TNIK MODULATORS, CONJUGATES, AND USES THEREOF

RELATED APPLICATION INFORMATION

[0001] This application claims the benefit of U.S. Provisional Application No. 62/573,616 filed October 17, 2017 and U.S. Provisional Application No. 62/643,060 filed March 14, 2018, the contents of each of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] One of the leading causes of death in the United States is cancer. The conventional methods of cancer treatment, like chemotherapy, surgery, and radiation therapy, tend to be either highly toxic or nonspecific to a cancer, or both, resulting in limited efficacy and harmful side effects. However, the immune system has the potential to be a powerful, specific tool in fighting cancers. In many cases tumors can specifically express genes whose products are required for inducing or maintaining the malignant state. These proteins may serve as antigen markers for the development and establishment of more specific anti-cancer immune response. The boosting of this specific immune response has the potential to be a powerful anti-cancer treatment that can be more effective than conventional methods of cancer treatment and can have fewer side effects.

SUMMARY OF THE INVENTION

[0003] The present disclosure provides compounds and conjugates, protein targeting modules, and formulations comprising the same, as well as methods of using these compositions in the treatment of patients including cancer patients.

[0004] In a first aspect, the present disclosure provides a compound represented by Formula

(I):

or a salt thereof, wherein:

W is selected from N and CH;

A is selected from an optionally substituted C ₃ . ₁₂ carbocycle and an optionally substituted 3- to 12-membered heterocycle, wherein substituents on A are independently selected at each occurrence from R ¹² ;

X ¹ and X ² are independently selected from -C(R ¹⁰ ) ₂ -, -0-, -S-, -N(R ¹⁰ )-, -C(O)-, -OC(0)0-, -C(0)N(R ¹⁰ )-, and -N(R ¹⁰ )C(O)-;

L is selected from C ₆ -Ci ₂ alkylene, C ₆ -Ci ₂ alkenylene, C ₆ -Ci ₂ alkynylene, a 6- to 12- membered heteroalkylene, a 6- to 12-membered heteroalkenylene, a C ₃ -C ₁₂ carbocyclene, a 3- to 12-membered heterocyclene, and an alkylene or a heteroalkylene interspersed with a C ₃ -C ₁₂ carbocyclene or a 3- to 12-membered heterocyclene, any of which is optionally substituted by one or more substituents independently selected at each occurrence from R ¹³ ;

R ¹ is selected at each occurrence from R ¹² ;

R ¹⁰ is independently selected at each occurrence from: hydrogen; C ₁ -C ₁₀ alkyl, C ₂ -Ci ₀ alkenyl, and C ₂ -Ci ₀ alkynyl, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , - H ₂ , =0, =S, -O-Ci-C ₁₀ alkyl, C ₃ -C ₁₂ carbocycle, and a 3- to 12-membered heterocycle; and a C ₃ -C ₁₂ carbocycle and a 3- to 12- membered heterocycle, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , =0, =S, C ₁ -C ₁₀ alkyl, -O-Ci-Cio alkyl, and -C ₁ -C ₁₀ haloalkyl;

R ¹² is independently selected at each occurrence from:

a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ -C(0)OR ¹⁰ , -OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , -S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ¹⁰ ), Ci _-6 alkylN(R ¹⁰ ) ₂ , and -CN;

C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, -O-Ci-C ₁₀ alkyl, and -C ₁ -C ₁₀ haloalkyl, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ -C(0)OR ¹⁰ , - OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , -S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ¹⁰ ), -CN, a C ₃ -C ₁₀ carbocycle, and a 3- to 10-membered heterocycle; and

a C ₃ -C ₁₀ carbocycle and a 3- to 10-membered heterocycle, each of which is

independently optionally substituted with one or more substituents independently selected from a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ -C(0)OR ¹⁰ , - OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , -S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ¹⁰ ), -CN, Ci-C ₆ alkyl, -Ci _-6 alkylN(R ¹⁰ ) ₂ , C ₂ -C ₆ alkenyl, and C ₂ -C ₆ alkynyl;

R ¹³ is independently selected at each occurrence from:

a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ -C(0)OR ¹⁰ , -OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , -S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ¹⁰ ), and -CN;

C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, -O-Ci-C ₁₀ alkyl, and -C ₁ -C ₁₀ haloalkyl, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ -C(0)OR ¹⁰ , - OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , -S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S,

=N(R ¹⁰ ), -CN, a C ₃ -C ₁₀ carbocycle, and a 3- to 10-membered heterocycle; and

a C ₃ -C ₁₀ carbocycle and a 3- to 10-membered heterocycle, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OR ¹⁰ , -SR ¹⁰ , - N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ -C(0)OR ¹⁰ , -OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , -S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ¹⁰ ), -CN, Ci-C ₆ alkyl, C ₂ -C ₆ alkenyl, and C ₂ -C ₆ alkynyl;

and

n is selected from 0, 1, 2, 3, 4, and 5.

[0005] In some embodiments, the compound or salt of Formula (I) is further covalently bound to L ³ , wherein L ³ is a linker.

[0006] In another aspect, the present disclosure provides a compound represented by Formula (II):

or a salt thereof, wherein:

W is selected from N and CH;

A is selected from an optionally substituted C ₃ -Ci ₂ carbocycle and an optionally substituted 3- to 12-membered heterocycle, wherein substituents on A are independently selected at each occurrence from R ²² ;

Z is selected from Ci-Ci ₂ alkyl, C ₂ -Ci ₂ alkenyl, a C ₃ -Ci ₂ carbocycle and a 3- to 12- membered heterocycle, any of which is optionally substituted by one or more substituents independently selected at each occurrence from R ²² ;

Q is selected from -0-, -S-, -N(R ²⁰ )-, -C(O)-, -C(0)0-, -OC(O)-, -OC(0)0-, - C(0)N(R ²⁰ )-, and -N(R ²⁰ )C(O)-; and -(C(R ²⁰ ) ₂ ) _m -, -O-(C(R ²⁰ ) ₂ ) _m -, and -(C(R ²⁰ ) ₂ ) _m -O-, wherein m is selected from 1, 2, 3, and 4;

R ² is selected at each occurrence from R ²² ;

R ²⁰ is independently selected at each occurrence from:

-X'-L ³ , hydrogen;

C ₁ -C ₁₀ alkyl, C ₂ -Ci ₀ alkenyl, and C ₂ -Ci ₀ alkynyl, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OH, - CN, -NO ₂ , - H ₂ , tert-butoxycarbonyl-NH- =0, =S, -O-Ci-Cio alkyl, C ₃ -C ₁₂ carbocycle, and a 3- to 12-membered heterocycle; and

a C ₃ -C ₁₂ carbocycle and a 3- to 12-membered heterocycle, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , tert-butoxycarbonyl-NH-, tert-butoxycarbonyl-, =0, =S, C1-C10 alkyl, -O-Ci-C 10 alkyl, and -C1-C10 haloalkyl;

R ²² is independently selected at each occurrence from:

-X'-L ³ , a halogen, -OR ²⁰ , -SR ²⁰ , -N(R ²⁰ ) ₂ , -C(0)R ²⁰ , -C(O)N(R ²⁰ ) ₂ , - N(R ²⁰ )C(O)R ²⁰ -C(0)OR ²⁰ , -OC(0)R ²⁰ , -S(0)R ²⁰ , -S(0) ₂ R ²⁰ , - S(O) ₂ N(R ²⁰ ) ₂ , -P(O)(OR ²⁰ ) ₂ , -OP(O)(OR ²⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ²⁰ ), and -CN;

C ₁ -C ₁₀ alkyl, C ₂ -Cio alkenyl, C ₂ -Cio alkynyl, -O-Ci-C ₁₀ alkyl, and -C ₁ -C ₁₀ haloalkyl, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OR ²⁰ , -SR ²⁰ , -N(R ²⁰ ) ₂ , -C(0)R ²⁰ , -C(O)N(R ²⁰ ) ₂ , - N(R ²⁰ )C(O)R ²⁰ -C(0)OR ²⁰ , -OC(0)R ²⁰ , -S(0)R ²⁰ , -S(0) ₂ R ²⁰ , - S(O) ₂ N(R ²⁰ ) ₂ , -P(O)(OR ²⁰ ) ₂ , -OP(O)(OR ²⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ²⁰ ), -CN, a C ₃ -C ₁₀ carbocycle, and a 3- to 10-membered heterocycle; and

a C ₃ -C ₁₀ carbocycle and a 3- to 10-membered heterocycle, each of which is independently optionally substituted with one or more substituents independently selected from a halogen, -OR ²⁰ , -SR ²⁰ , -N(R ²⁰ ) ₂ , -C(0)R ²⁰ , -C(O)N(R ²⁰ ) ₂ , - N(R ²⁰ )C(O)R ²⁰ -C(0)OR ²⁰ , -OC(0)R ²⁰ , -S(0)R ²⁰ , -S(0) ₂ R ²⁰ , -

S(O) ₂ N(R ²⁰ ) ₂ , -P(O)(OR ²⁰ ) ₂ , -OP(O)(OR ²⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ²⁰ ), -CN, C C ₆ alkyl, C ₂ -C ₆ alkenyl, and C ₂ -C ₆ alkynyl;

R ²³ is selected from H and L ³ ;

X' is independently selected at each occurrence from a bond, -0-, -S-, -NH-, -Ci-C ₆ alkylene, -Ci-C ₆ alkyl ene-NH-, -Ci-C ₆ alkylene-O-, and -Ci-C ₆ alkylene-S-, -saturated heterocycle, -O-saturated heterocycle, and aryl-alkylene-amine-;

n is selected from 0, 1, 2, 3, and 4;

and

3 · 20 22 · 3 23 · 3

L is a linker, wherein at least one R ^zu and R ^zz is -X'-U, or R" is ΙΛ

[0007] In another aspect, the present disclosure provides a compound represented by Formula

or a salt thereof, wherein:

W" is selected from CH, CR ^2' , and N;

B is selected from an optionally substituted 5- to 12-membered heterocycle, wherein at least one heteroatom in the heterocycle is a boron atom, and wherein substituents on B are independently selected at each occurrence from R ²² ;

Z' is selected from C1-C12 alkyl, C2-C12 alkenyl, a C3-C12 carbocycle, and a 3- to 12- membered heterocycle, any of which is optionally substituted by one or more substituents independently selected at each occurrence from R ²² ;

Q' is selected from -0-, -S-, -N(R ^20' )-, -C(O)-, -C(0)0-, -OC(O)-, -OC(0)0-, - C(0)N(R ^20' )-, and -N(R ^20' )C(O)-; and-(C(R ^20' ) ₂ ) _m -, -O-(C(R ^20' ) ₂ ) _m -, and -(C(R ^20' ) ₂ ) _m -O-, wherein m is selected from 1, 2, 3, and 4;

R ² is selected at each occurrence from R ^22' ;

R ²⁰ is independently selected at each occurrence from:

hydrogen;

C1-C10 alkyl, C ₂ -Cio alkenyl, and C ₂ -Cio alkynyl, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OH, - CN, -N0 ₂ , - H ₂ , fert-butoxycarbonyl-NH-, =0, =S, -O-Ci-Cio alkyl, C3-C0 carbocycle, and a 3- to 12-membered heterocycle; and

a C ₃ -Ci ₂ carbocycle and a 3- to 12-membered heterocycle, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , tert-butoxycarbonyl-NH-, tert-butoxycarbonyl-, =0, =S, C1-C10 alkyl, -O-Ci-Cio alkyl, and -C1-C10 haloalkyl;

R ²² is independently selected at each occurrence from:

a halogen, -OR ^20' , -SR ^20' , -N(R ^20' ) ₂ , -C(0)R ^20' , -C(O)N(R ^20' ) ₂ , - N(R ^20' )C(O)R ^20' -C(O)OR ^20' , -OC(0)R ^20' , -S(0)R ^20' , -S(0) ₂ R ^20' , - S(O) ₂ N(R ^20' ) ₂ , -P(O)(OR ^20' ) ₂ , -OP(O)(OR ^20' ) ₂ , -N0 ₂ , =0, =S, =N(R ^20' ), and -CN;

C1-C10 alkyl, C ₂ -C 10 alkenyl, C ₂ -C 10 alkynyl, -O-C i-C 10 alkyl, and -C1-C10 haloalkyl, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OR ^20' , -SR ^20' , -N(R ^20' ) ₂ , -C(0)R ^20' , - C(O)N(R ^20' ) ₂ , -N(R ^20' )C(O)R ^20' -C(0)OR ^20' , -OC(0)R ^20' , -S(0)R ^20' , -S(0) ₂ R ^20' , - S(O) ₂ N(R ^20' ) ₂ , -P(O)(OR ^20' ) ₂ , -OP(O)(OR ^20' ) ₂ , -N0 ₂ , =0, =S, =N(R ^20' ), -CN, a C ₃ -C ₁₀ carbocycle, and a 3- to 10-membered heterocycle; and

a C ₃ -Cio carbocycle and a 3- to 10-membered heterocycle each of which is optionally substituted with one or more substituents independently selected from a halogen, -OR ^20' , -SR ^20' , -N(R ^20' ) ₂ , -C(0)R ^20' , -C(O)N(R ^20' ) ₂ , -N(R ^20' )C(O)R ^20' _; - C(0)OR ^20' , -OC(0)R ^20' , -S(0)R ^20' , -S(0) ₂ R ^20' , -

S(O) ₂ N(R ^20' ) ₂ , -P(O)(OR ^20' ) ₂ , -OP(O)(OR ^20' ) ₂ , -N0 ₂ , =0, =S, =N(R ^20' ), -CN, Ci-C ₆ alkyl,

C ₂ -C ₆ alkenyl, and C ₂ -C ₆ alkynyl;

and

n is selected from 0, 1, 2, 3, and 4.

[0008] A compound or salt a described herein may be covalently attached to an antibody construct. Accordingly, in another aspect, the present disclosure provides a conjugate represented by the formula:

D— L ³ -†- Antibody

1 -8

in which wherein Antibody is an antibody construct, D is a compound or salt as described herein, and L ³ is a linker moiety as described herein, and/or D-L ³ is a compound or salt as described herein. The present disclosure further provides methods of preparing such antibody conjugates.

[0009] In a further aspect, the present disclosure provides protein targeting module and conjugate thereof. In some embodiments, a protein targeting module is represented by the formula: in which S is a spacer moiety, ULM is an E3 ubiqutin ligase binding moiety, and D is a compound or salt as described herein. A protein targeting module conjugate may be represented by a formula selected from:

and

in which Ab is an antibody construct, L ³ is a linker moiety, S is a spacer moiety, ULM is an E3 ubiqutin ligase binding moiety, and D is a compound or salt a described herein.

[0010] In another aspect, the present disclosure provides pharmaceutical compositions comprising a compound or salt as described herein, or a conjugate, protein targeting module, or protein targeting module conjugate comprising a compound or salt a described herein. The present disclosure further provides methods of killing tumor cells in vivo as well as methods for treatment (e.g., of cancer) comprising administering a compound or salt, conjugate, protein targeting module, protein targeting module conjugate, or pharmaceutical composition as described herein.

INCORPORATION BY REFERENCE

[0011] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative

embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also "Figure" and "FIG." herein), of which:

[0013] FIG. 1 shows activity for selected compounds.

DETAILED DESCRIPTION OF THE INVENTION

[0014] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0015] The present disclosure provides compounds and pharmaceutical compositions for use in the treatment of disease. In certain embodiments, the compounds of the disclosure are TNIK modulators. In certain embodiments, the compounds are TNIK inhibitors.

[0016] TNIK (TRAF2 and NCK Interacting Kinase) is a germinal center kinase that interacts with, among other things, TCF transciption factors. The TCF/LEF family of transcription factors bind to DNA and form a complex with the signalling molecule β-catenin in the cell nucleus. Stabilized β-catenin subsequently acts as a transcriptional co-activator to switch on Wnt target genes that contribute to, for example, cell proliferation. Mutations in Wnt signaling pathways have been implicated in a variety of cancers. TNIK interacts with both β-catenin and TCF4 to drive transcriptional activation of Wnt target genes. Inhibition of TNIK prevents Wnt3a mediated IL-10 production in monocytes. Accordingly, TNIK inhibitors provide an

immunomodulatory effect. Such modulators may be useful in the treatment of certain diseases in which immune modulation is desired.

[0017] The compounds of the present disclosure may act as TNIK inhibitors. The

compounds, salts thereof, and conjugates of the present disclosure may be useful for treatment and/or prevention, e.g., vaccination, of cancer, autoimmune diseases, inflammation, sepsis, allergy, asthma, graft rejection, graft-versus-host disease, immunodeficiencies, and infectious diseases.

[0018] In certain embodiments, the compounds, salts, and conjugates have utility in the treatment of cancer either as single agents or in combination therapy. In certain embodiments, the compounds, salts, and conjugates have utility as single agent immunomodulators, vaccine adjuvants and in combination with conventional cancer therapies. In certain embodiments, the compounds and salts are incorporated into a conjugate that can be utilized, for example, to enhance an immune response. In certain embodiments, the disclosure provides conjugates including a compound or salt described herein and an antibody construct.

Definitions

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

[0020] As used in the specification and claims, the singular form "a", "an" and "the" includes plural references unless the context clearly dictates otherwise. [0021] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive toward, a specific antigen. Antibody can include, for example, polyclonal, monoclonal, genetically engineered, and antigen binding fragments thereof. An antibody can be, for example, murine, chimeric, humanized,

heteroconjugate, bispecific, a diabody, a triabody, or a tetrabody. The antigen binding fragment can include, for example, Fab', F(ab')2, Fab, Fv, rlgG, and scFv.

[0022] As used herein, the abbreviations for the natural L-enantiomeric amino acids are conventional and can be as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine (C, Cys); glutamic acid (E, Glu); glutamine (Q, Gin); glycine (G, Gly); histidine (H, His); isoleucine (I, He); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Tip); tyrosine (Y, Tyr); and valine (V, Val). Unless otherwise specified, X can indicate any amino acid. In some embodiments, X can be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R).

[0023] The term "targeting moiety" refers to a structure that has a selective affinity for a target molecule relative to other non-target molecules. The targeting moiety binds to a target molecule. A targeting moiety may include, for example, an antibody, a peptide, a ligand, a receptor, or a binding portion thereof. The target molecule may be a biological receptor or other structure of a cell such as a tumor antigen.

[0024] As used herein, an "antigen binding domain" refers to a region of a molecule that binds to an antigen. An antigen binding domain may be a domain that can specifically bind to an antigen. An antigen binding domain can be an antigen-binding portion of an antibody or an antibody fragment. An antigen binding domain can be one or more fragments of an antibody that can retain the ability to specifically bind to an antigen. An antigen binding domain can be an antigen binding fragment. An antigen binding domain can recognize a single antigen.

[0025] As used herein, an "antibody construct" refers to a molecule, e.g., a protein, peptide, antibody or portion thereof, that contains an antigen binding domain and an Fc domain. An antibody construct can recognize, for example, multiple antigens.

[0026] As used herein, a "Fc domain" can be an Fc domain from an antibody or from a non- antibody that can bind to an Fc receptor.

[0027] As used herein, an "Fc null" refers to an Fc domain that exhibits weak to no binding to any of the Fcgamma receptors. In some embodiments, an Fc null domain or region exhibits a reduction in binding affinity (e.g., increase in Kd) to Fc gamma receptors of at least 1000-fold. [0028] "Conjugate", as used herein, refers to an antibody construct that is linked (e.g., conjugated) either directly or through a linker to a compound, e.g., a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), or (IIIc).

[0029] As used herein, an "Fc domain" can be an Fc domain from an antibody or from a non- antibody that can bind to an Fc receptor.

[0030] As used herein, "recognize" and "specifically bind" with regard to antibody or antibody construct interactions refer to the specific association or binding between an antigen binding domain of an antibody or portion thereof and an antigen, as compared with the binding of the antibody or antibody construct to a non-antigen.

[0031] As used herein, a "target binding domain" refers to a construct that contains an antigen binding domain from an antibody or from a non-antibody that can specifically bind to the antigen.

[0032] As used herein, a "tumor antigen" is an antigenic substance associated with a tumor or cancer cell, and can trigger an immune response in a host.

[0033] The term "salt" or "pharmaceutically acceptable salt" refers to salts derived from a variety of organic and inorganic counter ions well known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, ^-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropyl amine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts.

[0034] The term "C _x-y " when used in conjunction with a chemical moiety such as alkyl, alkenyl, or alkynyl is meant to include groups that contain from x to y carbons in the chain. For example, the term "Ci _-6 alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from 1 to 6 carbons. The term -C _x-y alkylene- refers to a substituted or unsubstituted alkylene chain with from x to y carbons in the alkylene chain. For example -Ci _-6 alkylene- may be selected from methylene, ethylene, propylene, butylene, pentylene, and hexylene, any one of which is optionally substituted.

[0035] The term "aryl" includes aromatic carbocycles with single ring structures or polycyclic structures. For a polycyclic aryl group, at least one of the rings of the polycycle is aromatic. Examples of aryl include phenyl, naphthyl, and dihydronaphthyl.

[0036] The terms "C _x-y alkenyl" and "C _x-y alkynyl" refer to substituted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond, respectively. The term -C _x-y alkenylene™ refers to a substituted or unsubstituted alkenylene chain with from x to y carbons in the alkenylene chain. For example, -C ₂ . ₆ alkenylene--- may be selected from ethenylene, propenylene, butenylene, pentenylene, and hexenylene, any one of which is optionally substituted. An alkenylene chain may have one double bond or more than one double bond in the alkenylene chain. The term -C _x-y alkynylene- refers to a substituted or unsubstituted alkynylene chain with from x to y carbons in the alkenylene chain. For example, ~-C ₂ .6 alkenylene- may be selected from ethynylene, propynylene, butynylene, pentynylene, and hexynylene, any one of which is optionally substituted. An alkenylene chain may have one triple bond or more than one triple bond in the alkenylene chain.

