claim 80, or a pharmaceutical composition of claim 81 in the manufacture of a medicament for treating a subject having or suspected of having cancer. Use of an antibody-drug conjugate of any one of claims 1 to 79, a composition of claim 80, or a pharmaceutical composition of claim 81 in the manufacture of a medicament for reducing or inhibiting the growth of a tumor in a subject. Use of an antibody-drug conjugate of any one of claims 1 to 79, a composition of claim 80, or a pharmaceutical composition of claim 81 in the manufacture of a medicament for reducing or suppressing a blood cancer in a subject. Use of an antibody-drug conjugate of any one of claims 1 to 79, a composition of claim 80, or a pharmaceutical composition of claim 81 in the manufacture of a medicament for reducing or slowing the expansion of a cancer cell population in a subject.