[0037] "Alkylene" refers to a straight divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing no

unsaturation, and preferably having from one to twelve carbon atoms, for example, methylene, ethylene, propylene, butylene, and the like. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group are through the terminal carbons respectively. In other embodiments, an alkylene comprises one to five carbon atoms (i.e., C ₁ -C ₅ alkylene). In other embodiments, an alkylene comprises one to four carbon atoms (i.e., C ₁ -C ₄ alkylene). In other embodiments, an alkylene comprises one to three carbon atoms (i.e., C ₁ -C ₃ alkylene). In other embodiments, an alkylene comprises one to two carbon atoms (i.e., C ₁ -C ₂ alkylene). In other embodiments, an alkylene comprises one carbon atom (i.e., Ci alkylene). In other embodiments, an alkylene comprises five to eight carbon atoms (i.e., C ₅ -C ₈ alkylene). In other embodiments, an alkylene comprises two to five carbon atoms (i.e., C ₂ -C5 alkylene). In other embodiments, an alkylene comprises three to five carbon atoms (i.e., C ₃ -C ₅ alkylene). Unless stated otherwise specifically in the specification, an alkylene chain is optionally substituted by one or more substituents such as those substituents described herein. [0038] "Alkenylene" refers to a straight divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing at least one carbon-carbon double bond, and preferably having from two to twelve carbon atoms. The alkenylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkenylene chain to the rest of the molecule and to the radical group are through the terminal carbons, respectively. In other embodiments, an alkenylene comprises two to five carbon atoms (i.e., C ₂ -C ₅ alkenylene). In other embodiments, an alkenylene comprises two to four carbon atoms (i.e., C ₂ -C ₄ alkenylene).

In other embodiments, an alkenylene comprises two to three carbon atoms (i.e., C ₂ -C ₃ alkenylene). In other embodiments, an alkenylene comprises two carbon atom (i.e., C ₂ alkenylene). In other embodiments, an alkenylene comprises five to eight carbon atoms (i.e., C ₅ -

C ₈ alkenylene). In other embodiments, an alkenylene comprises three to five carbon atoms (i.e. ,

C3-C ₅ alkenylene). Unless stated otherwise specifically in the specification, an alkenylene chain is optionally substituted by one or more substituents such as those substituents described herein.

[0039] "Alkynylene" refers to a straight divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing at least one carbon-carbon triple bond, and preferably having from two to twelve carbon atoms. The alkynylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkynylene chain to the rest of the molecule and to the radical group are through the terminal carbons, respectively. In other embodiments, an alkynylene comprises two to five carbon atoms (i.e., C ₂ -C ₅ alkynylene). In other embodiments, an alkynylene comprises two to four carbon atoms (i.e. , C ₂ -C ₄ alkynylene).

In other embodiments, an alkynylene comprises two to three carbon atoms (i.e. , C ₂ -C ₃ alkynylene). In other embodiments, an alkynylene comprises two carbon atom (i.e. , C ₂ alkynylene). In other embodiments, an alkynylene comprises five to eight carbon atoms (i.e., C ₅ -

C ₈ alkynylene). In other embodiments, an alkynylene comprises three to five carbon atoms (i.e.,

C ₃ -C ₅ alkynylene). Unless stated otherwise specifically in the specification, an alkynylene chain is optionally substituted by one or more substituents such as those substituents described herein.

[0040] "Heteroalkylene" refers to a straight divalent hydrocarbon chain including at least one heteroatom in the chain, containing no unsaturation, and preferably having from one to twelve carbon atoms and from one to 6 heteroatoms, e.g., -0-, -NH-, -S-. The heteroalkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the heteroalkylene chain to the rest of the molecule and to the radical group are through the terminal atoms of the chain. In other embodiments, a heteroalkylene comprises one to five carbon atoms and from one to three heteroatoms. In other embodiments, a heteroalkylene comprises one to four carbon atoms and from one to three heteroatoms. In other embodiments, a heteroalkylene comprises one to three carbon atoms and from one to two heteroatoms. In other embodiments, a heteroalkylene comprises one to two carbon atoms and from one to two heteroatoms. In other embodiments, a heteroalkylene comprises one carbon atom and from one to two heteroatoms. In other embodiments, a heteroalkylene comprises five to eight carbon atoms and from one to four heteroatoms. In other embodiments, a heteroalkylene comprises two to five carbon atoms and from one to three heteroatoms. In other embodiments, a heteroalkylene comprises three to five carbon atoms and from one to three heteroatoms. Unless stated otherwise specifically in the specification, a heteroalkylene chain is optionally substituted by one or more substituents such as those substituents described herein.

[0041] "Heteroalkenylene" refers to a straight divalent hydrocarbon chain including at least one heteroatom in the chain, containing at least one carbon-carbon double bond, and preferably having from one to twelve carbon atoms and from one to 6 heteroatoms, e.g., -0-, -NH-, -S-. The heteroalkenylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the heteroalkenylene chain to the rest of the molecule and to the radical group are through the terminal atoms of the chain. In certain embodiments, a heteroalkenylene comprises two to five carbon atoms and from one to three heteroatoms. In other embodiments, a heteroalkenylene comprises two to four carbon atoms and from one to three heteroatoms. In certain embodiments, a heteroalkenylene comprises two to three carbon atoms and from one to two heteroatoms. In certain embodiments, a heteroalkenylene comprises two carbon atoms and from one to two heteroatoms. In certain embodiments, a heteroalkenylene comprises five to eight carbon atoms and from one to four heteroatoms. In certain embodiments, a heteroalkenylene comprises two to five carbon atoms and from one to three heteroatoms. In other embodiments, a heteroalkenylene comprises three to five carbon atoms and from one to three heteroatoms. Unless stated otherwise specifically in the specification, a heteroalkenylene chain is optionally substituted by one or more substituents such as those substituents described herein.

[0042] The term "carbocycle" as used herein refers to a saturated, unsaturated, or aromatic ring or ring system in which each atom of the ring(s) is carbon. Carbocycle includes 3- to 10- membered monocyclic rings, 6- to 12-membered bicyclic rings, and 6- to 12-membered bridged rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated, and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. A bicyclic carbocycle includes any combination of saturated, unsaturated, and aromatic bicyclic rings, as valence permits. A bicyclic carbocycle includes any combination of ring sizes such as 4-5 fused ring systems, 5-5 fused ring systems, 5-6 fused ring systems, and 6-6 fused ring systems.

Exemplary carbocycles include cyclopentyl, cyclohexyl, cyclohexenyl, adamantyl, phenyl, indanyl, and naphthyl. The term "unsaturated carbocycle" refers to carbocycles with at least one degree of unsaturation and excluding aromatic carbocycles. Examples of unsaturated carbocycles include cyclohexadiene, cyclohexene, and cyclopentene. Further examples of carbocycles include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclopentadiene, cyclohexane, cycloheptane, cycloheptene, naphthalene, and adamantine.

[0043] The term "heterocycle" as used herein refers to a saturated, unsaturated, or aromatic ring or ring system including one or more heteroatoms. Exemplary heteroatoms include N, O, Si, P, B, and S atoms. Heterocycles include 3- to 10-membered monocyclic rings, 6- to 12- membered bicyclic rings, and 6- to 12-membered bridged rings. A bicyclic heterocycle includes any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits. In an exemplary embodiment, an aromatic ring, e.g., pyridyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, morpholine, piperidine or cyclohexene. A bicyclic heterocycle includes any combination of ring sizes such as 4-5 fused ring systems, 5-5 fused ring systems, 5-6 fused ring systems, and 6-6 fused ring systems. The term "unsaturated heterocycle" refers to heterocycles with at least one degree of unsaturation and excluding aromatic heterocycles. Examples of unsaturated heterocycles include dihydropyrrole, dihydrofuran, oxazoline, pyrazoline, and dihydropyridine. Additional examples of heterocycles include, but are not limited to, indole, benzothiophene, benzthiazole, benzoxazole,

benzimidazole, oxazolopyridine, imidazopyridine, thiazolopyridine, furan, oxazole, pyrrole, pyrazole, imidazole, thiophene, thiazole, isothiazole, and isoxazole. Further examples of heteroc cles include:

[0044] The term "heteroaryl" includes aromatic single ring structures, preferably 5- to 7- membered rings, more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The term "heteroaryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be aromatic or non-aromatic carbocyclic, or heterocyclic. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

Additional examples of heteroaryl groups include:

[0045] The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more carbons or substitutable heteroatoms, e.g., an H or N¾ of a compound. It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. In certain embodiments, substituted refers to moieties having substituents replacing two hydrogen atoms on the same carbon atom, such as substituting the two hydrogen atoms on a single carbon with an oxo, imino or thioxo group. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds.

[0046] In some embodiments, substituents may include any substituents described herein, for example: halogen, hydroxy, oxo (=0), thioxo (=S), cyano (-CN), nitro (-N0 ₂ ), imino (=N-H), oximo (=N-OH), hydrazino (=N-NH ₂ ), -R ^b -OR ^a , -R ^b -OC(0)-R ^a , -R ^b -OC(0)-OR ^a , -R ^b -OC(0)- N(R ^a ) ₂ , -R ^b -N(R ^a ) ₂ , -R ^b -C(0)R ^a , -R ^b -C(0)OR ^a , -R ^b -C(0)N(R ^a ) ₂ , -R ^b -0-R ^c -C(0)N(R ^a ) ₂ , -R ^b -N(R )C(0)OR ^a , -R ^b -N(R ^a )C(0)R ^a , -R ^b -N(R ^a )S(0) _t R ^a (where t is 1 or 2), -R ^b -S(0) _t R ^a (where t is 1 or 2), -R ^b -S(0) _t OR ^a (where t is 1 or 2), and -R ^b -S(0) _t N(R ^a ) ₂ (where t is 1 or 2); and alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, and heteroarylalkyl any of which may be optionally substituted by alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=0), thioxo (=S), cyano (-CN), nitro (-N0 ₂ ), imino (=N-H), oximo (=N-OH), hydrazine (=N- H ₂ ), -R ^b -OR ^a , -R ^b -

OC(0)-R ^a , -R ^b -OC(0)-OR ^a , -R ^b -OC(0)-N(R ^a ) ₂ , -R ^b -N(R ^a ) ₂ , -R ^b -C(0)R ^a , -R ^b -C(0)OR ^a , -R ^b -C(0 )N(R ^a ) ₂ , -R ^b -0-R ^c -C(0)N(R ^a ) ₂ , -R ^b -N(R ^a )C(0)OR ^a , -R ^b -N(R ^a )C(0)R ^a , -R ^b -N(R ^a )S(0) _t R ^a (where t is 1 or 2), -R ^b -S(0) _t R ^a (where t is 1 or 2), -R ^b -S(0) _t OR ^a (where t is 1 or 2) and -R ^b -S(0) _t N(R ^a ) ₂ (where t is 1 or 2); wherein each R ^a is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or

heteroarylalkyl, wherein each R ^a , valence permitting, may be optionally substituted with alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=0), thioxo (=S), cyano (- CN), nitro (-N0 ₂ ), imino (=N-H), oximo (=N-OH), hydrazine (=N- H ₂ ), -R ^b -OR ^a , -R ^b -OC(0)- R ^a , -R ^b -OC(0)-OR ^a , -R ^b -OC(0)-N(R ^a ) ₂ , -R ^b -N(R ^a ) ₂ , -R ^b -C(0)R ^a , -R ^b -C(0)OR ^a , -R ^b -C(0)N(R ^a ) ₂ , -R ^b -0-R ^c -C(0)N(R ^a ) ₂ , -R ^b -N(R ^a )C(0)OR ^a , -R ^b -N(R ^a )C(0)R ^a , -R ^b -N(R ^a )S(0) _t R ^a (where t is 1 or 2), -R ^b -S(0) _t R ^a (where t is 1 or 2), -R ^b -S(0) _t OR ^a (where t is 1 or 2) and -R ^b -S(0) _t N(R ^a ) ₂ (where t is 1 or 2); and wherein each R ^b is independently selected from a direct bond or a straight or branched alkyl ene, alkenylene, or alkynylene chain, and each R ^c is a straight or branched alkylene, alkenylene or alkynylene chain.

[0047] It will be understood by those skilled in the art that substituesnts can themselves be substituted, if appropriate. Unless specifically stated as "unsubstituted," references to chemical moieties herein are understood to include substituted variants. For example, reference to a "heteroaryl" group or moiety implicitly includes both substituted and unsubstituted variants, unless specified otherwise.

[0048] Chemical entities having carbon-carbon double bonds or carbon-nitrogen double bonds may exist in Z- or s- form (or cis- or trans- form). Furthermore, some chemical entities may exist in various tautomeric forms. Unless otherwise specified, compounds described herein are intended to include all Z-, E- and tautomeric forms as well.

[0049] A "tautomer" refers to a molecule wherein a proton shift from one atom of a molecule to another atom of the same molecule is possible. The compounds presented herein, in certain embodiments, exist as tautomers. In circumstances where tautomerization is possible, a chemical equilibrium of the tautomers will exist. The exact ratio of the tautomers depends on several factors, including physical state, temperature, solvent, and pH. Some examples of tautomeric equilibrium include:

[0050] The compounds disclosed herein, in some embodiments, are used in different enriched isotopic forms, e.g., enriched in the content of ² H, ³ H, ^U C, ¹³ C and/or ¹⁴ C. In certain

embodiments, the compound is deuterated in at least one position. Such deuterated forms can be made by the procedure described in, for example, U.S. Patent Nos. 5,846,514 and 6,334,997. As described in therein, deuteration can improve the metabolic stability and or efficacy, thus increasing the duration of action of drugs.

[0051] Unless otherwise stated, compounds described herein are intended to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by ¹³ C- or ¹⁴ C-enriched carbon are within the scope of the present disclosure.

[0052] The compounds of the present disclosure optionally contain unnatural proportions of atomic isotopes at one or more atoms that constitute such compounds. For example, the compounds may be labeled with isotopes, such as for example, deuterium ( ² H), tritium ( ³ H), iodine-125 ( ¹²⁵ I) or carbon-14 ( ¹⁴ C). Isotopic substitution with ² H, ^U C, ¹³ C, ¹⁴ C, ¹⁵ C, ¹² N, ¹³ N, ¹⁵ N, ¹⁶ N, ¹⁶ 0, ¹⁷ 0, ¹⁴ F, ¹⁵ F, ¹⁶ F, ¹⁷ F, ¹⁸ F, ³³ S, ³⁴ S, ³⁵ S, ³⁶ S, ³⁵ C1, ³⁷ C1, ⁷⁹ Br, ⁸¹ Br, and ¹²⁵ I are all contemplated. All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0053] In certain embodiments, the compounds disclosed herein have some or all of the 1H atoms replaced with ² H atoms. The methods of synthesis for deuterium-containing compounds are known in the art and include, by way of non-limiting example only, the following synthetic methods.

[0054] Deuterium substituted compounds are synthesized using various methods such as described in: Dean, Dennis C; Editor. Recent Advances in the Synthesis and Applications of Radiolabeled Compounds for Drug Discovery and Development. [In: Curr., Pharm. Des., 2000;

6(10)] 2000, 110 pp; George W.; Varma, Rajender S. The Synthesis of Radiolabeled Compounds via Organometallic Intermediates, Tetrahedron, 1989, 45(21), 6601-21; and Evans, E. Anthony. Synthesis of radiolabeled compounds, J. Radioanal. Chem., 1981, 64(1-2), 9-32.

[0055] Deuterated starting materials are readily available and are subjected to the synthetic methods described herein to provide for the synthesis of deuterium-containing compounds.

Large numbers of deuterium-containing reagents and building blocks are available commercially from chemical vendors, such as Aldrich Chemical Co.

[0056] Compounds of the present invention also include crystalline and amorphous forms of those compounds, pharmaceutically acceptable salts, and active metabolites of these compounds having the same type of activity, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms of the compounds, as well as mixtures thereof.

[0057] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0058] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0059] The phrase "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen- firee water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

Antibody Constructs

[0060] Disclosed herein are antibody constructs that may be used together with compounds of the disclosure. In certain embodiments, compounds of the disclosure are linked (e.g., conjugated) either directly or through a linker to a compound of the disclosure forming conjugates. In certain embodiments, conjugates are represented by:

A-(-L ³ — D ) _n wherein A is an antibody construct; L ³ is a linker; D is a compound or salt of any one of

Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), or (IIIc); and n is from 1 to 20. In certain embodiments, n is from 1 to 10, such as from 1 to 9, such as from 1 to 8, such as from 2 to 8, such as from 1 to 6, such as from 3 to 5, or such as from 1 to 3. In certain embodiments, n is 4. In certain embodiments, n is 1 to 50 or 1 to 100. In certain embodiments, conjugates may be refered to as antibody-compound conjugates.

[0061] In certain embodiments, a compound or salt of the disclosure, e.g., a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), or (IIIc), may be referred to herein as a drug, D, a heterocyclic compound, or a payload, particularly when referenced as part of a conjugate. "LP", "linker-payload", "D-L ³ ", or "compound-linker" may be used herein to refer to a compound or salt of the disclosure bound to a linker.

[0062] An antibody construct may contain, for example, two, three, four, five, six, seven, eight, nine, ten, or more antigen binding domains. An antibody construct may contain two antigen binding domains in which each antigen binding domain can recognize the same antigen. An antibody construct may contain two antigen binding domains in which each antigen binding domain can recognize different antigens. An antigen binding domain may be in a scaffold, in which a scaffold is a supporting framework for the antigen binding domain. An antigen binding domain may be in a non-antibody scaffold. An antigen binding domain may be in an antibody scaffold. An antibody construct may include an antigen binding domain in a scaffold. The antibody construct may include an Fc fusion protein. In some embodiments, the antibody construct is an Fc fusion protein. An antigen binding domain may specifically bind to a tumor antigen. An antigen binding domain may specifically bind to an antigen that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% homologous to a tumor antigen. An antigen binding domain may specifically bind to an antigen on an antigen presenting cell (APC). An antigen binding domain may specifically bind to an antigen that is at least 80%, at least 90%, at least 95%), at least 99%, or 100% homologous to an antigen on an immune cell, such as an antigen presenting cell (APC).

[0063] An antigen binding domain of an antibody may include one or more light chain (LC) CDRs and one or more heavy chain (HC) CDRs. For example, an antibody binding domain of an antibody may include one or more of the following: a light chain complementary determining region 1 (LC CDRl), a light chain complementary determining region 2 (LC CDR2), or a light chain complementary determining region 3 (LC CDR3). For another example, an antibody binding domain may include one or more of the following: a heavy chain complementary determining region 1 (HC CDRl), a heavy chain complementary determining region 2 (HC CDR2), or a heavy chain complementary determining region 3 (HC CDR3). As an additional example, an antibody binding domain of an antibody may include one or more of the following: LC CDRl, LC CDR2, LC CDR3, HC CDRl, HC CDR2, and HC CDR3. In some embodiments, an antibody binding domain of an antibody includes LC CDRl, LC CDR2, LC CDR3, HC CDRl, HC CDR2, and HC CDR3.

[0064] The antigen binding domain of an antibody construct may be selected from any domain that binds the antigen including, but not limited to, from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, or an antigen binding fragment thereof, for example, a heavy chain variable domain (V _H ) and a light chain variable domain (V _L ), a DARPin, an affimer, an avimer, a knottin, a monobody, an affinity clamp, an ectodomain, a receptor ectodomain, a receptor, a T cell receptor, or a recombinant T cell receptor.

[0065] The antigen binding domain of an antibody construct may be at least 80% homologous to an antigen binding domain selected from, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, or a functional fragment thereof, for example, a heavy chain variable domain (V _H ) and a light chain variable domain (V _L ), a DARPin, an affimer, an avimer, a knottin, a monobody, an affinity clamp, an ectodomain, a receptor ectodomain, a receptor, a cytokine, a ligand, an immunocytokine, a T cell receptor, or a recombinant T cell receptor.

[0066] In certain embodiments, an antibody construct comprises an Fc domain that may further comprise an Fc region, in which the Fc domain may be the part of an Fc region that interacts with Fc receptors. The Fc domain of an antibody construct may interact with Fc- receptors (FcRs) found on immune cells. The Fc domain may also mediate the interaction between effector molecules and cells, which can lead to activation of the immune system. The Fc domain may be derived from IgG, IgA, or IgD antibody isotypes, and may comprise two identical protein fragments, which are derived from the second and third constant domains of the antibody's heavy chains. In an Fc domain derived from an IgG antibody isotype, the Fc domain may comprise a highly-conserved N-glycosylation site, which may be essential for FcR- mediated downstream effects. The Fc domain may be derived from IgM or IgE antibody isotypes, in which the Fc region may comprise three heavy chain constant domains. In some embodiments, the Fc domain is an Fc null (i.e., exhibiting reduced or substantially no specific binding to Fey receptors).

[0067] An Fc domain may interact with different types of FcRs. The different types of FcRs may include, for example, FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, FcyRIIIB, FcaRI, FcμR, FcsRI, FcsRII, and FcRn. FcRs may be located on the membrane of certain immune cells including, for example, B lymphocytes, natural killer cells, macrophages, monocytes, neutrophils, follicular dendritic cells, eosinophils, basophils, platelets, and mast cells. Once the FcR is engaged by the Fc domain, the FcR may initiate functions including, for example, clearance of an antigen- antibody complex via receptor-mediated endocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody dependent cell-mediated phagocytosis (ADCP), and ligand- triggered transmission of signals across the plasma membrane that can result in alterations in secretion, exocytosis, and cellular metabolism. FcRs may deliver signals when FcRs are aggregated by antibodies and multivalent antigens at the cell surface. The aggregation of FcRs with immunoreceptor tyrosine-based activation motifs (ITAMs) may sequentially activate SRC family tyrosine kinases and SYK family tyrosine kinases. IT AM comprises a twice-repeated YxxL sequence flanking seven variable residues. The SRC and SYK kinases may connect the transduced signals with common activation pathways.

[0068] An antibody may consist of two identical light protein chains and two identical heavy protein chains, all held together covalently by disulfide linkages. The N-terminal regions of the light and heavy chains together may form the antigen recognition site of an antibody.

Structurally, various functions of an antibody may be confined to discrete protein domains. The sites that can recognize and can bind antigen may consist of three complementarities determining regions (CDRs) that may lie within the variable heavy chain region and variable light chain region at the N-terminal end of the heavy chain and the light chain. The constant domains may provide the general framework of the antibody and may not be involved directly in binding the antibody to an antigen, but may be involved in various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity, and may bind Fc receptors. The constant domains may be in an Fc region. The constant domains may include an Fc domain. The variable regions of natural light and heavy chains may have the same general structures, and each domain may comprise four framework regions, whose sequences can be somewhat conserved, connected by three hyper-variable regions or CDRs. The four framework regions (FR) may largely adopt a β-sheet conformation and the CDRs can form loops connecting, and in some aspects forming part of, the β-sheet structure. The CDRs in each chain may be held in close proximity by the framework regions and, with the CDRs from the other chain, may contribute to the formation of the antigen binding site.

[0069] An antibody construct may comprise a light chain of an amino acid sequence having at least one, two, three, four, five, six, seven, eight, nine or ten modifications and in certain embodiments, not more than 40, 35, 30, 25, 20, 15 or 10 modifications of the amino acid sequence relative to the natural or original amino acid sequence. An antibody construct may comprise a heavy chain of an amino acid sequence having at least one, two, three, four, five, six, seven, eight, nine or ten modifications and in certain embodiments, not more than 40, 35, 30, 25, 20, 15 or 10 modifications of the amino acid sequence relative to the natural or original amino acid sequence.

[0070] An antibody of an antibody construct may include an antibody of any type, which may be assigned to different classes of immunoglobins, e.g., IgA, IgD, IgE, IgG, and IgM. Several different classes may be further divided into isotypes, e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. An antibody may further comprise a light chain and a heavy chain, often more than one chain. The heavy-chain constant regions (Fc) that corresponds to the different classes of immunoglobulins may be α, δ, ε, γ, and μ, respectively. The light chains may be one of either kappa (κ) or lambda (λ), based on the amino acid sequences of the constant domains. The Fc domain may further comprise an Fc region. An Fc receptor may bind an Fc domain. Antibody constructs may also include any fragment or recombinant forms thereof, including but not limited to, single chain variable fragments (scFvs), 'T-bodies', anti-calins, centyrins, affibodies, domain antibodies, or peptibodies.

[0071] An antibody construct may comprise an antibody fragment. An antibody fragment may include (i) a Fab fragment, a monovalent fragment consisting of the V _L , V _H, C _L and C _HI domains; (ii) a F(ab') ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a Fv fragment consisting of the V _L and V _H domains of a single arm of an antibody. Although the two domains of the Fv fragment, V _L and V _H , may be coded for by separate genes, they may be linked by a synthetic linker to be made as a single protein chain in which the V _L and V _H regions pair to form monovalent molecules.

[0072] F(ab') ₂ and Fab' moieties may be recombinantly produced or produced by treating immunoglobulin (e.g., monoclonal antibody) with a protease such as pepsin and papain, and may include an antibody fragment generated by digesting immunoglobulin near the disulfide bonds existing between the hinge regions in each of the two H chains. The Fab fragment may also contain the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments may differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain C _H i domain including one or more cysteine(s) from the antibody hinge region.

[0073] An Fv may be the minimum antibody fragment which contains a complete antigen- recognition and antigen-binding site. This region may consist of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. In this configuration, the CDRs of each variable domain may interact to define an antigen-binding site on the surface of the VH-VL dimer. A single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) may recognize and bind antigen, although the binding can be at a lower affinity than the affinity of the entire binding site.

[0074] An antibody may include an Fc domain comprising an Fc region. The Fc domain of an antibody may interact with FcRs found on immune cells. The Fc domain may also mediate the interaction between effector molecules and cells, which may lead to activation of the immune system. In the IgG, IgA, and IgD antibody isotypes, the Fc domain or region may comprise two identical protein fragments, which can be derived from the second and third constant domains of the antibody's heavy chains. In the IgM and IgE antibody isotypes, the Fc regions may comprise three heavy chain constant domains. In the IgG antibody isotype, the Fc regions may comprise a highly-conserved N-glycosylation site, which may be important for FcR-mediated downstream effects.

[0075] An antibody used herein may be "chimeric" or "humanized." Humanized forms of non-human (e.g., murine) antibodies can be chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂ or other target-binding subdomains of antibodies), which may contain minimal sequences derived from non-human immunoglobulin. In general, the humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence.

[0076] An antibody may be a human antibody. As used herein, "human antibodies" can include antibodies having, for example, the amino acid sequence of a human immunoglobulin and may include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins that do not express endogenous immunoglobulins. Human antibodies may be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which may express human immunoglobulin genes. Completely human antibodies that recognize a selected epitope may be generated using guided selection. In this approach, a selected non-human monoclonal antibody, e.g., a mouse antibody, may be used to guide the selection of a completely human antibody recognizing the same epitope.

[0077] An antibody may be a bispecific antibody or a dual variable domain antibody (DVD). Bispecific and DVD antibodies may be monoclonal, often human or humanized, antibodies that can have binding specificities for at least two different antigens.

[0078] An antibody may be a derivatized antibody. For example, derivatized antibodies may be modified by glycosylation, deglycosylation, defucosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein.

[0079] In certain embodiments, a targeting moiety or antibody construct comprises an Fc domain or an Fc region. The Fc domain or Fc region may interact with one or more Fc receptors (FcRs). An Fc domain or Fc region of an antibody construct may interact with one or more Fc receptors. An Fc domain or Fc region may interact with Fc receptors found on immune cells. An Fc domain or Fc region may also mediate the interaction between effector molecules and cells, which can lead to activation of the immune system. An Fc domain or Fc region may be derived from IgG, IgA, or IgD antibody isotypes, and may comprise two identical protein fragments, which are derived from the second and third constant domains of the antibody's heavy chains. In an Fc domain or region derived from an IgG antibody isotype, the Fc domain or region may comprise a highly-conserved N-glycosylation site, which may be essential for FcR-mediated downstream effects. The Fc domain or region may be derived from IgM or IgE antibody isotypes, in which the Fc domain or region may comprise three heavy chain constant domains.

[0080] An Fc domain may interact with different types of FcRs. The different types of FcRs may include, for example, FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, FcyRIIIB, FcaRI, FcμR, FcsRI, FcsRII, and FcRn. FcRs may be located on the membrane of certain immune cells including, for example, B lymphocytes, natural killer cells, macrophages, neutrophils, follicular dendritic cells, eosinophils, basophils, platelets, and mast cells. Once the FcR is engaged by the Fc domain, the FcR may initiate functions including, for example, clearance of an antigen-antibody complex via receptor-mediated endocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody dependent cell-mediated phagocytosis (ADCP), and ligand-triggered transmission of signals across the plasma membrane that can result in alterations in secretion, exocytosis, and cellular metabolism. FcRs may deliver signals when FcRs are aggregated by antibodies and multivalent antigens at the cell surface. The aggregation of FcRs with immunoreceptor tyrosine- based activation motifs (ITAMs) may sequentially activate SRC family tyrosine kinases and SYK family tyrosine kinases. IT AM comprises a twice-repeated YxxL sequence flanking seven variable residues. The SRC and SYK kinases may connect the transduced signals with common activation pathways.

[0081] In some embodiments, an Fc domain or region of the antibody construct of a conjugate can exhibit increased binding affinity to one or more Fc receptors. In some embodiments, an Fc domain or region can exhibit increased binding affinity to one or more Fcgamma receptors. In some embodiments, an Fc domain or region can exhibit increased binding affinity to FcRn receptors. In some embodiments, an Fc domain or region can exhibit increased binding affinity to Fcgamma and FcRn receptors.

[0082] In some embodiments, an Fc domain or region of the antibody construct of a conjugate can exhibit reduced binding affinity to one or more Fc receptors. In some embodiments, an Fc domain or region can exhibit reduced binding affinity to one or more Fcgamma receptors. In some embodiments, an Fc domain or region can exhibit reduced binding affinity to FcRn receptors. In some embodiments, an Fc domain or region can exhibit reduced binding affinity to Fcgamma and FcRn receptors. In some embodiments, an Fc domain is an Fc null domain or region. In some embodiments, an Fc domain or region can exhibit reduced binding affinity to FcRn receptors, but have the same or increased binding affinity to one or more Fcgamma receptors as compared to a wildtype IgG. In some embodiments, an Fc domain or region can exhibit increased binding affinity to FcRn receptors, but have the same or decreased binding affinity to one or more Fcgamma receptors.

[083] The Fc domain may have one or more, two or more, three or more, or four or more amino acid substitutions that decrease binding of the Fc domain to an Fc receptor. In certain embodiments, an Fc domain has decreased binding affinity for one or more of FcyRI (CD64), FcyRIIA (CD32), FcyRIIIA (CD 16a), FcyRIIIB (CD 16b), or any combination thereof. In order to decrease binding affinity of an Fc domain or region to an Fc receptor, the Fc domain or region may comprise one or more amino acid substitutions that reduces the binding affinity of the Fc domain or region to an Fc receptor.

[084] In certain embodiments, the one or more substitutions comprise any one or more of IgGl heavy chain mutations corresponding to E233P, L234V, L234A, L235A, L235E, AG236, G237A, E318A, K320A, K322A, A327G, A330S, or P331 S according to the EU index of Kabat numbering.

[0085] In some embodiments, the Fc domain or region can comprise a sequence of an IgG isoform that has been modified from the wild-type IgG sequence. In some embodiments, the Fc domain or region can comprise a sequence of the IgGl isoform that has been modified from the wild-type IgGl sequence. In some embodiments, the modification comprises substitution of one or more amino acids that reduce binding affinity of an IgG Fc domain or region to all Fey receptors. A modification can be substitution of E233, L234 and L235, such as

E233P/L234V/L235A or E233P/L234V/L235A/AG236, according to the EU index of Kabat. A modification can be a substitution of P238, such as P238A, according to the EU index of Kabat. A modification can be a substitution of D265, such as D265A, according to the EU index of Kabat. A modification can be a substitution of N297, such as N297A, according to the EU index of Kabat. A modification can be a substitution of A327, such as A327Q, according to the EU index of Kabat. A modification can be a substitution of P329, such as P239A, according to the EU index of Kabat.

[0086] In some embodiments, an IgG Fc domain or region comprises at least one amino acid substitution that reduces its binding affinity to FcyRl, as compared to a wild-type or reference IgG Fc domain. A modification can comprise a substitution at F241, such as F241 A, according to the EU index of Kabat. A modification can comprise a substitution at F243, such as F243A, according to the EU index of Kabat. A modification can comprise a substitution at V264, such as V264A, according to the EU index of Kabat. A modification can comprise a substitution at D265, such as D265A according to the EU index of Kabat.

[0087] In some embodiments, an IgG Fc domain or region comprises at least one amino acid substitution that increases its binding affinity to FcyRl, as compared to a wild-type or reference IgG Fc domain. A modification can comprise a substitution at A327 and P329, such as

A327Q/P329A, according to the EU index of Kabat.

[0088] In some embodiments, the modification comprises substitution of one or more amino acids that reduce binding affinity of an IgG Fc domain or region to FcyRII and FcyRIIIA receptors. A modification can be a substitution of D270, such as D270A, according to the EU index of Kabat. A modification can be a substitution of Q295, such as Q295A, according to the EU index of Kabat. A modification can be a substitution of A327, such as A237S, according to the EU index of Kabat.

[0089] In some embodiments, the modification comprises substitution of one or more amino acids that increases binding affinity of an IgG Fc domain or region to FcyRII and FcyRIIIA receptors. A modification can be a substitution of T256, such as T256A, according to the EU index of Kabat. A modification can be a substitution of K290, such as K290A, according to the EU index of Kabat.

[0090] In some embodiments, the modification comprises substitution of one or more amino acids that increases binding affinity of an IgG Fc domain or region to FcyRII receptor. A modification can be a substitution of R255, such as R255A, according to the EU index of Kabat.

A modification can be a substitution of E258, such as E258A, according to the EU index of

Kabat. A modification can be a substitution of S267, such as S267A, according to the EU index of Kabat. A modification can be a substitution of E272, such as E272A, according to the EU index of Kabat. A modification can be a substitution of N276, such as N276A, according to the

EU index of Kabat. A modification can be a substitution of D280, such as D280A, according to the EU index of Kabat. A modification can be a substitution of H285, such as H285A, according to the EU index of Kabat. A modification can be a substitution of N286, such as N286A, according to the EU index of Kabat. A modification can be a substitution of T307, such as

T307A, according to the EU index of Kabat. A modification can be a substitution of L309, such as L309A, according to the EU index of Kabat. A modification can be a substitution of N315, such as N315 A, according to the EU index of Kabat. A modification can be a substitution of

K326, such as K326A, according to the EU index of Kabat. A modification can be a substitution of P331, such as P331 A, according to the EU index of Kabat. A modification can be a substitution of S337, such as S337A, according to the EU index of Kabat. A modification can be a substitution of A378, such as A378A, according to the EU index of Kabat. A modification can be a substitution of E430, such as E430, according to the EU index of Kabat.

[0091] In some embodiments, the modification comprises substitution of one or more amino acids that increases binding affinity of an IgG Fc domain or region to FcyRII receptor and reduces the binding affinity to FcyRIIIA receptor. A modification can be a substitution of H268, such as H268A, according to the EU index of Kabat. A modification can be a substitution of

R301, such as R301 A, according to the EU index of Kabat. A modification can be a substitution of K322, such as K322A, according to the EU index of Kabat.

[0092] In some embodiments, the modification comprises substitution of one or more amino acids that decreases binding affinity of an IgG Fc domain or region to FcyRII receptor but does not affect the binding affinity to FcyRIIIA receptor. A modification can be a substitution of R292, such as R292A, according to the EU index of Kabat. A modification can be a substitution of K414, such as K414A, according to the EU index of Kabat.

[0093] In some embodiments, the modification comprises substitution of one or more amino acids that decreases binding affinity of an IgG Fc domain or region to FcyRII receptor and increases the binding affinity to FcyRIIIA receptor. A modification can be a substitution of S298, such as S298A, according to the EU index of Kabat. A modification can be substitution of S239, 1332 and A330, such as S239D/I332E/A330L. A modification can be substitution of S239 and 1332, such as S239D/I332E. [0094] In some embodiments, the modification comprises substitution of one or more amino acids that decreases binding affinity of an IgG Fc domain or region to FcyRIIIA receptor. A modification can be substitution of F241 and F243, such as F241 S/F243S or F241I/F243I, according to the EU index of Kabat.

[0095] In some embodiments, the modification comprises substitution of one or more amino acids that decreases binding affinity of an IgG Fc domain or region to FcyRIIIA receptor and does not affect the binding affinity to FcyRII receptor. A modification can be a substitution of S239, such as S239A, according to the EU index of Kabat. A modification can be a substitution of E269, such as E269A, according to the EU index of Kabat. A modification can be a substitution of E293, such as E293A, according to the EU index of Kabat. A modification can be a substitution of Y296, such as Y296F, according to the EU index of Kabat. A modification can be a substitution of V303, such as V303A, according to the EU index of Kabat. A modification can be a substitution of A327, such as A327G, according to the EU index of Kabat. A modification can be a substitution of K338, such as K338A, according to the EU index of Kabat. A modification can be a substitution of D376, such as D376A, according to the EU index of Kabat.

[0096] In some embodiments, the modification comprises substitution of one or more amino acids that increases binding affinity of an IgG Fc domain or region to FcyRIIIA receptor and does not affect the binding affinity to FcyRII receptor. A modification can be a substitution of E333, such as E333A, according to the EU index of Kabat. A modification can be a substitution of K334, such as K334A, according to the EU index of Kabat. A modification can be a substitution of A339, such as A339T, according to the EU index of Kabat. A modification can be substitution of S239 and 1332, such as S239D/I332E.

[0097] In some embodiments, the modification comprises substitution of one or more amino acids that increases binding affinity of an IgG Fc domain or region to FcyRIIIA receptor. A modification can be substitution of L235, F243, R292, Y300 and P396, such as

L235V/F243L/R292P/Y300L/P396L (IgGl VLPLL) according to the EU index of Kabat. A modification can be substitution of S298, E333 and K334, such as S298A/E333 A/K334A, according to the EU index of Kabat. A modification can be substitution of K246, such as K246F, according to the EU index of Kabat.

[0098] Other substitutions in an IgG Fc domain that affect its interaction with one or more Fey receptors are disclosed in U.S. Patent Nos. 7,317,091 and 8,969,526 (the disclosures of which are incorporated by reference herein).

[0099] In some embodiments, an IgG Fc domain or region comprises at least one amino acid substitution that reduces the binding affinity to FcRn, as compared to a wild-type or reference IgG Fc domain. A modification can comprise a substitution at H435, such as H435A according to the EU index of Kabat. A modification can comprise a substitution at 1253, such as I253A according to the EU index of Kabat. A modification can comprise a substitution at H310, such as H310A according to the EU index of Kabat. A modification can comprise substitutions at

1253, H310 and H435, such as I253A/H310A/H435A according to the EU index of Kabat.

[0100] A modification can comprise a substitution of one amino acid residue that increases the binding affinity of an IgG Fc domain for FcRn, relative to a wildtype or reference IgG Fc domain. A modification can comprise a substitution at V308, such as V308P according to the

EU index of Kabat. A modification can comprise a substitution at M428, such as M428L according to the EU index of Kabat. A modification can comprise a substitution at N434, such as N434A according to the EU index of Kabat or N434H according to the EU index of Kabat. A modification can comprise substitutions at T250 and M428, such as T250Q and M428L according to the EU index of Kabat. A modification can comprise substitutions at M428 and

N434, such as M428L and N434S, N434A or N434H according to the EU index of Kabat. A modification can comprise substitutions at M252, S254 and T256, such as M252Y/S254T/T256E according to the EU index of Kabat. A modification can be a substitution of one or more amino acids selected from P257L, P257N, P257I, V279E, V279Q, V279Y, A281 S, E283F, V284E,

L306Y, T307V, V308F, Q31 IV, D376V, and N434H. Other substitutions in an IgG Fc domain that affect its interaction with FcRn are disclosed in U.S. Patent No. 9,803,023 (the disclosure of which is incorporated by reference herein).

[0101] In certain embodiments, the antibody construct comprises an antigen binding domain and an IgG Fc domain, wherein a K _d for binding of the antigen binding domain to an antigen in a presence of a heterocyclic compound is less than about 100 nM and no greater than about 100 times a K _d for binding of the antigen binding domain to the antigen in the absence of

heterocyclic compound. In certain embodiments, the antibody construct comprises a K _d for binding of the IgG Fc domain to an Fc receptor in the presence of the heterocyclic compound is no greater than about 100 times a K _d for binding the IgG Fc domain to the Fc receptor in the absence of the heterocyclic compound. In certain embodiments, the antigen is selected from CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC (PD-L2), HLD-DR, carcinoembryonic antigen (CEA), TAG-72, EpCAM, MUCl, folate-binding protein, A33, G250, prostate-specific membrane antigen (PSMA), ferritin, GD2, GD3, GM2, Ley, CA-125, CA19-9 (MUCl sLe(a)), epidermal growth factor, pl85HER2, IL-2 receptor, EGFRvIII (de2-7 EGFR), fibroblast activation protein, tenascin, a

metalloproteinase, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, UPV E6, HPV E7, Her-2/neu, MAGE A3, p53 nonmutant, NY-ESO-1, Mel an A/MART 1 , Ras mutant, gplOO, p53 mutant, PR1, bcr-abl, tyronsinase, survivin, PSA, hTERT, a Sarcoma translocation breakpoint protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B 1, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GMl, mesothelin (MSLN), PSCA, MAGE Al, sLe(a), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TESL Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, Legumain, Tie 3, Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAIL 1, MUCl 6, MAGE A4, MAGE C2, GAGE, EGFR, CMET, HER3, MUCl, MUCl 5, CA6, NAPI2B, TROP2, CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, or Fos-related antigen 1. In certain embodiments, the antigen is expressed on an immune cell. In certain embodiments, the antigen is CEA. In certain embodiments, the antigen binding domain specifically binds to CEA.

[0102] In certain embodiments, an antigen binding domain specifically binds to an antigen, such as those selected from CD5, CD25, CD37, CD33, CD45, BCMA, CS-1, PD-L1, B7-H3, B7-DC (PD-L2), HLD-DR, carcinoembryonic antigen (CEA), TAG-72, EpCAM, MUCl, folate- binding protein (FOLR1), A33, G250 (carbonic anhydrase IX), prostate-specific membrane antigen (PSMA), GD2, GD3, GM2, Ley, CA-125, CA19-9 (MUCl sLe(a)), epidermal growth factor, HER2, IL-2 receptor, EGFRvIII (de2-7 EGFR), fibroblast activation protein (FAP), a tenascin, a metalloproteinase, endosialin, avB3, LMP2, EphA2, PAP, AFP, ALK, polysialic acid, TRP-2, fucosyl GMl, mesothelin (MSLN), PSCA, sLe(a), GM3, BORIS, Tn, TF, GloboH, STn, CSPG4, AKAP-4, SSX2, Legumain, Tie 2, Tim 3, VEGFR2, PDGFR-B, ROR2, TRAIL 1, MUCl 6, EGFR, CMET, HER3, MUCl, MUCl 5, CA6, NAPI2B, TROP2, CLDN18.2, RON, LY6E, FRAlpha, DLL3, PTK7, LIV1, ROR1, CLDN6, GPC3, ADAM 12, LRRC15, CDH6, TMEFF2, TMEM238, GPNMB, ALPPL2, UPK1B, UPK2, LAMP-1, LY6K, EphB2, STEAP, ENPP3, CDH3, Nectin4, LYPD3, EFNA4, GPA33, SLITRK6 or HAVCR1. In certain embodiments, the antigen is LRRC15. In certain embodiments, the antigen binding domain specifically binds to LRRC15.

[0103] In certain embodiments, an antigen binding domain specifically binds to a non- proteinaceous or glycoantigen, such as GD2, GD3, GM2, Ley, polysialic acid, fucosyl GMl, GM3, Tn, STn, sLe(animal), or GloboH.

[0104] In certain embodiments, the antigen is selected from MUCl 6, UPK1B, VTCN1, TMPRSS3, TMEM238, Clorfl86, TMPRSS4, CLDN6, CLDN8 and STRA6. In certain embodiments, the antigen is selected from MUCl 6, UPK1B, VTCN1, TMPRSS3, TMEM238, Clorfl86 and TMPRSS4. In certain embodiments, the antigen is MUC16. In certain

embodiments, the antigen is UPKIB. In certain embodiments, the antigen is VTCN1. In certain embodiments, the antigen is TMPRSS3. In certain embodiments, the antigen is TMEM238. In certain embodiments, the antigen is Clorfl86. In certain embodiments, the antigen is

TMPRSS4. In certain embodiments, the antigen is CLDN6. In certain embodiments, the antigen is CLDN8. In certain embodiments, the antigen is STRA6.

[0105] In certain embodiments, the antigen is selected from ACSL5, AP1M2, AREG, CDH1, CDH17, CEACAM5, CEACAM6, CEACAM7, CLCA1, CLDN3, DPEP1, ERBB3, GPA33, GPRC5A, ITGA6, KRTCAP3, LSR, MUC13, NOX1, PLOD3, PLPP2, SLC12A2, SLC44A4, SLC52A2, SMIM22, ST14, TFRC, TMPRSS4, sLE(x) and TSPAN6. In certain embodiments, the antigen is selected from CLDN4, CLDN7, EPCAM, PIGR, TMEM141, TMEM54, TSPAN1 LRG5, and TSPAN8. In certain embodiments, the antigen is selected from ACSL5, AP1M2, AREG, CDH1, CDH17, CEACAM5, CEACAM6, CEACAM7, CLCA1, CLDN3, DPEP1, ERBB3, GPA33, GPRC5A, ITGA6, KRTCAP3, LSR, MUC13, NOX1, PLOD3, PLPP2, SLC12A2, SLC44A4, SLC52A2, SMIM22, ST14, TFRC, TMPRSS4, sLE(x), TSPAN6, CLDN4, CLDN7, EPCAM, PIGR, TMEM141, TMEM54, TSPAN1, LRG5 and TSPAN8. In certain embodiments, the antigen is any one of ACSL5, AP1M2, AREG, CDH1, CDH17, CEACAM5, CEACAM6, CEACAM7, CLCA1, CLDN3, DPEP1, ERBB3, GPA33, GPRC5A, ITGA6, KRTCAP3, LSR, MUC13, NOX1, PLOD 3, PLPP2, SLC12A2, SLC44A4, SLC52A2, SMIM22, ST 14, TFRC, TMPRSS4, sLE(x), TSPAN6, CLDN4, CLDN7, EPCAM, PIGR, TMEM141, TMEM54, TSPAN1, LRG5 and TSPAN8.

[0106] An antibody construct may comprise an antibody with modifications of at least one amino acid residue. Modifications may be substitutions, additions, mutations, deletions, or the like. An antibody modification can be an insertion of an unnatural amino acid.

[0107] In certain embodiments, the antibody construct comprises a HER2 antibody, e.g., pertuzumab, trastuzumab, or an antigen binding portion thereof. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of pertuzumab (SEQ ID NOs: 1 and 2). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of pertuzumab (SEQ ID NO:2), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of pertuzumab (SEQ ID NO: 1), as determined by the Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of pertuzumab (SEQ ID NO:2), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of pertuzumab (SEQ ID NO: 1), as determined by FMGT. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of trastuzumab (SEQ ID NOs:3 and 4). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of trastuzumab (SEQ ID NO:4), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of trastuzumab (SEQ ID NO:3), as determined by the Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of trastuzumab (SEQ ID NO:4), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of trastuzumab (SEQ ID NO:3), as determined by EVIGT.

[0108] In certain embodiments, the antibody construct comprises a MUC16 antibody, e.g., sofituzumab, 4H11 (US2013/0171152), 4H5 (US2013/0171152), MORab-009, RG7787 or an antigen binding portion thereof. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of sofituzumab (SEQ ID NOs:9 and 10). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of sofituzumab (SEQ ID NO: 10), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of sofituzumab (SEQ ID NO: 9), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of sofituzumab (SEQ ID NO: 10), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of sofituzumab (SEQ ID NO:9), as determined by EVIGT. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of antibody 4H11 (SEQ ID NOs:5 and 6). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of antibody 4H11 (SEQ ID NO:6), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody 4H11 (SEQ ID NO:5), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of antibody 4H11 (SEQ ID NO:6), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody 4H11 (SEQ ID NO:5), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of antibody 4H11 (SEQ ID NO:6), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of 4H11 (SEQ ID NO:5), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of antibody 4H11 (SEQ ID NO:5), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of 4H11 (SEQ ID NO:6), as determined by EVIGT. In certain

embodiments, the antibody construct comprises the heavy and light chain variable region sequences of antibody 4A5 (SEQ ID NOs:7 and 8). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of antibody 4A5 (SEQ ID NO: 8), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of 4A5 (SEQ ID NO:7), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or an antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of antibody 4A5 (SEQ ID NO: 8), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody 4A5 (SEQ ID NO: 7), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of antibody 4A5 (SEQ ID NO:8), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody 4A5 (SEQ ID NO: 7), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of 4A5 (SEQ ID NO: 8), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of 4A5 (SEQ ID NO: 7), as determined by EVIGT. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of antibody MORab-009 (SEQ ID NOs: 11 and 12). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of antibody MORab-009 (SEQ ID NO: 12), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody 4H11 (SEQ ID NO: 11), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of antibody MORab-009 (SEQ ID NO: 12), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody MORab-009 (SEQ ID NO: 11), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of antibody MORab-009 (SEQ ID NO: 12), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of MORab-009 (SEQ ID NO: 11), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of antibody MORab-009 (SEQ ID NO: 12), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of MORab-009 (SEQ ID NO: 11), as determined by EVIGT. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of antibody RG7787 (SEQ ID NOs: 13 and 14). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of antibody RG7787 (SEQ ID NO: 14), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody RG7787 (SEQ ID NO: 13), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of antibody RG7787 (SEQ ID NO: 14), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody RG7787 (SEQ ID NO: 13), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of antibody RG7787 (SEQ ID NO: 14), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of RG7787 (SEQ ID NO: 13), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of antibody RG7787 (SEQ ID NO: 14), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of RG7787 (SEQ ID NO: 13), as determined by EVIGT.

[0109] In certain embodiments, the antibody construct comprises a PD-L1 antibody, e.g., atezolizumab, MDX-1105 (WO 2007/005874) or an antigen binding portion thereof. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of atezolizumab (SEQ ID NOs: 15 and 16). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of atezolizumab (SEQ ID NO: 16), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of atezolizumab (SEQ ID NO: 15), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of atezolizumab (SEQ ID NO: 16), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of atezolizumab (SEQ ID NO: 15), as determined by EVIGT. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of MDX-1105 (SEQ ID NOs: 17 and 18). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of MDX-1105 (SEQ ID NO: 18), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of MDX-1105 (SEQ ID NO: 17), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of MDX-1105 (SEQ ID NO: 18), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of MDX-1105 (SEQ ID NO: 17), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of MDX-1105 (SEQ ID NO: 18), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of MDX-1105 (SEQ ID NO: 17), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of MDX-1105 (SEQ ID NO: 18), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of MDX-1105 (SEQ ID NO: 17), as determined by IMGT. [0110] In certain embodiments, the antibody construct comprises an antibody to FAP, e.g., an antibody to FAP variant 1, antibody to FAP variant 2, antibody to FAP variant 3, antibody to FAP variant 4, antibody to FAP variant 5, antibody to FAP variant 6, antibody to FAP variant 7, sibrotuzumab, or an antigen binding portion thereof. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of an antibody to FAP variant 1 (SEQ ID NOs: 19 and 21). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 1 (SEQ ID NO:21), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 1 (SEQ ID NO: 19), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 1 (SEQ ID NO:21), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of an antibody to FAP variant 1 (SEQ ID NO: 19), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 1 (SEQ ID NO:21), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of an antibody to FAP variant 1 (SEQ ID NO: 19), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 1 (SEQ ID NO:21), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 1 (SEQ ID NO: 19), as determined by EVIGT. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of an antibody to FAP variant 1 (SEQ ID NOs:20 and 21). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 1 (SEQ ID NO:21), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 1 (SEQ ID NO:20), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 1 (SEQ ID NO:21), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of an antibody to FAP variant 1 (SEQ ID NO:20), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 1 (SEQ ID NO:21), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of FAP variant 1 (SEQ ID NO:20), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 1 (SEQ ID NO:21), and HC CDRl,

HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 1 (SEQ

ID NO:20), as determined by EVIGT.

[0111] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of an antibody to FAP variant 2 (SEQ ID NOs:22 and 23). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 2 (SEQ ID NO:23), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 2 (SEQ ID NO:22), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 2 (SEQ ID NO:23), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of an antibody to FAP variant 2 (SEQ ID NO:22), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 2 (SEQ ID NO:23), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 2 (SEQ ID NO:22), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 2 (SEQ ID NO:23), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 2 (SEQ ID NO:22), as determined by EVIGT.

[0112] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of an antibody to FAP variant 3 (SEQ ID NOs:24 and 25). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 3 (SEQ ID NO:25), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 3 (SEQ ID NO:24), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 3 (SEQ ID NO:25), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of an antibody to FAP variant 3 (SEQ ID NO:24), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 3 (SEQ ID NO:25), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 3 (SEQ ID NO:24), as determined by IMGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 3 (SEQ ID NO:25), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 3 (SEQ ID NO:24), as determined by IMGT.

[0113] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of an antibody to FAP variant 4 (SEQ ID NOs:26 and 27). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 4 (SEQ ID NO:27), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 4 (SEQ ID NO:26), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 4 (SEQ ID NO:27), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of an antibody to FAP variant 4 (SEQ ID NO:26), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 4 (SEQ ID NO:27), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 4 (SEQ ID NO:26), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 4 (SEQ ID NO:27), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 4 (SEQ ID NO:26), as determined by EVIGT.

[0114] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of an antibody to FAP variant 5 (SEQ ID NOs:28 and 29). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 5 (SEQ ID NO:29), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 5 (SEQ ID NO:28), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 5 (SEQ ID NO:29), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of an antibody to FAP variant 5 (SEQ ID NO:28), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 5 (SEQ ID NO:29), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 5 (SEQ ID

NO:28), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and

LC CDR3 of the light chain variable region of an antibody to FAP variant 5 (SEQ ID NO:29), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 5 (SEQ ID NO:28), as determined by EVIGT.

[0115] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of an antibody to FAP variant 6 (SEQ ID NOs:30 and 31). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 6 (SEQ ID NO: 31), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 6 (SEQ ID NO:30), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 6 (SEQ ID NO: 31), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of an antibody to FAP variant 6 (SEQ ID NO:30), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 6 (SEQ ID NO: 31), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 6 (SEQ ID NO:30), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 6 (SEQ ID NO: 31), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 6 (SEQ ID NO: 30), as determined by EVIGT.

[0116] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of an antibody to FAP variant 7 (SEQ ID NOs:32 and 35). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:35), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:32), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:35), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of an antibody to FAP variant 7 (SEQ ID NO:32), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:35), and HC CDRl, HC

CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID

NO:32), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and

LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO: 35), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:32), as determined by EVIGT.

[0117] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of an antibody to FAP variant 7 (SEQ ID NOs:33 and 35). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:35), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:33), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:35), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of an antibody to FAP variant 7 (SEQ ID NO:33), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:35), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:33), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO: 35), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:33), as determined by EVIGT.

[0118] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of an antibody to FAP variant 7 (SEQ ID NOs:34 and 35). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:35), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:34), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:35), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of an antibody to FAP variant 7 (SEQ ID NO:34), as determined by Kabat index. In certain embodiments, the antibody constmct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:35), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:34), as determined by IMGT. In certain embodiments, the antibody constmct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO: 35), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:34), as determined by EVIGT.

[0119] In certain embodiments, the antibody constmct comprises the heavy and light chain variable region sequences of an antibody to FAP variant 7 (SEQ ID NOs:32 and 36). In certain embodiments, the antibody constmct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:36), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:32), as determined by Kabat index. In certain embodiments, the antibody constmct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:36), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of an antibody to FAP variant 7 (SEQ ID NO:32), as determined by Kabat index. In certain embodiments, the antibody constmct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:36), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:32), as determined by EVIGT. In certain embodiments, the antibody constmct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO: 36), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:32), as determined by EVIGT.

[0120] In certain embodiments, the antibody constmct comprises the heavy and light chain variable region sequences of an antibody to FAP variant 7 (SEQ ID NOs:33 and 36). In certain embodiments, the antibody constmct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:36), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:33), as determined by Kabat index. In certain embodiments, the antibody constmct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:36), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of an antibody to FAP variant 7 (SEQ ID NO:33), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:36), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:33), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO: 36), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:33), as determined by EVIGT.

[0121] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of an antibody to FAP variant 7 (SEQ ID NOs:34 and 36). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:36), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:34), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:36), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of an antibody to FAP variant 7 (SEQ ID NO:34), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO:36), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:34), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of an antibody to FAP variant 7 (SEQ ID NO: 36), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of antibody to FAP variant 7 (SEQ ID NO:34), as determined by EVIGT.

[0122] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of sibrotuzumab (SEQ ID NOs:69 and 70). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of sibrotuzumab (SEQ ID NO:70), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of sibrotuzumab (SEQ ID NO: 69), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of sibrotuzumab (SEQ ID NO:70), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of sibrotuzumab (SEQ ID NO: 69), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of sibrotuzumab (SEQ ID NO: 70), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of sibrotuzumab (SEQ ID NO: 69), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of sibrotuzumab (SEQ ID NO:70), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of sibrotuzumab (SEQ ID NO: 69), as determined by EVIGT.

[0123] In certain embodiments, the antibody construct comprises an antibody to LRRC15, e.g., antibody huM25 to LRRC15, antibody huAD208.4.1 to LRRC15, antibody huAD208.12.1 to LRRC15, antibody huAD208.14.1 to LRRC15, antibody hul39.10 to LRRC15, or an antigen binding portion thereof. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of huM25 (SEQ ID NOs:37 and 38). In certain

embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of huM25 (SEQ ID NO: 38), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of huM25 (SEQ ID NO:37), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of huM25 (SEQ ID NO:38), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of huM25 (SEQ ID NO:37), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of huM25 (SEQ ID NO: 38), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of huM25 (SEQ ID NO:37), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of huM25 (SEQ ID NO:38), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of huM25 (SEQ ID NO:37), as determined by EVIGT.

[0124] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of huAD208.4.1 (SEQ ID NOs:39 and 40). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of huAD208.4.1 (SEQ ID NO:40), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of huAD208.4.1 (SEQ ID NO:39), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of huAD208.4.1 (SEQ ID NO:40), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of huAD208.4.1 (SEQ ID NO:39), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of huAD208.4.1 (SEQ ID NO:40), and HC CDRl, HC CDR2 and

HC CDR3 of the heavy chain variable region of huAD208.4.1 (SEQ ID NO:39), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of huAD208.4.1 (SEQ ID NO:40), and HC CDRl, HC CDR2 and HC

CDR3 of the heavy chain variable region of huAD208.4.1 (SEQ ID NO:39), as determined by

EVIGT.

[0125] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of huAD208.12.1 (SEQ ID NOs:41 and 42). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of huAD208.12.1 (SEQ ID NO:42), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of huAD208.12.1 (SEQ ID NO:41), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of huAD208.12.1 (SEQ ID NO:42), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of huAD208.12.1 (SEQ ID NO:41), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of huAD208.12.1 (SEQ ID NO:42), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of huAD208.12.1 (SEQ ID NO:41), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of huAD208.12.1 (SEQ ID NO:42), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of huAD208.12.1 (SEQ ID NO:41), as determined by EVIGT.

[0126] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of huAD208.14.1 (SEQ ID NOs:43 and 44). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of huAD208.14.1 (SEQ ID NO:44), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of huAD208.14.1 (SEQ ID NO:43), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of huAD208.14.1 (SEQ ID NO:44), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of huAD208.142.1 (SEQ ID NO:43), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of huAD208.14.1 (SEQ ID NO:44), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of huAD208.14.1 (SEQ ID NO:43), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of huAD208.14.1 (SEQ ID NO:44), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of huAD208.14.1 (SEQ ID NO:43), as determined by EVIGT.

[0127] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of hul39.10 (SEQ ID NOs:45 and 46). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of hul39.10 (SEQ ID NO:46), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of hul39.10 (SEQ ID NO:45), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of hul39.10 (SEQ ID NO:46), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of hul39.10 (SEQ ID NO:45), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of hul39.10 (SEQ ID NO:46), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of hul39.10 (SEQ ID NO:45), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of hul39.10 (SEQ ID NO:46), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of hul39.10 (SEQ ID NO:45), as determined by EVIGT.

[0128] In certain embodiments, the antibody construct comprises an antibody to EGFR or EGFRvIII, e.g., cetuximab, panitumumab, nimotuzumab, zalutumumab, AMG595, ABT806, or an antigen binding portion thereof. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of cetuximab (SEQ ID NOs:47 and 48). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of cetuximab (SEQ ID NO:48), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of cetuximab (SEQ ID NO:47), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of cetuximab (SEQ ID NO:48), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of cetuximab (SEQ ID NO:47), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of cetuximab (SEQ ID NO:48), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of cetuximab (SEQ ID NO:47), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of cetuximab (SEQ ID NO:48), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of cetuximab (SEQ ID NO:47), as determined by EVIGT.

[0129] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of panitumumab (SEQ ID NOs:49 and 50). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of panitumumab (SEQ ID NO:50), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of panitumumab (SEQ ID NO:49), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of panitumumab (SEQ ID NO:50), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of panitumumab (SEQ ID NO:49), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of panitumumab (SEQ ID NO:50), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of panitumumab (SEQ ID NO:49), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of panitumumab (SEQ ID NO:50), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of cetuximab (SEQ ID NO:49), as determined by EVIGT.

[0130] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of nimotuzumab (SEQ ID NOs:51 and 52). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of nimotuzumab (SEQ ID NO: 52), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of nimotuzumab (SEQ ID NO:51), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of nimotuzumab (SEQ ID NO: 52), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of nimotuzumab (SEQ ID NO:51), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of nimotuzumab (SEQ ID NO:52), and HC CDRl, HC CDR2 and

HC CDR3 of the heavy chain variable region of nimotuzumab (SEQ ID NO:51), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of nimotuzumab (SEQ ID NO:52), and HC CDRl, HC CDR2 and HC

CDR3 of the heavy chain variable region of nimotuzumab (SEQ ID NO:51), as determined by

EVIGT.

[0131] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of zalutumumab (SEQ ID NOs:53 and 54). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of zalutumumab (SEQ ID NO:54), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of zalutumumab (SEQ ID NO:53), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of zalutumumab (SEQ ID NO:54), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of zalutumumab (SEQ ID NO:53), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of zalutumumab (SEQ ID NO:54), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of zalutumumab (SEQ ID NO:53), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of zalutumumab (SEQ ID NO: 54), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of zalutumumab (SEQ ID NO:53), as determined by EVIGT.

[0132] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of AMG595 (SEQ ID NOs:65 and 66). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of AMG595 (SEQ ID NO:66), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of AMG595 (SEQ ID NO:65), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of AMG595 (SEQ ID NO:66), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of AMG595 (SEQ ID NO:65), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of AMG595 (SEQ ID NO:66), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of AMG595 (SEQ ID NO:65), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of AMG595 (SEQ ID NO:66), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of AMG595 (SEQ ID NO:65), as determined by EVIGT.

[0133] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of AMG595 (SEQ ID NOs:65 and 66). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of AMG595 (SEQ ID NO:66), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of AMG595 (SEQ ID NO:653), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of AMG595 (SEQ ID NO:66), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of AMG595 (SEQ ID NO:65), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of AMG595 (SEQ ID NO:66), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of AMG595 (SEQ ID NO:65), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of AMG595 (SEQ ID NO:66), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of AMG595 (SEQ ID NO:65), as determined by EVIGT.

[0134] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of ABT806 (SEQ ID NOs:67 and 68). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of ABT806 (SEQ ID NO:68), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of ABT806 (SEQ ID NO: 67), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of ABT806 (SEQ ID NO:68), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of ABT806 (SEQ ID NO: 67), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of ABT806 (SEQ ID NO: 68), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of ABT806 (SEQ ID NO: 67), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of ABT806 (SEQ ID NO:68), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of ABT806 (SEQ ID NO: 67), as determined by IMGT.

[0135] In certain embodiments, the antibody construct comprises an antibody to mesothelin, e.g., anetumab, or an antigen binding portion thereof. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of anetumab (SEQ ID

NOs:55 and 56). In certain embodiments, the antibody construct comprises LC CDR1, LC

CDR2 and LC CDR3 of the light chain variable region of anetumab (SEQ ID NO:56), and HC

CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of anetumab (SEQ ID

NO:55), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDR1, LC

CDR2 and LC CDR3 of the light chain variable region of anetumab (SEQ ID NO:56), and HC

CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of anetumab (SEQ ID

NO:55), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of anetumab

(SEQ ID NO:56), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of anetumab (SEQ ID NO:55), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC

CDR1, LC CDR2 and LC CDR3 of the light chain variable region of anetumab (SEQ ID

NO:56), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of anetumab (SEQ ID NO: 55), as determined by EVIGT.

[0136] In certain embodiments, the antibody construct comprises an antibody to TROP2, e.g., sacituzumab, or an antigen binding portion thereof. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of sacituzumab (SEQ ID NOs:57 and 58). In certain embodiments, the antibody construct comprises LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of sacituzumab (SEQ ID NO:58), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of sacituzumab (SEQ ID NO:57), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of sacituzumab (SEQ ID NO:58), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of sacituzumab (SEQ ID NO:57), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of sacituzumab (SEQ ID NO:58), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of sacituzumab (SEQ ID NO:57), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of sacituzumab (SEQ ID

NO: 58), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of sacituzumab (SEQ ID NO:57), as determined by EVIGT.

[0137] In certain embodiments, the antibody construct comprises an antibody to CEA, e.g., PR1 A3, humanized PA1A3, arcitumomab, labetuzumab, or an antigen binding portion thereof. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of PR1 A3 (SEQ ID NOs:59 and 60). In certain embodiments, the antibody construct comprises LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of PR1 A3 (SEQ ID NO:60), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of PR1 A3 (SEQ ID NO:59), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of PR1 A3 (SEQ ID NO:60), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of PR1 A3 (SEQ ID NO:59), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of PR1 A3 (SEQ ID NO:60), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of PR1 A3 (SEQ ID NO:59), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of PR1 A3 (SEQ ID NO:60), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of PR1 A3 (SEQ ID NO:59), as determined by EVIGT. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of humanized PR1A3 (SEQ ID NOs:61 and 62).

[0138] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of labetuzumab (SEQ ID Nos:81 and 82). In certain embodiments, the antibody construct comprises LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of labetuzumab (SEQ ID NO:82), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of labetuzumab (SEQ ID NO:81), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of labetuzumab (SEQ ID NO:82), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of labetuzumab (SEQ ID NO:81), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of labetuzumab (SEQ ID NO: 82), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of labetuzumab (SEQ ID NO: 81), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of labetuzumab (SEQ ID NO: 82), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of labetuzumab (SEQ ID NO: 81), as determined by EVIGT.

[0139] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of arcitumomab (SEQ ID NOs:71 and 72). In certain embodiments, the antibody construct comprises LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of arcitumomab (SEQ ID NO:72), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of arcitumomab (SEQ ID NO:71), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of arcitumomab (SEQ ID NO:72), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of arcitumomab (SEQ ID NO:71), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of arcitumomab (SEQ ID NO:72), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of arcitumomab (SEQ ID NO:71), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of arcitumomab (SEQ ID NO:72), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of arcitumomab (SEQ ID NO:71), as determined by EVIGT.

[0140] In certain embodiments, the antibody construct comprises an antibody to claudin 18.2, e.g., claudiximab, or an antigen binding portion thereof. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of claudiximab (SEQ ID NOs:63 and 64). In certain embodiments, the antibody construct comprises LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of claudiximab (SEQ ID NO:64), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of claudiximab (SEQ ID NO:63), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of claudiximab (SEQ ID NO:64), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of claudiximab (SEQ ID NO:63), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDR1, LC CDR2 and LC CDR3 of the light chain variable region of claudiximab (SEQ ID NO:64), and HC CDR1, HC CDR2 and HC CDR3 of the heavy chain variable region of claudiximab (SEQ ID NO:63), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of claudiximab (SEQ ID NO: 64), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of claudiximab (SEQ ID NO:63), as determined by EVIGT.

[0141] In certain embodiments, the antibody construct comprises an antibody to VTCN1, e.g., hlDl 1 Vhl .9 VAR C2, or an antigen binding portion thereof. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of hlDl 1 Vhl .9 VAR C2b (SEQ ID NOs:73 and 74). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of hlDl 1 Vhl .9 VAR C2 (SEQ ID NO:74), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of hlDl 1 Vhl .9 VAR C2 (SEQ ID NO:73), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of hlDl 1 Vhl .9 VAR C2 (SEQ ID NO:74), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of hlDl 1 Vhl .9 VAR C2 (SEQ ID NO:73), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of hlDl 1 Vhl .9 VAR C2 (SEQ ID NO:74), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of hlDl 1 Vhl .9 VAR C2 (SEQ ID NO:73), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of hlDl 1 Vhl .9 VAR C2 (SEQ ID NO: 74), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of hlDl 1 Vhl .9 VAR C2 (SEQ ID NO:73), as determined by EVIGT.

[0142] In certain embodiments, the antibody construct comprises an antibody to LRG5, e.g., BNClOl, or an antigen binding portion thereof. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of BNClOl (SEQ ID NOs:75 and 76). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of BNClOl (SEQ ID NO:76), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of BNClOl (SEQ ID NO: 75), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of BNClOl (SEQ ID NO:76), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of BNClOl (SEQ ID NO: 75), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of BNClOl (SEQ ID NO:76), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of BNClOl (SEQ ID NO: 75), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of BNClOl (SEQ ID NO:76), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of BNClOl (SEQ ID NO: 75), as determined by EVIGT.

[0143] In certain embodiments, the antibody construct comprises an antibody to TMPRSS4, e.g., T2-6C, T2-6G, or an antigen binding portion thereof. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of T2-6C (SEQ ID

Nos:77 and 78). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of T2-6C (SEQ ID NO:78), and HC CDRl, HC

CDR2 and HC CDR3 of the heavy chain variable region of T2-6C (SEQ ID NO:77), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and

LC CDR3 of the light chain variable region of T2-6C (SEQ ID NO:78), and HC CDRl, HC

CDR2 and HC CDR3 of the heavy chain variable region of T2-6C (SEQ ID NO:77), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC

CDRl, LC CDR2 and LC CDR3 of the light chain variable region of T2-6C (SEQ ID NO:78), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of T2-6C (SEQ ID

NO:77), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and

LC CDR3 of the light chain variable region of T2-6C (SEQ ID NO:78), and HC CDRl, HC

CDR2 and HC CDR3 of the heavy chain variable region of T2-6C (SEQ ID NO:77), as determined by EVIGT.

[0144] In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of T2-6G (SEQ ID Nos:79 and 80). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of T2-6G (SEQ ID NO:80), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of T2-6G (SEQ ID NO:79), as determined by Kabat index. In certain

embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of T2-6G (SEQ ID NO:80), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of T2-6G (SEQ ID NO:79), as determined by Kabat index. In certain

embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of T2-6G (SEQ ID NO:80), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of T2-6G (SEQ ID NO:79), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of T2-6G (SEQ ID NO:80), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of T2-6G (SEQ ID NO:79), as determined by EVIGT.

[0145] In certain embodiments, the antibody construct comprises an antibody to EphA2, or an antigen binding portion thereof. In certain embodiments, the antibody construct comprises the heavy and light chain variable region sequences of EphA2 Ab (SEQ ID Nos:83 and 84). In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of EphA2 Ab (SEQ ID NO:84), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of EphA2 Ab (SEQ ID NO:83), as determined by Kabat index. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of EphA2 Ab (SEQ ID NO: 84), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of EphA2 Ab (SEQ ID NO:83), as determined by Kabat index. In certain embodiments, the antibody construct comprises LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of EphA2 Ab (SEQ ID NO: 84), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of EphA2 Ab (SEQ ID NO:83), as determined by EVIGT. In certain embodiments, the antibody construct comprises a humanized antibody or antigen binding fragment thereof comprising LC CDRl, LC CDR2 and LC CDR3 of the light chain variable region of EphA2 Ab (SEQ ID NO:84), and HC CDRl, HC CDR2 and HC CDR3 of the heavy chain variable region of EphA2 Ab (SEQ ID NO:83), as determined by EVIGT.

[0146] The exemplary antibody construct V _H sequences and V _L sequences are illustrated in Table A below.

Table A: Exemplary Antibody Construct VH sequences and VL sequences

Antibody Region SEQ ID Sequence

NO:

SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

v _L 4 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGK

APKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYY CQQHYTTPPTFGQGTKVEIK

4H11 V _H 5 EVKLQESGGGFVKPGGSLKVSCAASGFTFSSYAMSWVRLSPE

MRLEWVATISSAGGYIFYSDSVQGRFTISRDNAKNTLHLQMGS

LRSGDTAMYYCARQGFGNYGDYYAMDYWGQGTTVTVSS

V _L 6 DIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNQLAWYQ

QKPGQSPELLIYWASTRQSGVPDRFTGSGSGTDFTLTISSVQAE DLAVYYCQQSYNLLTFGPGTKLEVK

4A5 V _H 7 EVKLEESGGGFVKPGGSLKISCAASGFTFRNYAMSWVRLSPE

MRLEWVATISSAGGYIFYSDSVQGRFTISRDNAKNTLHLQMGS

LRSGDTAMYYCARQGFGNYGDYYAMDYWGQGTTVTVSS

v _L 8 DIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNQLAWYQ

QKTGQSPELLIYWASTRQSGVPDRFTGSGSGTDFTLTISSVQAE DLAVYYCQQSYNLLTFGPGTKLEIK

Sofituzumab V _H 9 EVQLVESGGGLVQPGGSLRLSCAASGYSITNDYAWNWVRQA

PGKGLEWVGYISYSGYTTYNPSLKSRFTISRDTSKNTLYLQMN SLRAEDTAVYYCARWTSGLDYWGQGTLVTVSS

v _L 10 DIQMTQSPSSLSASVGDRVTITCKASDLIHNWLAWYQQKPGK

APKLLIYGATSLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYY

CQQYWTTPFTFGQGTKVEIK

MORab-009 V _H 11 QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHG

KSLE WIGLITPYNG AS S YNQKFRGKATLT VDKS S ST AYMDLL S LTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS

V _L 12 DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSP

KRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYY CQQWSKHPLTFGSGTKVEIK

RG7787 V _H 13 MQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQ

APGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVY MELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS

v _L 14 MDIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGK

APKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYY

CQQWSKHPLTFGQGTKLEIK

Atezolizumab V _H 15 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPG

KGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMN SLRAEDT AVYYC ARRH WPGGFD Y WGQGTL VT VS S

V _L 16 DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGK

APKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY Antibody Region SEQ ID Sequence

NO:

CQQYLYHPATFGQGTKVEIK

MDX-1105 V _H 17 QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPG

QGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSS LRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS

v _L 18 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQA

PRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPTFGQGTKVEIK

Antibody to V _H 19 QVQLQESGPGLVKPSQTLSLTCAISGDSVSSNSVTWNWIRQSP FAP variant 1 SRGLEWLGRTYYRSKWYNDYAVSVKGRITINPDTSKNQFYLQ

LKSVTPEDAAVYYCARDSSILYGDYWGQGTLVTVSS

V _H 20 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSVTWNWIRQSP

SRGLEWLGRTYYRSKWYNDYAVSVKGRITINPDTSKNQFYLQ LKSVTPEDAAVYYCARDSSILYGDYWGQGTLVTVS

V _L 21 QAVLTQPSSLSASPGASASLTCTLPSGINVGTYRIFWFQQKPGS

PPQYLLSYKSDSDNHQGSGVPSRFSGSKDASANAGILLISGLQS EDEAD YYCMIWHS S AWVFGGGTKLTVL

Antibody to V _H 22 QVQLVQSGAEVKKPGASVKVSCKTSGYTFTDYYIHWVRQAP FAP variant 2 GQGLEWMGWINPNRGGTNYAQKFQGRVTMTRDTSIATAYM

ELSRLRSDDTAVYYCATASLKIAAVGTFDCWGQGTLVTVSS

V _L 23 SYELTQPPSVSVSPGQTARITCSGDALSKQYAFWFQQKPGQAP

ILVIYQDTKRPSGIPGRFSGSSSGTTVTLTISGAQADDEADYYC

QSADSSGTYVFGTGTKVTVL

Antibody to V _H 24 EVQLVETGGGVVQPGRSLRLSCAASGFSFSTHGMYWVRQPPG FAP variant 3 KGLEWVAVISYDGSDKKYADSVKGRFTISRDNSKNTVYLEMS

S VR AEDT ALYYCFCRRD AFDL WGQGTMVT VS S

V _L 25 SYVLTQPPSVSVSPGQTARITCSGDALPKKYAYWYQQKSGQA

PVLVIYEDTKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYY CYSTDSSGNYWVFGGGTEVTVL

Antibody to V _H 26 EVQLVESGGGLVEPGGSLRLSCAASGFTFSDAWMNWVRQAP FAP variant 4 GKGLEWVGRIKTKSDGGTTDYAAPVRGRFSISRDDSKNTLFLE

MNSLKTEDTAIYYCFITVIVVSSESPLDHWGQGTLVTVSS v _L 27 SYELTQPPSVSVSPGQTARITCSGDELPKQYAYWYQQKPGQAP

VLVIYKDRQRPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYC

QSAYSINTYVIFGGGTKLTVL

Antibody to V _H 28 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPG FAP variant 5 KGLE WIS YI S SGS S YTNYAD S VKGRFTISRDN AKKS VYLE VNG

LTVEDTAVYYCARVRYGDREMATIGGFDFWGQGTLVTVSS Antibody Region SEQ ID Sequence

NO:

v _L 29 SYELTQPPSVSVSPGQTARITCSGDALPKQYAYWYQQSPGQAP

VLVIYKDSERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYC

QSADSGGTSRIFGGGT LTVL

Antibody to V _H 30 QVQLQESGPGLVRSTETLSLTCLVSGDSINSHYWSWLRQSPGR FAP variant 6 GLEWIGYIYYTGPTNYNPSLKSRVSISLGTSKDQFSLKLSSVTA

ADTARYYCARNKVFWRGSDFYYYMDVWGKGTTVTVSS v _L 31 EIVLTQSPGTLSLSLGERATLSCRASQSLAN YLAWYQQKPGQ

APRLLMYDASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVY YCQQFVTSHHMYIFGQGTKVEIK

Antibody to V _H 32 HVQLQESGPGLVKPSETLSLTCTVSGGSISSN YYWGWIRQTP FAP variant 7 GKGLEWIGSIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSV

TAADTAVYYCARGARWQARPATRIDGVAFDIWGQGTMVTVS S

V _H 33 QVQLQESGPGLVKPSETLSLTCTVSGGSISSN YYWGWIRQTP

GKGLEWIGSIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSV TAADTAVYYCARGARWQARPATRIDGVAFDIWGQGTMVTVS S

V _H 34 EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPG

QGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYME

LRSLRSDDTAVYYCARDWSRSGYYLPDYWGQGTLVTVSS

V _L 35 ETTLTQSPGTLSLSPGERATLSCRASQTVTRNYLAWYQQKPGQ

APRLLMYGASNRAAGVPDRFSGSGSGTDFTLTISRLEPEDFAV

YYCQQFGSPYTFGQGTKVEIK

v _L 36 DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSNGYNYLDWYLQ

RPGQSPHLLIFLGSNRASGVPDRFSGSGSGTDFTLKISRVEAED

VGIYYCMQALQTPPTFGQGTKVEIK

Antibody V _H 37 EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAP huM25 to GQGLEWIGEILPGSDTTNYNEKFKDRATFTSDTSINTAYMELS LRRC15 RLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSS v _L 38 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGA

VKFLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDF

ATYFCQQGEALPWTFGGGTKVEIK

Antibody V _H 39 EVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYYIHWVKQAPG huAD208.4.1 QGLEWIGLVYPYIGGTNYNQKFKGKATLTVDTSTTTAYMEMS to LRRC15 SLRSEDTAVYYCARGDNKYDAMDYWGQGTTVTVSS

V _L 40 DIVLTQSPDSLAVSLGERATINCRASQSVSTSSYSYMHWYQQK

PGQPPKLLIKYASSLESGVPDRFSGSGSGTDFTLTISSLQ

AEDVAVYYCEQSWEIRTFGGGTKVEIK Antibody Region SEQ ID Sequence

NO:

Antibody V _H 41 EVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMHWVKQA huAD208.12.1 PGQGLEWIGMIHPNSGSTKHNEKFRGKATLTVDESTTTAYME to LRRC15 LSSLRSEDTAVYYCARSDFGNYRWYFDVWGQGTTVTVSS v _L 42 EIVLTQSPATLSLSPGERATLSCRASQSSSNNLHWYQQKPGQA

PRVLIKYVSQSISGIPARFSGSGSGTDFTLTISSLEPEDFA

VYFCQQSNSWPFTFGQGT LEIK

Antibody V _H 43 EVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYYIHWVKQAPG huAD208.14.1 QGLEWIGLVYPYIGGSSYNQQFKGKATLTVDTSTSTAYMELSS to LRRC15 LRSEDT AVY YC ARGDNNYD AMD Y WGQGTT VT VS S v _L 44 DIVLTQSPDSLAVSLGERATISCRASQSVSTSTYNYMHWYQQK

PGQPPKLLVKYASNLESGVPDRFSGSGSGTDFTLTISSL

QAEDVAVYYCHHTWEIRTFGGGTKVEIK

Antibody V _H 45 EVQLVESGGGLVQPGGSLRLSCAVSGFSLTSYGVHWVRQATG hul39.10 to KGLEWLGVIWAGGSTNYNSALMSRLTISKENAKSSVYLQMNS LRRC15 LRAGDT AMY YCATHMITEDYYGMD Y WGQGTT VTVS S

V _L 46 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQ

QKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISS

LQAEDVAVYYCKQSYNLPTFGGGTKVEIK

Cetuximab V _H 47 QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGK (EGFR) GLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSL

QSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA

v _L 48 DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPR

LLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQN NNWPTTFGAGTKLELK

Panitumumab V _H 49 QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSP (EGFR) GKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSV

TAADT AIYYC VRDRVTGAFDI WGQGTMVT VS S

v _L 50 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKA

PKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFC

QHFDHLPLAFGGGTKVEIK

Nimotuzumab V _H 51 QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAP (EGFR) GQGLEWIGGINPTSGGSNFNEKFKTRVTITVDESTNTAYMELS

SLRSEDTAFYFCARQGLWFDSDGRGFDFWGQGSTVTVSS

V _L 51 DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQ

TPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDI

ATYYCFQYSHVPWTFGQGTKLEIK

Zalutumumab V _H 53 QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAP (EGFR) GKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQ

MNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVT Antibody Region SEQ ID Sequence

NO:

vss

v _L 54 AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAP

KLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQ QFNSYPLTFGGGTKVEIK

Anetumab V _H 55 QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPG (MSLN) KGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSL

KASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS

V _L 56 DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGK

APKLMIYGVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEAD YYCS SYDIES ATP VFGGGT LTVL

Sacituzumab V _H 57 QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAP (TROP2) GQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQI

SSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS

v _L 58 DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKA

PKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYY CQQHYITPLTFGAGTKVEIK

PR1A3 V _H 59 QVKLQQSGPELKKPGETVKISCKASGYTFTEFGMNWVKQAPG (CEA) KGLKWMGWINTKTGEATYVEEFKGRFAFSLETSATTAYLQIN

NLKNEDTAKYFCARWDFYDYVEAMDYWGQGTTVTVSS

v _L 60 DIVMTQSQRFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPG

QSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAE YFCHQYYTYPLFTFGSGTKLEMK

Humanized V _H 61 QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAP PR1A3 GQGLEWMGWINTKTGEATYVEEFKGRVTFTTDTSTSTAYME (CEA) LRSLRSDDTAVYYCARWDFAYYVEAMDYWGQGTTVTVSS

V _L 62 DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQKPGK

APKLLIYSASYRKRGVPSRFSGSGSGTDFTLTISSLQPEDFATY

YCHQYYTYPLFTFGQGTKLEIK

CLAUDIXIM V _H 63 QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRP

AB GQGLEWIGNIYPSD S YTNYNQKFKDK ATLT VDKS S ST AYMQL

(CLND18.2) SSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVSS

v _L 64 DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWY

QQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQA

EDLAVYYCQNDYSYPFTFGSGTKLEIK

AMG595 V _H 65 QVQLVESGGGVVQSGRSLRLSCAASGFTFRNYGMHWVRQAP (EGFRvIII) GKGLEWVAVIWYDGSDKYYADSVRGRFTISRDNSKNTLYLQ

MNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVS S

V _L 66 DTVMTQTPLSSHVTLGQPASISCRSSQSLVHSDGNTYLSWLQQ Antibody Region SEQ ID Sequence

NO:

RPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEISRVEAEDV GVYYCMQSTHVPRTFGQGTKVEIK

ABT806 V _H 67 EVQLQESGPGLVKPSQTLSLTCTVSGYSISRDFAWNWIRQPPG (EGFRvIII) KGLEWMGYISYNGNTRYQPSLKSRITISRDTSKNQFFLKLNSV

TAADTATYYCVTASRGFPYWGQGTLVTVSS

V _L 68 DIQMTQSPSSMSVSVGDRVTITCHSSQDINSNIGWLQQKPGKS

FKGLIYHGTNLDDGVPSRFSGSGSGTDYTLTISSLQPEDFATYY

CVQYAQFPWTFGGGTKLEIK

Sibrotuzumab V _H 69 QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPG (FAP) QRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSS

LRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSS

v _L 70 DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWY

QQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQA EDVAVYYCQQYFSYPLTFGQGTKVEIK

Arcitumomab V _H 71 EVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMNWVRQPPG (CEA) KALEWLGFIGNKANGYTTEYSASVKGRFTISRDKSQSILYLQM

NTLRAEDSATYYCTRDRGLRFYFDYWGQGTTLTVSS

V _L 72 QTVLSQSPAILSASPGEKVTMTCRASSSVTYIHWYQQKPGSSP

KSWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYY CQHWSSKPPTFGGGTKLEIK

hlDl l Vhl.9 V _H 73 EVQLVQSGAEVKKPGASVKVSCKASGVTFTSYWIGWVRQAP VAR C2 GQGLEWIGDIYPGGGYTNYNEKFKGRVTITRDTSTSTAYLELS (VTCN1) SL ASEDT AVY YC ARL AGS S YRG AMD S WGQGTL VT VS S

v _L 74 MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCK

ASQGFNKYVAWYQQKPGKAPKLLIYYTSTLQPGVPSRFSGSG SGRDYTLTISSLQPEDFATYYCLQYGDLLYAFGQGTKVEIKR

BNC101 V _H 75 MEWSWVFLFFLSVTTGVHSEVQLVQSGAEVKKPGESLRISCK (LRG5) GSGYSFTAYWIEWVRQAPGKGLEWIGEILPGSDSTNYNEKFK

GHVTISADKSISTAYLQWSSLKASDTAVYYCARSGYYGSSQY WGQGTL VTVSS

v _L 76 DIVLTQSPASLAVSPGQRATITCRASESVDSYGNSFMHWYQQK

PGQPPKLLIYLTSNLESGVPDRFSGSGSGTDFTLTINPVEANDA

ATYYCQQNAEDPRTFGGGTKLEIK

T2-6C V _H 77 QMQLVESGGGLVQPGRSLRLSCAASGFTFDDYAIHWVRQAPG (TMPRSS4) KGLEWVSGISWNSEIVGYGDSVKGRFTISRDNAKNSLDLQMN

SLRAEDTAVYYCARGSSGRAFDIWGQGTMVTVSS

V _L 78 SGVGSDIQMTQSPSSVSASVGDRITITCRASQSISTYLNWYQQK

PGKAPKLLIYGATSLQSGVPSKFSGSGSGTOFTLTIRGLQPDDF

GTYYCQQSYNL1PRTFGQGTKLDIKR Antibody Region SEQ ID Sequence

NO:

T2-6G V _H 79 QVQLVESGGGVVQPGRSLRLSCVGSFTFSNYGMHWVRQAPG (TMPRSS4) KGLQWVAVXSYDGSLKKYYADSVKGRFTISRDNSKNTLYLQ

MNSLRSEDTAVYYCARGTTMDVWGQGKGTTVTVSS

v _L 80 SGVGSQSALTQPPSASGTPGQRVTISCSGSNSNIGSNTVNWYQ

QFPGKAPQLLIFGHNQRPSGVPDRFSGSKSGTSASLSISGLQSE DEAHYYCASWDDTVSGPKWVFGGGTKVDIKR

Labetuzumab V _H 81 EVQLVESGGGVVQPGRSLRLSCSSSGFDFTTYWMSWVRQAPG (CEACAM5) KGLEWVAEIHPDS STINY APSLKDRFTISRDNSKNTLFLQMD SL

RPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS

v _L 82 DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGK

APKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYY

CQQYSLYRSFGQGTKVEIK

Ab to EPHA2 V _H 83 EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYMMAWVRQAP

GKGLEWVSRIGPSGGPTHYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCAGYDSGYDYVAVAGPAEYFQHWGQGTL VTVSS

V _L 84 DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPGKA

PKLLIYKASNLHTGVPSRFSGSGSGTEFSLTISGLQPDDFATYY

CQQYNSYSRTFGQGTKVEIK

Target Binding Domain

[0147] An antibody construct may further comprise a target binding domain. A target binding domain comprises a domain that specifically binds to a target. A target binding domain may comprise an antigen binding domain. A target binding domain may be a domain that can specifically bind to an antigen. A target binding domain may be an antigen-binding portion of an antibody or an antibody fragment. A target binding domain may be one or more fragments of an antibody that can retain the ability to specifically bind to an antigen. A target binding domain may be any antigen binding fragment. A target binding domain may be in a scaffold, in which a scaffold is a supporting framework for the antigen binding domain. A target binding domain may comprise an antigen binding domain in a scaffold.

[0148] A target binding domain may comprise an antigen binding domain which can refer to a portion of an antibody comprising the antigen recognition portion, i.e., an antigenic determining variable region of an antibody sufficient to confer recognition and specific binding of the antigen recognition portion to a target, such as an antigen, i.e., the epitope. A target binding domain may comprise an antigen binding domain of an antibody.

[0149] An Fv can be the minimum antibody fragment which contains a complete antigen- recognition and antigen-binding site. This region may consist of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. In this configuration, the three

CDRs of each variable domain may interact to define an antigen-binding site on the surface of the V _H -V _L dimer. A single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can recognize and bind antigen, although at a lower affinity than the entire binding site.

[0150] A target binding domain may be at least 80% homologous to a specific antigen binding domain selected from, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, or a functional fragment thereof, for example, a heavy chain variable domain (V _H ) and a light chain variable domain (V _L ), a single chain variable fragment (scFv), or a DARPin, an affimer, an avimer, a knottin, a monobody, an affinity clamp, an ectodomain, a receptor ectodomain, a receptor, a cytokine, a ligand, an immunocytokine, a T cell receptor, or a recombinant T cell receptor.

[0151] A target binding domain may be attached to an antibody construct. For example, an antibody construct may be fused with a target binding domain to create an antibody construct target binding domain fusion. The antibody construct-target binding domain fusion may be the result of the nucleic acid sequence of the target binding domain being expressed in frame with the nucleic acid sequence of the antibody construct. The antibody construct-target binding domain fusion may be the result of an in-frame genetic nucleotide sequence encoding the antibody construct, or a or a contiguous peptide sequence of the antibody construct, with the target binding domain. As another example, a target binding domain may be linked to an antibody construct. A target binding domain may be linked to an antibody construct by a chemical conjugation. A target binding domain may be attached to a terminus of an Fc domain or Fc region. A target binding domain may be attached to a terminus of an Fc domain or Fc region. A target binding domain may be attached to a terminus of an antibody construct. A target binding domain may be attached to a terminus of an antibody. A target binding domain may be attached to a light chain of an antibody. A target binding domain may be attached to a terminus of a light chain of an antibody. A target binding domain may be attached to a heavy chain of an antibody. A target binding domain may be attached to terminus of a heavy chain of an antibody. The terminus may be a C-terminus. An antibody construct may be attached to 1, 2, 3, and/or 4 target binding domains. The target binding domain may direct the antibody construct to, for example, a particular cell or cell type. A target binding domain of an antibody construct may be selected in order to recognize an antigen, e.g., an antigen expressed on an immune cell. An antigen can be a peptide or fragment thereof. An antigen may be expressed on an immune cell, such as an antigen -presenting cell. An antigen may be expressed on a dendritic cell, a

macrophage, or a B cell. As another example, an antigen may be a tumor antigen. The tumor antigen may be any tumor antigen. When multiple target binding domains are attached to an antibody construct, the target binding domains may bind to the same antigen. When multiple target binding domains are attached to an antibody construct, the target binding domains may bind different antigens.

[0152] In certain embodiments, an antibody construct specifically binds a second antigen. In certain embodiments, the target binding domain is linked to said antibody construct at a C- terminal end of said Fc domain.

Attachment of Linkers to Antibody Construct

[0153] The conjugates may comprise a linker, e.g., a cleavable (e.g., peptide) linker or a non- cleavable linker. Linkers of the conjugates and methods described herein may not affect the binding of active portions of a conjugate (e.g., active portions include antigen binding domains, Fc domains, target binding domains, antibodies, compounds or salts of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), or (IIIc)) to a target, which can be a cognate binding partner such as an antigen. A linker can form a linkage between different parts of a conjugate, e.g., between an antibody construct and a heterocyclic compound or a salt thereof. In certain embodiments, a conjugate includes multiple linkers. In certain embodiments, wherein a conjugate includes multiple linkers, the linkers may be the same linkers or different linkers.

[0154] A linker may be covalently bound to an antibody construct by a bond between the antibody construct and the linker. A linker may be bound to an antibody construct by a bond between antibody construct and the linker. A linker may be bound to a terminus of an amino acid sequence of an antibody construct, or may be bound to a side chain modification to the antibody construct, such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, a non-natural amino acid residue, glutamine or glutamic acid residue. A linker may be bound to a terminus of an amino acid sequence of an Fc domain or Fc region of an antibody construct, or may be bound to a side chain modification of an Fc domain or Fc region of an antibody construct, such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, a non-natural amino acid residue, glutamine or glutamic acid residue. A linker may be bound to a terminus of an amino acid sequence of an Fc domain of an antibody construct, or may be bound to a side chain modification of an Fc domain of an antibody construct, such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, a non-natural amino acid residue, glutamine, or glutamic acid residue.

[0155] A linker may be bound to an antibody construct at a hinge cysteine. A linker may be bound to an antibody construct at a light chain constant domain lysine. A linker may be bound to an antibody construct at a heavy chain constant domain lysine. A linker may be bound to an antibody constmct at an engineered cysteine in the light chain. A linker may be bound to an antibody construct at an Fc region lysine. A linker may be bound to an antibody construct at an

Fc domain lysine. A linker may be bound to an antibody construct at an Fc region cysteine. A linker may be bound to an antibody construct at an Fc domain cysteine. A linker may be bound to an antibody construct at a glutamine residue. A linker may be bound to an antibody construct at an engineered light chain glutamine. A linker may be bound to an antibody construct at an unnatural amino acid engineered into the light chain. A linker may be bound to an antibody construct at an unnatural amino acid engineered into the heavy chain. Amino acids can be engineered into an amino acid sequence of an antibody construct, for example, a linker of a conjugate. Engineered amino acids may be added to a sequence of existing amino acids.

Engineered amino acids may be substituted for one or more existing amino acids of a sequence of amino acids.

[0156] A linker may be conjugated to an antibody construct via a sulfhydryl group on the antibody construct. A linker may be conjugated to an antibody construct via a primary amine on the antibody construct. A linker may be conjugated to an antibody construct via a residue of an unnatural amino acid on an antibody construct, e.g., a ketone moiety.

[0157] In certain embodiments, when one or more linkers are bound to an antibody construct at the sites described herein, an Fc domain of the antibody construct can bind to Fc receptors. In certain embodiments, an antibody construct bound to a linker or an antibody construct bound to a linker bound to a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ha), (lib), (He), (III), (Ilia), (Illb), or (IIIc), retains the ability of the Fc domain of the antibody to bind to Fc receptors. In certain embodiments, when a linker is connected to an antibody construct at the sites described herein, the antigen binding domain of an antibody construct bound to a linker or an antibody construct bound to a linker bound to a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), or (IIIc) can bind its antigen. In certain embodiments, when a linker is connected to an antibody construct at the sites described herein, a target binding domain of an antibody construct bound to a linker or an antibody construct bound to a linker bound to a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), or (IIIc) can bind its antigen.

[0158] In certain embodiments, a linker or linker bound to a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), or (IIIc) disclosed herein is attached to an amino acid residue of an IgG Fc domain selected from: 221, 222, 224, 227, 228, 230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 258, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 283, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 302, 305, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335 336, 396, 428, or any subset thereof wherein numbering of amino acid residues in the Fc domain is according to the EU index as in Kabat.

[0159] In certain embodiment, a linker or linker bound to a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), or (IIIc) disclosed herein may not be attached to an amino acid residue of an IgG Fc domain selected from: 221, 222, 224, 227, 228, 230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 258, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 283, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 302, 305, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335 336, 396, 428, or any subset thereof wherein numbering of amino acid residues in the Fc domain is according to the EU index as in Kabat.

Compounds

[0160] The following is a discussion of compounds and salts thereof that may be used in the conjugates and/or methods of the disclosure. The compounds and salts described in Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc) may be covalently bound to a linker, L ³ , that may further be covalently bound to an antibody construct. The compounds and salts described in Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc) may be referred to herein as "heterocyclic compounds".

[0161] In certain embodiments, the present disclosure provides a compound represented by the structure of Formula (I):

or a salt thereof, in which:

W is selected from N and CH;

A is selected from an optionally substituted C ₃ -Ci ₂ carbocycle and an optionally substituted 3- to 12-membered heterocycle, wherein substituents on A are independently selected at each occurrence from R ¹² ;

X ¹ and X ² are independently selected from -C(R ¹⁰ ) ₂ -, -0-, -S-, -N(R ¹⁰ )-, -C(O)-,

-OC(0)0-, -C(0)N(R ¹⁰ )-, and -N(R ¹⁰ )C(O)-; L is selected from C ₁ -C ₁₂ alkylene, C ₂ -Ci ₂ alkenylene, C _2- Ci ₂ alkynylene, a 6- to 12- membered heteroalkylene or heteroalkenylene, a C ₃ -Ci ₂ carbocyclene, a 3- to 12-membered heterocyclene, and an alkylene or a heteroalkylene interspersed with a C ₃ -Ci ₂ carbocyclene or a 3- to 12-membered heterocyclene, any of which is optionally substituted by one or more substituents independently selected at each occurrence from R ¹³ ;

R ¹ is selected at each occurrence from R ¹² ;

R ¹⁰ is independently selected at each occurrence from: hydrogen; C ₁ -C ₁₀ alkyl, C ₂ -Cio alkenyl, and C ₂ -Ci ₀ alkynyl, each of which is optionally substituted at each occurrence with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , =0, =S, -O-Ci-Cio alkyl, C ₃ -Ci ₂ carbocycle, and a 3- to 12-membered heterocycle; and a C ₃ -Ci ₂ carbocycle and a 3- to 12-membered heterocycle, each of which is optionally substituted at each occurrence with one or more substituents independently selected from a halogen, -OH, -CN, - N0 ₂ , -NH ₂ , =0, =S, C ₁ -C ₁₀ alkyl, -O-Ci-Cio alkyl, and -Ci-Cio haloalkyl;

R ¹² is independently selected at each occurrence from: a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ -C(0)OR ¹⁰ , -OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , - S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ¹⁰ ), and -CN; d-do alkyl, C ₂ - C ₁₀ alkenyl, C ₂ -C.i ₀ alkynyl, -O-Ci-Cio alkyl, and -C ₁ -C ₁₀ haloalkyl, each of which is optionally substituted at each occurrence with one or more substituents independently selected from a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ -C(0)OR ¹⁰ , - OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , -S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ¹⁰ ), -CN, a C ₃ -Cio carbocycle, and a 3- to 10-membered heterocycle; and a C ₃ -Ci ₀ carbocycle and a 3- to 10-membered heterocycle, wherein each C ₃ -Cio carbocycle and 3- to 10- membered heterocycle in R ¹² is independently optionally substituted with one or more substituents independently selected from a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , - C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ -C(0)OR ¹⁰ , -OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , - S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ¹⁰ ), -CN, Ci-C ₆ alkyl, C ₂ -C ₆ alkenyl, and C ₂ -C ₆ alkynyl;

R ¹³ is independently selected at each occurrence from: a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ -C(0)OR ¹⁰ , -OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , - S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ¹⁰ ), and -CN; C ₁ -C ₁₀ alkyl, C ₂ - C ₁₀ alkenyl, C ₂ -Cio alkynyl, -O-Ci-Cio alkyl, and -C ₁ -C ₁₀ haloalkyl, each of which is optionally substituted at each occurrence with one or more substituents independently selected from a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ -C(0)OR ¹⁰ , - OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , -S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ¹⁰ ), -CN, a C ₃ -Cio carbocycle, and a 3- to 10-membered heterocycle; and a C ₃ -Cio carbocycle and a 3- to 10-membered heterocycle, wherein each C3-C10 carbocycle and 3- to 10- membered heterocycle in R ¹³ is independently optionally substituted with one or more substituents independently selected from a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , - C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ -C(0)OR ¹⁰ , -OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , - S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ¹⁰ ), -CN, d-C ₆ alkyl, C ₂ -C ₆ alkenyl, and C ₂ -C ₆ alkynyl; and

n is selected from 0, 1, 2, 3, 4, and 5.

[0162] In certain embodiments for a compound of Formula (I), R ¹² is independently selected at each occurrence from:

a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ - C(0)OR ¹⁰ , -OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , -S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ¹⁰ ), and -CN;

Ci-Cio alkyl, C ₂ -Ci ₀ alkenyl, and C ₂ -C.i ₀ alkynyl, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OR ¹⁰ , -SR ¹⁰ , - N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ -C(0)OR ¹⁰ , -OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , -S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ¹⁰ ), -CN, a C ₃ -C ₁₀ carbocycle, and a 3- to 10-membered heterocycle; and

a C ₃ -Cio carbocycle and a 3- to 10-membered heterocycle, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OR ¹⁰ , -SR ¹⁰ , - N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ -C(0)OR ¹⁰ , -OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , -S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ¹⁰ ), -CN, d-C ₆ alkyl, C ₂ -C ₆ alkenyl, and C ₂ -C ₆ alkynyl;

[0163] In certain embodiments for a compound of Formula (I), R ¹³ is independently selected from:

a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ - C(0)OR ¹⁰ , -OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , -S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ¹⁰ ), and -CN;

Ci-C io alkyl, C ₂ -Ci ₀ alkenyl, C ₂ -Ci ₀ alkynyl, -O-C i-Cio alkyl, and -Ci-Ci ₀ haloalkyl, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ -C(0)OR ¹⁰ , - OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , -S(O) ₂ N(R ¹⁰ ) ₂ , -P(O)(OR ¹⁰ ) ₂ , -OP(O)(OR ¹⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ¹⁰ ), -CN, a C ₃ -Cio carbocycle, and a 3- to 10-membered heterocycle; and

C ₃ -Cio carbocycle and a 3- to 10-membered heterocycle, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OR ¹⁰ , -SR ¹⁰ , - N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ -C(0)OR ¹⁰ , -OC(0)R ¹⁰ , -S(0)R ¹⁰ , -S(0) ₂ R ¹⁰ , -S(0) ₂ N(R ^iU ) ₂ , -P(0)(OR ^iU ) ₂ , -OP(0)(OR ^iU ) ₂ , -N0 ₂ , =0, =S, =N(R ^iU ), -CN, d-C ₆ alkyl, C ₂ -C ₆ alkenyl, and C ₂ -C ₆ alkynyl.

[0164] In some embodiments, when R ¹² is selected from C ₃ -C ₁₀ carbocycle and 3- to 10- membered heterocycle, the substituents on C ₃ -C ₁₀ carbocycle and 3- to 10-membered

heterocycle may be further selected from -Ci _-6 alkylN(R ¹⁰ ) ₂ .

[0165] In some embodiments, a compound or salt represented by Formula (I) is represented by Formula (la):

[0166] In some embodiments, a compound or salt represented by Formula (I) is represented by Formula (lb):

[0167] In some embodiments, for a compound or salt represented by Formula (I), W is N. In other embodiments, W is CH. In some embodiments, W is CR ¹ , where R ¹ is selected from R ¹² .

[0168] In some embodiments, for a compound or salt represented by any one of Formulas (I), (la), and (lb), A is selected from a heterocycle optionally substituted with one or more substituents independently selected at each occurrence from R ¹² . As described herein, heterocycles may include one or more heteroatoms (e.g., 1, 2, 3, or more heteroatoms) and may be saturated, unsatured, or aromatic. A heterocycle may be monocyclic or multi cyclic (e.g., bicyclic or tricyclic). In some embodiments, A is selected from an optionally substituted bicyclic heterocycle. Examples of bicyclic heterocycles include, but are not limited to, quinazoline, a quinoline, an isoquinoline, a benzimidazole, an indazole, a dihydrobenzoxaborole, a tetrahydroisoquinoline, a benzothiophene, a benzoxazole, a benzothiazole, an indole, a benzotriazole, a benzofuran, a dihydrobenzofuran, a benzodioxoline, a benzodioxin, an indolizine, an imidazopyridine, a purine, a benzothiadiazole, a benzoxadiazole, a

tetrahydroindole, an azaindole, an indazoline, a pyrrolopyrimidine, and a pyrazolopyrimidine. In some embodiments, A is an optionally substituted 8-12 membered bicyclic heterocycle. In certain embodiments, A is selected from a quinazoline, a quinoline, an isoquinoline, a benzimidazole, an indazole, a dihydrobenzoxaborole, a tetrahydroisoquinoline, a

benzothiophene, a benzoxazole, a benzothiazole, an indole, and a benzotriazole, any of which may be optionally substituted with one or more substituents independently selected at each occurrence from R ¹² . In some embodiments, A is selected from a quinazoline, an isoquinoline, a benzimidazole, and a dihydrobenzoxaborole. For example, A may be an optionally substituted quinazoline. In certain embodiments, A is re resented by:

in which A is optionally substituted with one or more substituents independently selected at each occurrence from R ¹² .

[0169] In certain embodiments, for a compound or salt represented by any one of Formulas (I), (la), and (lb), A is selected from an optionally substituted monocyclic heterocycle, wherein substituents on A are independently selected at each occurrence from R ¹² . Examples of monocyclic heterocycles include, but are not limited to, pyrazole, imidazole, pyrimidine, pyridine, piperidine, pyrrole, furan, and tetrahydrofuran.

[0170] In certain embodiments, A is selected from an optionally substituted C3-C ₁₂ carbocycle, wherein substituents on A are independently selected at each occurrence from R ¹² . As described herein, carbocycles may be saturated, unsaturated, or aromatic. Examples of carbocycles include, but are not limited to, cyclooctane, cycloheptane, cyclohexane,

cyclopentane, benzene, naphthalene, tetralin, decalin, cyclooctene, cycloheptene, cyclohexene, and cyclopentene.

[0171] In some embodiments, for a compound or salt represented by any one of Formulas (I), (la), and (lb), X ¹ and X ² are independently selected from -C(R ¹⁰ ) ₂ -, -0-, -S-, -N(R ¹⁰ )-, -C(O)-, - C(0)N(R ¹⁰ )-, and -N(R ¹⁰ )C(O)-. In some embodiments, X ¹ and X ² are independently selected from -C(R ¹⁰ ) ₂ -, -0-, -S-, -N(R ¹⁰ )-, -C(O)-, -C(0)N(R ¹⁰ )-, and -N(R ¹⁰ )C(O)-, wherein R ¹⁰ is independently selected at each occurrence from: hydrogen; C ₁ -C3 alkyl optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , =0, =S, -O-C ₁ -C3 alkyl; and C3-C6 cycloalkyl optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , =0, =S, -O-C ₁ -C3 alkyl. For example, X ¹ and X ² may be independently selected from -C(R ¹⁰ ) ₂ -, -0-, -S-, and - N(R ¹⁰ )-. In some embodiments, X ¹ is -0-. In other embodiments, X ² is -0-. In certain embodiments, X ¹ and X ² are each -0-. [0172] In some embodiments, for a compound or salt represented by any one of Formulas (I),

(la), and (lb), L is selected from C1-C12 alkylene and C2-C12 alkenylene, any of which is optionally substituted by one or more substituents independently selected at each occurrence from R ¹³ . As described herein, an alkenylene may include 1, 2, or more double bonds. In certain embodiments, L is selected from optionally substituted C ₆ -Ci ₂ alkylene and C ₆ -Ci ₂ alkenylene.

In some embodiments, L is selected from C4-C9 alkenylene, any of which is optionally substituted by one or more substituents independently selected at each occurrence from R ¹³ . For example, L may be represented by: example, the L may be selected from:

[0173] In other embodiments, L is selected from a 6- to 12-membered heteroalkylene or heteroalkenylene optionally substituted by one or more substituents independently selected at each occurrence from R ¹³ . Examples of heteroalkylenes and heteroalkenylenes include, but are not limited to, ethers, thioethers, and amines. In some embodiments, L is an optionally substituted C1-C12 alkylene chain in which at least one methylene is replaced with -0-, -N(R ¹⁰ )-, or -S-. In certain embodiments, the heteroalkylene or heteroalkenylene is selected from -(C1-C3 alkylene or C1-C3 alkenylene)-(G-Ci-C ₃ alkylene or Ci-C ₃ alkenylene) _0- 4-G-(Ci-C ₃ alkylene or Ci-C ₃ alkenylene)- in which G is a heteroatom selected from O, S, and N, where N may be represented by H or N(R ¹⁰ ), and the heteroalkylene or heteroalkenylene is optionally substituted by one or more substituents independently selected at each occurrence from R ¹³ . For example, L may be represented by:

[0174] In other embodiments, L is selected from an alkylene or a heteroalkylene interspersed with a C ₃ -Ci2 carbocyclene or a 3- to 12-membered heterocyclene, any of which is optionally substituted by one or more substituents independently selected at each occurrence from R ¹³ . As used herein, "interspersed" means placed between and encompasses any possible sequence of alkylene, C ₃ -C ₁₂ carbocyclene, and a 3- to 12-membered heterocyclene or heteroalkylene, C ₃ -C ₁₂ carbocyclene, and a 3- to 12-membered heterocyclene. For example, L may be represented by - (alkylene)-(C ₃ -Ci ₂ carbocyclene)-, -(alkylene)-(C ₃ -Ci ₂ carbocyclene)-(alkylene)-, - (heteroalkylene)-(C ₃ -Ci2 carbocyclene)-, -(heteroalkylene)-(C ₃ -Ci ₂ carbocyclene)-(alkylene)-, or similar sequences including heterocyclenes in placeof carbocyclenes.

[0175] In certain embodiments, for a compound or salt represented by any one of Formulas (I), (la), and (lb), n is selected from 1, 2, 3, 4, and 5. For example, n may be selected from 1, 2, and 3. In some embodiments, n is 1. In certain embodiments, n is 2. In other embodiments, n is 0.

[0176] In some embodiments, for a compound or salt of any one of Formulas (I), (la), and (lb), R ¹ is independently selected at each occurrence from a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , - C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -CN, -N0 ₂ , -C1-C10 alkyl, -OC1-C10 alkyl, and -C1-C10 haloalkyl.

[0177] In a first aspect, for a compound of Formula (I) or a salt thereof:

W is N;

A is selected from an optionally substituted bicyclic heterocycle, preferably a bicyclic heteroaryl, wherein substituents on A are independently selected at each occurrence from R ¹² ;

X ¹ and X ² are independently selected from -O- and -N(R ¹⁰ )-;

L is selected from C ₆ -C ₉ alkylene, C ₆ -C ₉ alkenylene, and C ₆ -C ₉ alkynylene, any of which is optionally substituted by one or more substituents independently selected at each occurrence from R ¹³ ;

R ¹ is selected at each occurrence from R ¹² ;

R ¹⁰ is independently selected at each occurrence from: hydrogen; and Ci-C ₃ alkyl, C ₃ -C ₆ carbocycle, or 3-7 membered heterocycle, each of which are optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , and -Ci-C ₃ alkyl-NH ₂ ;

R ¹² is independently selected at each occurrence from: a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ and -CN; Ci-C ₃ alkyl optionally substituted with one or more substituents independently selected from a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , - N0 ₂ , -CN, a C ₃ -C ₆ carbocycle, and a 3- to 6-membered heterocycle; and C ₃ -C ₆ carbocycle optionally substituted with one or more substituents independently selected from a

halogen, -OH, -CN, -N0 ₂ , -NH ₂ , -C C ₃ alkyl-NH ₂ ; R ¹³ is independently selected at each occurrence from: a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -NO2, =0, =S, =N(R ¹⁰ ), and -CN; and C1-C10 alkyl and -C1-C10 haloalkyl; and

n is selected from 0, 1 or 2.

[0178] In a second aspect, for a compound of Formula (I), or a salt thereof:

W is N;

A is selected from an optionally substituted bicylic aromatic heterocycle, such as a quinazoline, wherein substituents on A are independently selected at each occurrence from R ¹² ;

X ¹ is selected from, -0-, -and - H-;

X ² is -0-;

L is selected from unsubstituted C ₆ -C alkylene and unsubstituted C ₆ -C alkenylene;

R ¹⁰ is independently selected at each occurrence from: hydrogen; and C ₁ -C ₃ alkyl, C ₃ -C ₆ carbocycle, or 5-7 membered heterocycle, each of which are optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , and -C ₁ -C ₃ alkyl-NH ₂ ;

R ¹² is independently selected at each occurrence from: a halogen, -OR ¹⁰ , -SR ¹⁰ , -N(R ¹⁰ ) ₂ , -C(0)R ¹⁰ , -C(O)N(R ¹⁰ ) ₂ , -N(R ¹⁰ )C(O)R ¹⁰ and -CN; and phenyl optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , -C ₁ -C ₃ alkyl-NH ₂ ;

n is 0.

[0179] In a third aspect, for a compound of Formula (I), or a salt thereof: W is N;

A is a quinazoline that is optionally substituted with one substituent selected from R ¹² ; X ¹ is selected from, -0-, -and -NH-;

X ² is selected from -0-;

L is unsubstituted C ₆ -Ci ₂ alkylene or unsubstituted C ₆ -Ci ₂ alkenylene, such as

unsubstituted C ₆ -C alkylene or unsubstituted C ₆ -C ₈ alkenylene;

R ¹² is -O-piperidine or a phenyl optionally substituted with -C ₁ -C3 alkyl-NH ₂ ; and n is 0.

[0180] In such first, second, or third as ect of a compound of Formula (I), A can be

represented by the following formula , wherein A is optionally substituted with one substituent selected from R ¹² . In such first, second, or third aspect of a compound of Formula (I), A can be represented by the following formula . In such first, second, or third aspect of a compound of Formula (I), L may be unsubstituted C ₇ alkenylene. In such first, second, or third aspect of a compound of Formula (I), L may be represented by:

or third aspect of a compound of Formula (I), L may be represented by:

[0181] In certain embodiments, a compound or salt of Formula (I) is represented by:

where the compound or salt may optionally include one or more substituents independently selected at each occurrence from R ¹² . In other embodiments, a compound or salt of Formula (I) is represented by:

where i is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0182] In some embodiments, a compound or salt of Formula (I) is selected from:

[0183] In some embodiments a compound or salt of Formula (I) is represented by:

[0184] In other embodiments, a compound or salt of Formula (I) is represented by:

where i is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. For example, i may be 7.

[0185] A compound or salt of any one of Formulas (I), (la), and (lb) may be covalently bound to a linker, L ³ . A linker may, for example, link a compound or salt of any one of Formulas (I), (la), and (lb) to a residue of an antibody construct. [0186] In some embodiments, for a compound or salt of any one of Formulas (I), (la), and (lb), the linker L ³ is covalently bound to an atom in A, X ¹ , X ² , or L or covalently bound to a substituent on A, X ¹ , X ² , or L. In certain embodiments, the linker L ³ is covalently bound to an atom in A or X ¹ or covalently bound to a substituent on A or X ¹ . In certain embodiments, the linker L ³ is not bound to X ² or a substituent on X ² .

[0187] The compound or salt of any one of Formulas (I), (la), and (lb), wherein the compound of Formula (I), (la), or (lb) is further bound to a linker and may be represented by:

in which X is selected from -0-, -NH-, -S-, saturated heterocycle, -O-saturated heterocycle-, a 5- or 6-membered aryl or heteroaryl ring, and -(aryl or heteroaryl)-alkylene-amine- and L ³ is a linker. In an example, X ¹⁰ is - H- and is linked to A through a methylene group. In certain embodiments, X ¹⁰ is selected from -(saturated heterocycle)-, such as -(saturated 5- or 6- membered heterocycle)-, e.g., piperizine, or piperidine. In certain embodiments, X ¹⁰ is selected from -0-(saturated heterocycle)-, such as -0-(saturated 5- or 6- membered heterocycle)-, e.g., - O-piperizine-, or -O-piperidine-. In certain embodiments, X ¹⁰ is selected from -(aryl or heteroaryl)-alkylene-amine-, such as phenyl-Ci-C ₃ alkylene-amine-, e.g., -phenyl-methylene- H- . In some embodiments, -X ¹⁰ -L ³ is selected from: In certain embodiments when L ³ is

cted from -C(R ¹⁰ ) ₂ - , -N(R ¹⁰ )-, -C(0)N(R ¹⁰ )-, and -N(R ¹⁰ )C(O)-, wherein L ³ replaces R ¹⁰ for -N(R ¹⁰ )-, -C(0)N(R ¹⁰ )-, and -N(R ¹⁰ )C(O)- and L ³ replaces one R ¹⁰ for -C(R ¹⁰ ) ₂ -.

[0188] In certain embodiments, the present disclosure provides a compound represented by the structure of Formula (II):

or a salt thereof, in which:

W is selected from CH, CR ² , and N;

A is selected from an optionally substituted C ₃ -C ₁₂ carbocycle and an optionally substituted 3- to 12-membered heterocycle, wherein substituents on A are independently selected at each occurrence from R ²² ;

Z is selected from Ci-Ci ₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₃ -Ci ₂ carbocycle, and a 3- to 12- membered heterocycle, any of which is optionally substituted by one or more substituents independently selected at each occurrence from R ²² ;

Q is selected from -0-, -S-, -N(R ²⁰ )-, -C(O)-, -C(0)0-, -OC(O)-, -OC(0)0-, - C(0)N(R ²⁰ )-, and -N(R ²⁰ )C(O)-; and -(C(R ²⁰ ) ₂ ) _m -, -O-(C(R ²⁰ ) ₂ ) _m -, and -(C(R ²⁰ ) ₂ ) _m -O-, wherein m is selected from 1, 2, 3, and 4;

R ² is selected at each occurrence from R ²² : R ²⁰ is independently selected at each occurrence from: -X'-L ³ , hydrogen; Ci-Cio alkyl, C ₂ -C ₁₀ alkenyl, and C ₂ -C ₁₀ alkynyl, each of which is optionally substituted at each occurrence with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , =0, =S, -O-Ci-Cio alkyl, C ₃ -C ₁₂ carbocycle, and a 3- to 12-membered heterocycle; and a C ₃ -C ₁₂ carbocycle and a 3- to 12-membered heterocycle, each of which is optionally substituted at each occurrence with one or more substituents independently selected from a halogen, -OH, -CN, - N0 ₂ , -NH ₂ , =0, =S, C ₁ -C ₁₀ alkyl, -O-Ci-C ₁₀ alkyl, and -Ci-Cio haloalkyl;

R 22 is independently selected at each occurrence from: -X'-U 3, a halogen, -OR 20 , -SR 20 , - N(R ²⁰ ) ₂ , -C(0)R ²⁰ , -C(O)N(R ²⁰ ) ₂ , -N(R ²⁰ )C(O)R ²⁰ -C(0)OR ²⁰ , -OC(0)R ²⁰ , -S(0)R ²⁰ , -S(0) ₂ R ² °, -S(O) ₂ N(R ²⁰ ) ₂ , -P(O)(OR ²⁰ ) ₂ , -OP(O)(OR ²⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ²⁰ ), and -CN; C1-C10 alkyl, C ₂ - C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, -O-Ci-Cio alkyl, and -C ₁ -C ₁₀ haloalkyl, each of which is optionally substituted at each occurrence with one or more substituents independently selected from a halogen, -OR ²⁰ , -SR ²⁰ , -N(R ²⁰ ) ₂ , -C(0)R ²⁰ , -C(O)N(R ²⁰ ) ₂ , -N(R ²⁰ )C(O)R ²⁰ -C(0)OR ²⁰ , - OC(0)R ²⁰ , -S(0)R ²⁰ , -S(0) ₂ R ²⁰ , -S(O) ₂ N(R ²⁰ ) ₂ , -P(O)(OR ²⁰ ) ₂ , -OP(O)(OR ²⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ²⁰ ), -CN, a C ₃ -C ₁₀ carbocycle, and a 3- to 10-membered heterocycle; and a C ₃ -C ₁₀ carbocycle and a 3- to 10-membered heterocycle, wherein each C ₃ -C ₁₀ carbocycle and 3- to 10- membered heterocycle in R ²² is independently optionally substituted with one or more substituents independently selected from a halogen, -OR ²⁰ , -SR ²⁰ , -N(R ²⁰ ) ₂ , -C(0)R ²⁰ , - C(O)N(R ²⁰ ) ₂ , -N(R ²⁰ )C(O)R ²⁰ -C(0)OR ²⁰ , -OC(0)R ²⁰ , -S(0)R ²⁰ , -S(0) ₂ R ² °, - S(O) ₂ N(R ²⁰ ) ₂ , -P(O)(OR ²⁰ ) ₂ , -OP(O)(OR ²⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ²⁰ ), -CN, C C ₆ alkyl, C ₂ -C ₆ alkenyl, and C ₂ -C ₆ alkynyl;

R ²³ is H;

X' is independently selected at each occurrence from a bond, -0-, -S-, NH-, -Ci-C ₆ alkylene, -Ci-C ₆ alkylene-NH-, -Ci-C ₆ alkylene-O-, and -Ci-C ₆ alkylene-S-;

n is selected from 0, 1, 2, 3, and 4; and

L 3 is a linker, wherei ·n at least one R 20 , or R 2"2 is -X'-L 3, or at least one substituent on A or Z is -X'-L ³ .

[0189] In certain embodiments, R ²² is independently selected at each occurrence from: -X'- L ³ , a halogen, -OR ²⁰ , -SR ²⁰ , -N(R ²⁰ ) ₂ , -C(0)R ²⁰ , -C(O)N(R ²⁰ ) ₂ , -N(R ²⁰ )C(O)R ²⁰ _, -C(0)OR ²⁰ , - OC(0)R ²⁰ , -S(0)R ²⁰ , -S(0) ₂ R ²⁰ , -S(O) ₂ N(R ²⁰ ) ₂ , -P(O)(OR ²⁰ ) ₂ , -OP(O)(OR ²⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ²⁰ ), and -CN; C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, and C ₂ -C ₁₀ alkynyl, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OR ²⁰ , -SR ²⁰ , - N(R ²⁰ ) ₂ , -C(0)R ²⁰ , -C(O)N(R ²⁰ ) ₂ , -N(R ²⁰ )C(O)R ²⁰ -C(0)OR ²⁰ , -OC(0)R ²⁰ , -S(0)R ²⁰ , -S(0) ₂ R ² °, -S(O) ₂ N(R ²⁰ ) ₂ , -P(O)(OR ²⁰ ) ₂ , -OP(O)(OR ²⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ²⁰ ), -CN, a C3-C10 carbocycle, and a 3- to 10-membered heterocycle; and a C ₃ -C ₁₀ carbocycle and a 3- to 10-membered heterocycle, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OR ²⁰ , -SR ²⁰ , -N(R ²⁰ ) ₂ , -C(0)R ²⁰ , -C(O)N(R ²⁰ ) ₂ , -N(R ²⁰ )C(O)R ²⁰ - C(0)OR ²⁰ , -OC(0)R ²⁰ , -S(0)R ²⁰ , -S(0) ₂ R ²⁰ , -S(O) ₂ N(R ²⁰ ) ₂ , -P(O)(OR ²⁰ ) ₂ , -OP(O)(OR ²⁰ ) ₂ , -N0 ₂ , =0, =S, =N(R ²⁰ ), -CN, Ci-C ₆ alkyl, C ₂ -C ₆ alkenyl, and C ₂ -C ₆ alkynyl.

[0190] In certain embodiments for a compound or salt of Formula (II), substituents on R ²⁰ when R ²⁰ is Ci-Cio alkyl, C ₂ -Cio alkenyl, C ₂ -Cio alkynyl, C ₃ -Ci ₂ carbocycle and 3- to 12- membered heterocycle may be further selected from tert-butyloxycarbonyl- H-. In certain embodiments, substituents on R ²⁰ when R ²⁰ is 3- to 12-membered heterocycle may be further selected from tert-butyloxycarbonyl-. In certain embodiments, for a compound or salt of

Formula (II), substituents on R ²⁰ when R ²⁰ is Ci-Cio alkyl, C ₂ -Cio alkenyl, C ₂ -Cio alkynyl, C ₃ -Ci ₂ carbocycle and 3- to 12-membered heterocycle are not selected from tert-butyloxycarbonyl- H-. In certain embodiments, substituents on R ²⁰ when R ²⁰ is 3- to 12-membered heterocycle are not selected from tert-butyloxycarbonyl-.

[0191] In certain embdoiments, R ²³ is further selected from L ³ . In certain embodiments for a

20 22 3 23 3

compound or salt of Formula (II), at least one R ^zu and R ^zz is -X'-U, or R" is ΙΛ In some embodiments, a compound or salt of Formula II) is represented by Formula (Ila):

[0192] In some embodiments, a compound or salt of Formula (II) is represented by Formula

(lib):

[0193] In some embodiments, a compound or salt of Formula (II) is represented by Formula (lie):

[0194] In some embodiments, for a compound or salt of any one of Formulas (II), (Ila), (lib), and (lie), W is N. In some embodiments, for a compound or salt of any one of Formulas (II), (Ila), (lib), and (He), W is CH. In some embodiments, W is CR ² , where R ² is selected from R ²² .

[0195] In some embodiments, for a compound or salt represented by any one of Formulas (II), (Ila), (lib), and (He), A is selected from a heterocycle optionally substituted with one or more substituents independently selected at each occurrence from R ²² . As described herein, heterocycles may include one or more heteroatom (e.g., 1, 2, 3, or more heteroatoms) and may be saturated, unsaturated, or aromatic. A heterocycle may be monocyclic or multi cyclic (e.g., bicyclic or tricyclic). In some embodiments, A is selected from an optionally substituted bicyclic heterocycle. Examples of bicyclic heterocycles include, but are not limited to, quinazoline, a quinoline, an isoquinoline, a benzimidazole, an indazole, a dihydrobenzoxaborole, a tetrahydroisoquinoline, a benzothiophene, a benzoxazole, a benzothiazole, an indole, a benzotriazole, a benzofuran, a dihydrobenzofuran, a benzodioxoline, a benzodioxin, an indolizine, an imidazopyridine, a purine, a benzothiadiazole, a benzoxadiazole, a

tetrahydroindole, an azaindole, an indazoline, a pyrrolopyrimidine, a pyrazolopyrimidine, a dihydrobenzoxaborinin, a dihydrobenzoxaborepin, a benzoxazaborinin, a benzodiazaborinin, a dihydrobenzodiazaborinin, and a benzoxazaborinin.

[0196] In some embodiments, for a compound or salt represented by any one of Formulas (II), (Ila), (lib), and (lie), A is an optionally substituted 8-12 membered bicyclic heterocycle. In certain embodiments, A is selected from a quinazoline, a quinoline, an isoquinoline, a

benzimidazole, an indazole, a dihydrobenzoxaborole, a tetrahydroisoquinoline, a

benzothiophene, a benzoxazole, a benzothiazole, an indole, and a benzotriazole, any of which may be optionally substituted with one or more substituents independently selected at each occurrence from R ²² . In some embodiments, for a compound or salt represented by any one of Formulas (II), (Ila), (lib), and (lie), A is selected from a quinazoline, a benzimidazole, and a dihydrobenzoxaborole. For example, A may be an optionally substituted benzimidazole. In certain embodiments, A is represented by: in which A is optionally substituted with one or more substituents independently selected at each occurrence from R ²² .

[0197] In other embodiments, A is a boron-containing heterocycle optionally substituted with one or more substituents independently selected at each occurrence from R ²² . For example, A may be selected from a dihydrobenzoxaborole, a dihydrobenzoxaborinin, a

dihydrobenzoxaborepin, a benzoxazaborinin, a benzodiazaborinin, a dihydrobenzodiazaborinin, and a benzoxazaborinin, any of which is optionally substituted with one or more substituents in :

[0198] In some embodiments, A is selected from:

which is optionally substituted with one or more substituents independently selected at each occurrence fromo R ²² . For example, A may be selected from:

[0199] In other embodiments, for a compound or salt represented by any one of Formulas (II), (Ila), (lib), and (He), A is selected from an optionally substituted monocyclic heterocycle, wherein substituents on A are independently selected at each occurrence from R ²² . Examples of monocyclic heterocycles include, but are not limited to, pyrazole, imidazole, pyrimidine, pyridine, piperidine, pyrrole, furan, and tetrahydrofuran. [0200] In certain embodiments, for a compound or salt represented by any one of Formulas (II), (Ila), (lib), and (lie), A is selected from an optionally substituted C ₃ -C ₁₂ carbocycle, wherein substituents on A are independently selected at each occurrence from R ²² . As described herein, carbocycles may be saturated, unsatured or aromatic and may be monocyclic or multi cyclic (e.g., bicyclic or tricyclic). Examples of carbocycles include, but are not limited to, cyclooctane, cycloheptane, cyclohexane, cyclopentane, benzene, naphthalene, tetralin, decalin, cyclooctene, cycloheptene, cyclohexene, and cyclopentene.

[0201] In some embodiments, for a compound or salt represented by any one of Formulas (II), (Ila), (lib), and (He), Z is selected from a C ₁ -C ₁₂ alkyl, a C ₂ -Ci ₂ alkenyl, an aryl group, and a 3- to 12-membered heterocycle, any of which is optionally substituted with substituents

independently selected at each occurrence from R ²² . In some embodiments, Z is an optionally substituted C ₂ -Ci ₀ alkylene, wherein substituents on Z are independently selected at each occurrence from R ²² . For example, Z may be selected from:

[0202] In other embodiments, for a compound or salt represented by any one of Formulas (II), (Ila), (lib), and (He), Z is selected from an optionally substituted C ₃ -C ₁₂ heterocycle, wherein substituents on Z are independently selected at each occurrence from R ²² . For example, Z may be a pyrrolidine, a tetrahydrofuran, a tetrahydrothiophene, an imidazolidine, a pyrazolidine, an oxazolidine, an isoxazolidine, a thiazolidine, an isothiazolidine, a dioxolane, a dithiolane, a piperidine, a tetrahydropyran, a thiane, a piperazine, a morpholine, a thiomorpholine, a dioxane, a dithiane, an azepane, an oxepane, a thiepane, and a diazepane, in which Z is optionally substituted with one or more substituents independently selected at each occurrence from R ²² . In certain embodiments, Z is an optionally substituted piperidine. For instance, Z may be represented by:

[0203] In other embodiments, for a compound or salt represented by any one of Formulas (II), (Ila), (lib), and (lie), Z is selected from a pyrrole, a furan, a thiophene, an imidazole, a pyrazole, an oxazole, a thiazole, a pyridine, a pyran, a thiopyran, an azepine, an oxepine, a thiepine, a diazepine, and a thiazepine.

[0204] In other embodiments, for a compound or salt represented by any one of Formulas (II), (Ila), (lib), and (He), Z is selected from an optionally substituted C3-C ₁₂ carbocycle, wherein substituents on Z are independently selected at each occurrence from R ²² . In certain embodiments, Z is selected from an optionally substituted C3-C12 cycloalkyl group. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutanyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, Z is an optionally substituted cyclohexyl group, in which substituents on Z are independently selected at each occurrence from

20 20

halogen, -OR , -SR , and -N sented by:

[0205] In some embodiments, for a compound or salt of any one of Formulas (II), (Ila), (lib), and (lie), at least one substituent on A is -X'-L ³ . In some embodiments, at least one substituent on Z is -X'-L ³ . A linker L ³ may be covalently bound to an atom in A or Z or a substituent on A or Z. For example, a compound or salt of any one of Formulas (II), (Ila), (lib), and (lie) may be represented by:

In some embodiments, at least one R ² is -X'-L ³ . In some embodiments, at least one R ²⁰ is -X'- L ³ . In some embodiments, at least one R ²² is -X'-L ³ .

[0206] In some embodiments, for a compound or salt represented by any one of Formulas (II), (Ila), (lib), and (lie), Q is selected from -0-, -S-, -N(R ²⁰ )-, -C(O)-, -N(R ²⁰ )C(O)-,

and -(C(R ²⁰ ) ₂ ) _m -, and m is selected from 1, 2, 3, and 4. In certain embodiments, Q is selected from -0-, -S-, -N(R ²⁰ )-, and -C(R ²⁰ ) ₂ -. For example, Q may be -0-. In some embodiments, Q is selected from -S-, -N(R ²⁰ )-, -C(O)-, -C(0)0-, -OC(O)-, -OC(0)0-, -C(0)N(R ²⁰ )-, and - N(R ²⁰ )C(O)-; and-(C(R ²⁰ ) ₂ ) _m -, -O-(C(R ²⁰ ) ₂ ) _m -, and-(C(R ²⁰ ) ₂ ) _m -O-, wherein m is selected from 1, 2, 3, and 4.

[0207] In some embodiments, for a compound or salt represented by any one of Formulas (II), (Ila), (lib), and (He), n is 1. In other embodiments, n is 2. In some embodiments, n is 0.

[0208] In some embodiments, for a compound or salt represented by any one of Formulas (II), (Ila), (lib), and (He), R ² is independently selected at each occurrence from a halogen, -OR ²⁰ , - SR ²⁰ , -N(R ²⁰ ) ₂ , -C(0)R ²⁰ , -C(O)N(R ²⁰ ) ₂ , -CN, -N0 ₂ , -Ci-Cio alkyl, -OCi-Cio alkyl, and -Ci-Cio haloalkyl.

[0209] In a fourth aspect, for a compound of Formula (II), or a salt thereof:

wherein A is optionally substituted with one or more substituents indepedently selected from

R ²² ;

Z is selected from C ₁ -C ₁₂ alkyl, C ₂ -Ci ₂ alkenyl, a C ₃ -Ci ₂ carbocycle, and a 3- to 12- membered heterocycle, any of which is optionally substituted by one or more substituents independently selected at each occurrence from R ²² ;

Q is -0-;

R ²⁰ is independently selected at each occurrence from: -X'-L ³ , hydrogen; C ₁ -C ₁₀ alkyl, C ₂ -Cio alkenyl, and C ₂ -Cio alkynyl, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , - H ₂ , tert-butoxycarbonyl- H-, =0, =S, -O-Ci-C ₁₀ alkyl, C ₃ -Ci ₂ carbocycle, and a 3- to 12-membered heterocycle; and a C ₃ -Ci ₂ carbocycle and a 3- to 12-membered heterocycle, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , tert-butoxycarbonyl-NH-, tert-butoxycarbonyl-, =0, =S, C ₁ -C ₁₀ alkyl, -O-Ci-Cio alkyl, and -Ci- Cio haloalkyl;

R 22 is independently selected at each occurrence from: -X'-U 3, a halogen, -OR 20 , -SR 20 , - N(R ²⁰ ) ₂ , -C(0)R ²⁰ , -S(0) ₂ R ²⁰ , and -CN; and Ci-C ₃ alkyl optionally substituted with one or more substituents independently selected from a halogen, -OR ²⁰ , -N(R ²⁰ ) ₂ , and -N0 ₂ ;

R ²³ is H or L ³ ;

X' is independently selected at each occurrence from a bond, -0-, -S-, NH-, -Ci-C ₆ alkylene, -Ci-C ₆ alkylene-NH-, -Ci-C ₆ alkylene-O-, and -Ci-C ₆ alkylene-S-;

n is 0; and

L 3 is a linker, wherei ·n at least one R 2 ^z 0 ^u and R 2 ^z 2 ^z i ·s -X'-L 3", or R 2"3 i ·s -V 3.

[0210] In a fifth aspect, for a compound of Formula (II), or a salt thereof:

wherein A is optionally substituted with one or more substituents indepedently selected from - X'-L ³ , -C(0)R ²⁰ and -S(0) ₂ R ²⁰ ; and Ci-C ₃ alkyl substituted with -N(R ²⁰ ) ₂ ; Z is selected from C ₂ -C ₆ alkyl, C ₅ -C ₆ cycloalkyl, and a 5- or 6-membered heterocycle, any of which is optionally substituted by one or more substituents independently selected from-X'-L ³ and -OR ²⁰ ;

Q is -0-;

R ²⁰ is independently selected from: -X'-L ³ ; hydrogen; Ci-Cio alkyl optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , tert-butoxycarbonyl-NH-, =0, =S, -O-Ci-Cio alkyl, C ₃ -Ci ₂ carbocycle, and a 3- to 12-membered heterocycle; and a C ₃ -C ₁₂ carbocycle and a 3- to 12-membered heterocycle, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , fert-butoxycarbonyl-NH-, fert-butoxycarbonyl-, =0, =S, Ci-Cio alkyl, -O-Ci- Cio alkyl, and -Ci-Cio haloalkyl;

R ²³ is H or L ³ ;

X' is independently selected at each occurrence from a bond, -0-, -S-, NH-, -Ci-C ₆ alkylene, -Ci-C ₆ alkylene-NH-, -Ci-C ₆ alkylene-O-, and -Ci-C ₆ alkylene-S-;

n is 0; and

L 3 is a linker, wherein at least one R 20 or one substituent on A or 3 , or R 23

Z is -X'-L is -

L ³ .

[0211] In a sixth aspect, for a compound of Formula (II), or a salt thereof:

wherein A is optionally substituted with one or more substituents independently selected from - X'-L ³ ; -C(0)R ²⁰ and -S(0) ₂ R ² °; and Ci-C ₄ alkyl, preferably Ci alkyl, substituted with -NH ₂ , - N0 ₂ , or -NHC(0)CH ₃ ;

Z is selected from -C ₁ -C ₁₂ alkyl-OH (preferably C ₂ -C ₇ alkyl-OH or C ₃ -C ₅ alkyl-OH), cyclohexyl, -cyclohexyl-OH, and piperidine, any of which is optionally substituted by -X'-L ³ ;

Q is -0-;

R ²⁰ is independently selected from: -X'-L ³ ; methyl optionally substituted with -NH ₂ or tert-butoxycarbonyl-NH-; and pyrrolidine and piperidine, each of which is optionally substituted with tert-butoxycarbonyl-;

R ²³ is H or L ³ ;

X' is independently selected at each occurrence from a bond, -0-, -S-, NH-, -Ci-C ₆ alkylene, -Ci-C ₆ alkylene-NH-, -Ci-C ₆ alkylene-O-, and -Ci-C ₆ alkylene-S-;

n is 0; and L 3 is a linker, wherei ·n at least one R 20 or one substi ·tuent on A or Z is -X'-I 3, or R 2"3 is

-L ³ .

[0212] In some embodiments, a compound or salt of any one of Formulas (II), (Ila), (lib), and (lie is represented by:

any of which may be optionally substituted by one of more substituents independently selected at each occurrence from R ²² , where at least one substituent on A or Z is -X'-L ³ , or at least one R ² ,

20 22 3 3

R , or R is -X'-L . For example, any of the compounds shown above may include -X'-L on A. In some embodiments, X' in any of the compounds shown above is -CH ₂ H-. In some embodiments, a compound or salt of any one of Formulas (II), (Ila), (lib), and (lie) is

represented b :

any of which may be optionally substituted by one of more substituents independently selected at each occurrence from R ²² , where X' is selected from a bond, -0-, -S-, H-, -Ci-C ₆ alkylene, -Ci- C ₆ alkylene-NH-, -Ci-C ₆ alkylene-O-, and -Ci-C ₆ alkylene-S- and L ³ is any linker. In some embodiments, X' in any of the compounds shown above is selected from -Ci-C ₆ alkylene-NH-. For example, X' may be -CH ₂ NH-.

[0213] In certain embodiments, the disclosure provides a compound represented by the structure of Formula (III):

or a salt thereof, in which:

W" is selected from CH, CR ^2' , and N;

B is selected from an optionally substituted 5- to 12-membered heterocycle, wherein at least one heteroatom in the heterocycle is a boron atom, and wherein substituents on B are independently selected at each occurrence from R ²² ;

Z' is selected from C _\ -Cu alkyl, C ₂ -C ₁₂ alkenyl, a C ₃ -Ci ₂ carbocycle, and a 3- to 12- membered heterocycle, any of which is optionally substituted by one or more substituents independently selected at each occurrence from R ²² ;

Q' is selected from -0-, -S-, -N(R ^20' )-, -C(O)-, -C(0)0-, -OC(O)-, -OC(0)0-, - C(0)N(R ^20' )-, and -N(R ^20' )C(O)-; and-(C(R ^20' ) ₂ ) _m -, -O-(C(R ^20' ) ₂ ) _m -, and-(C(R ^20' ) ₂ ) _m -O-, wherein m is selected from 1, 2, 3, and 4;

R ² is selected at each occurrence from R ^22' ; R ²⁰ is independently selected at each occurrence from: hydrogen; Ci-Cio alkyl, C ₂ -Ci ₀ alkenyl, and C ₂ -Cio alkynyl, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , - H ₂ , =0, =S, -O-Ci-Cio alkyl, C3-C12 carbocycle, and a 3- to 12-membered heterocycle; and a C3-C12 carbocycle and a 3- to 12- membered heterocycle, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , =0, =S, C1-C10 alkyl, -O-Ci-Cio alkyl, and -C1-C10 haloalkyl;

22' 20' 20'

R is independently selected at each occurrence from: a halogen, -OR ^zu , -SR ^ZU , - N(R ^20' ) ₂ , -C(0)R ^20' , -C(O)N(R ^20' ) ₂ , -N(R ^20' )C(O)R ^20' -C(0)OR ^20' , -OC(0)R ^20' , -S(0)R ^20' , - S(O) ₂ R ^20' , -S(O) ₂ N(R ^20' ) ₂ , -P(O)(OR ^20' ) ₂ , -OP(O)(OR ^20' ) ₂ , -N0 ₂ , =0, =S, =N(R ^20' ), and -CN; Ci- C10 alkyl, C ₂ -Cio alkenyl, C ₂ -Cio alkynyl, -O-Ci-Cio alkyl, and -C1-C10 haloalkyl, each of which is optionally substituted with one or more substituents independently selected from a halogen, - OR ^20' , -SR ^20' , -N(R ^20' ) ₂ , -C(0)R ^20' , -C(O)N(R ^20' ) ₂ , -N(R ^20' )C(O)R ^20' -C(0)OR ^20' , -OC(0)R ^20' , - S(0)R ^20' , -S(0) ₂ R ^20' , -S(O) ₂ N(R ^20' ) ₂ , -P(O)(OR ^20' ) ₂ , -OP(O)(OR ^20' ) ₂ , -N0 ₂ , =0, =S, =N(R ^20' ), - CN, a C3-C10 carbocycle, and a 3- to 10-membered heterocycle; and a C3-C10 carbocycle and a 3- to 10-membered heterocycle, wherein each C3-C10 carbocycle and 3- to 10-membered heterocycle in R ²² is independently optionally substituted with one or more substituents independently selected from a halogen, -OR ^20' , -SR ^20' , -N(R ^20' ) ₂ , -C(0)R ^20' , -C(O)N(R ^20' ) ₂ , - N(R ^20' )C(O)R ^20' , -C(O)OR ^20' , -OC(0)R ^20' , -S(0)R ^20' , -S(0) ₂ R ^20' , -

S(O) ₂ N(R ^20' ) ₂ , -P(O)(OR ^20' ) ₂ , -OP(O)(OR ^20' ) ₂ , -N0 ₂ , =0, =S, =N(R ^20' ), -CN, C C ₆ alkyl, C ₂ -C ₆ alkenyl, and C ₂ -C ₆ alkynyl; and

n is selected from 0, 1, 2, 3, and 4.

[0214] In certain embodiments for a compound or salt of Formula (III), R ²² is independently selected at each occurrence from: a halogen, -OR ^20' , -SR ^20' , -N(R ^20' ) ₂ , -C(0)R ^20' , -C(O)N(R ^20' ) ₂ , -N(R ^20' )C(O)R ^20' -C(0)OR ^20' , -OC(0)R ^20' , -S(0)R ^20' , -S(0) ₂ R ^20' , -

S(O) ₂ N(R ^20' ) ₂ , -P(O)(OR ^20' ) ₂ , -OP(O)(OR ^20' ) ₂ , -N0 ₂ , =0, =S, =N(R ^20' ), and -CN; C1-C10 alkyl, C ₂ -Cio alkenyl, C ₂ -Cio alkynyl, -O-Ci-Cio alkyl, and -C1-C10 haloalkyl, each of which is optionally substituted with one or more substituents independently selected from a halogen, - OR ^20' , -SR ^20' , -N(R ^20' ) ₂ , -C(0)R ^20' , -C(O)N(R ^20' ) ₂ , -N(R ^20' )C(O)R ^20' -C(0)OR ^20' , -OC(0)R ^20' , - S(0)R ^20' , -S(0) ₂ R ^20' , -S(O) ₂ N(R ^20' ) ₂ , -P(O)(OR ^20' ) ₂ , -OP(O)(OR ^20' ) ₂ , -N0 ₂ , =0, =S, =N(R ^20' ), - CN, a C3-C10 carbocycle, and a 3- to 10-membered heterocycle; and a C3-C10 carbocycle and a 3- to 10-membered heterocycle, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OR ^20' , -SR ^20' , -N(R ^20' ) ₂ , -C(0)R ^20' , - C(O)N(R ^20' ) ₂ , -N(R ^20' )C(O)R ^20' -C(0)OR ^20' , -OC(0)R ^20' , -S(0)R ^20' , -S(0) ₂ R ^20' , - S(O) ₂ N(R ^20' ) ₂ , -P(O)(OR ^20' ) ₂ , -OP(O)(OR ^20' ) ₂ , -N0 ₂ , =0, =S, =N(R ^20' ), -CN, d-C ₆ alkyl, C ₂ -C ₆ alkenyl, and C ₂ -C ₆ alkynyl.

[0215] In certain embodiments for a compound or salt of Formula (III), substituents on R ²⁰ when R ²⁰ is Ci-Ci ₀ alkyl, C ₂ -Ci ₀ alkenyl, C ₂ -Ci ₀ alkynyl, C ₃ -Ci ₂ carbocycle and 3- to 12- membered heterocycle may be further selected from tert-butyloxycarbonyl- H-. In certain embodiments, substituents on R ²⁰ when R ²⁰ is 3- to 12-membered heterocycle may be further selected from tert-butyloxycarbonyl-. In certain embodiments, for a compound or salt of Formula (III), substituents on R ²⁰ when R ²⁰ is Ci-Ci ₀ alkyl, C ₂ -Ci ₀ alkenyl, C ₂ -Ci ₀ alkynyl, C ₃ - Ci ₂ carbocycle and 3- to 12-membered heterocycle are not selected from tert-butyloxycarbonyl- NH-. In certain embodiments, substituents on R ²⁰ when R ²⁰ is 3- to 12-membered heterocycle are not selected from tert-butyloxycarbonyl-.

[0216] In some embodiments, a compound or salt of Formula (III) is represented by Formula

(Ilia):

[0217] In some embodiments, a compound or salt of Formula (III) is represented by Formula

(Illb):

[0218] In some embodiments, a compound or salt of Formula (III) is represented by Formula

(IIIc):

[0219] In some embodiments, for a compound or salt represented by any one of Formulas

(III), (Ilia), (Illb), and (IIIc), W" is CH. In some embodiments, W" is CR ² , where R ^2' is selected from R ²² . In some embodiments, for a compound or salt represented by any one of

Formulas (III), (Ilia), (Illb), and (IIIc), W" is N.

[0220] In some embodiments, for a compound or salt represented by any one of Formulas (III), (Ilia), (Illb), and (IIIc), B is selected from a dihydrobenzoxaborole, a

dihydrobenzoxaborinin, a dihydrobenzoxaborepin, a benzoxazabonnin, a benzodiazaborinin, a dihydrobenzodiazaborinin, and a benzoxazabonnin, any of which is optionally substituted with one or more substituents independently selected at each occurrence from R ^22' . In some embodiments, B is selected from:

substituted by one or more R

[0221] In some embodiments, for a compound or salt represented by any one of Formulas (III), (Ilia), (Illb), and (IIIc), Z' is selected from a C ₁ -C ₁₂ alkyl, a C ₂ -C ₁₂ alkenyl, an aryl group, and a 3- to 12-membered heterocycle, any of which is optionally substituted with substituents independently selected at each occunence from R ^22' . In some embodiments, Z' is an optionally substituted C ₂ -Cio alkylene, wherein substituents on Z' are independently selected at each occunence from R ²² . For example, Z' may be selected from:

[0222] In other embodiments, for a compound or salt represented by any one of Formulas (III), (Ilia), (Illb), and (IIIc), Z' is selected from an optionally substituted 3- to 10-membered heterocycle, wherein substituents on Z' are independently selected at each occurrence from R ^22' In certain embodiments, Z' is selected from an optionally substituted saturated 3- to 10- membered heterocycle, e.g., optionally substituted saturated 5- to 8-membered heterocycle. In certain embodiments, Z' is an optionally substituted saturated N-containing heterocycle. For example, Z' may be a pyrrolidine, a tetrahydrofuran, a tetrahydrothiophene, an imidazolidine, a pyrazolidine, an oxazolidine, an isoxazolidine, a thiazolidine, an isothiazolidine, a dioxolane, a dithiolane, a piperidine, a tetrahydropyran, a thiane, a piperazine, a morpholine, a

thiomorpholine, a dioxane, a dithiane, an azepane, an oxepane, a thiepane, and a diazepane, in which Z' is optionally substituted with one or more substituents independently selected at each occurrence from R ²² . In certain embodiments, Z' is an optionally substituted piperidine. For example, Z' may be represented by:

[0223] In other embodiments, for a compound or salt represented by any one of Formulas (III), (Ilia), (Illb), and (IIIc), Z' is selected from a pyrrole, a furan, a thiophene, an imidazole, a pyrazole, an oxazole, a thiazole, a pyridine, a pyran, a thiopyran, an azepine, an oxepine, a thiepine, a diazepine, and a thiazepine.

[0224] In other embodiments, for a compound or salt represented by any one of Formulas (III), (Ilia), (Illb), and (IIIc), Z' is selected from an optionally substituted C ₃ -Ci ₂ carbocycle, wherein substituents on Z' are independently selected at each occurrence from R ²² . In certain embodiments, Z' is selected from an optionally substituted C ₃ -Ci ₂ cycloalkyl group. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutanyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, Z' is an optionally substituted cyclohexyl group, in which substituents on Z' are independently selected at each occurrence

20' 20' 20'

from halogen, -OR , -SR , a -N(R ) ₂ . For example, Z' may be represented by:

[0225] In some embodiments, for a compound or salt represented by any one of Formulas (III), (Ilia), (Illb), and (IIIc), Q' is selected from -0-, -S-, -N(R ^20' )-, -C(O)-, -N(R ^20' )C(O)-, and -(C(R ²⁰ ) ₂ ) _m -, and m is selected from 1, 2, 3, and 4. In certain embodiments, Q' is selected from -0-, -S-, -N(R ^20' )-, and -C(R ^20' ) ₂ -. For example, Q' may be -0-. In some embodiments, Q' is selected from -S-, -N(R ^20' )-, -C(O)-, -C(0)0-, -OC(O)-, -OC(0)0-, -C(0)N(R ^20' )-, and - N(R ^20' )C(O)-; and -(C(R ^20' ) ₂ )m-, -O-(C(R ^20' ) ₂ ) _m -, and -(C(R ^20' ) ₂ ) _m -O-, wherein m is selected from 1, 2, 3, and 4. In some embodiments, Q' is selected from -S-, -N(R ^20' )-, -C(O)-, -C(0)0-, - C(0)N(R ^20' )-, and -N(R ^20' )C(O)-; and -(C(R ^20' ) ₂ ) _m -, -O-(C(R ^20' ) ₂ ) _m -, and -(C(R ^20' ) ₂ ) _m -O-, wherein m is selected from 1, 2, 3, and 4.

[0226] In some embodiments, for a compound or salt represented by any one of Formulas (III), (Ilia), (Illb), and (IIIc), n is 1. In other embodiments, n is 2. In some embodiments, n is 0.

[0227] In some embodiments, for a compound or salt represented by any one of Formulas (III), (Ilia), (Illb), and (IIIc), R ² is independently selected at each occurrence from a halogen, - OR ^20' , -SR ^20' , -N(R ^20' ) ₂ , -C(0)R ^20' , -C(O)N(R ^20' ) ₂ , -CN, -N0 ₂ , -Ci-Cio alkyl, -OCi-Cio alkyl, and -Ci-Cio haloalkyl.

[0228] In a seventh aspect for a compound of Formula (III), or a salt thereof: " is N;

wherein B is optionally substituted with one or more substituents independently selected from

R ^22' ;

Z' is selected from C _\ -Cu alkyl, C ₂ -C ₁₂ alkenyl, C ₃ -Ci ₂ carbocycle, and a 3- to 12- membered heterocycle, any of which is optionally substituted by one or more substituents independently selected at each occurrence from R ²² ;

Q' is -0-;

R ²⁰ is independently selected at each occurrence from: hydrogen; Ci-Ci ₀ alkyl, C ₂ -Ci ₀ alkenyl, and C ₂ -Cio alkynyl, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , tert-butoxycarbonyl-NH-, =0, =S, -O-Ci-Cio alkyl, C ₃ -C ₁₂ carbocycle, and a 3- to 12-membered heterocycle; and a C ₃ -C ₁₂ carbocycle and a 3- to 12-membered heterocycle, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , tert- butoxycarbonyl-NH-, tert-butoxycarbonyl-, =0, =S, Ci-Cio alkyl, -O-Ci-Cio alkyl, and -Ci-Cio haloalkyl;

22' 20 20

R is independently selected at each occurrence from: a halogen, -OR , -SR , - N(R ²⁰ ) ₂ , -C(0)R ²⁰ , -S(0) ₂ R ²⁰ , and -CN; and Ci-C ₃ alkyl optionally substituted with one or more substituents independently selected from a halogen, -OR ²⁰ , -N(R ²⁰ ) ₂ , and -N0 ₂ ; and

n is 0. [0229] In a eighth aspect, for a compound of Formula (III), or a salt thereof: " is N;

wherein B is optionally substituted with one or more substituents independently selected from - C(0)R ^20' and -S(0) ₂ R ^20' ; and C ₁ -C ₃ alkyl, preferably Ci alkyl, substituted with -N(R ^20' ) ₂ ^' ;

Z' is selected from C ₂ -C ₆ alkyl, C5-C6 cycloalkyl, and a 5- or 6-membered heterocycle, any of which is optionally substituted by one or more substituents independently selected from -

OR ^20' ;

Q' is -0-;

R ²⁰ is independently selected from hydrogen; C ₁ -C ₁₀ alkyl optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , - H ₂ , tert- butoxycarbonyl- H-, =0, =S, -O-Ci-Cio alkyl, C ₃ -C ₀ carbocycle, and a 3- to 12-membered heterocycle; and a C ₃ -Ci ₂ carbocycle and a 3- to 12-membered heterocycle, each of which is optionally substituted with one or more substituents independently selected from a halogen, -OH, -CN, -N0 ₂ , -NH ₂ , fert-butoxycarbonyl-NH-, fert-butoxycarbonyl-, =0, =S, C ₁ -C ₁₀ alkyl, -O-Ci- C ₁₀ alkyl, and -C ₁ -C ₁₀ haloalkyl; and

n is 0.

[0230] In a ninth aspect, for a compound of Formula (III), or a salt thereof: " is N;

wherein B is optionally substituted with one or more substituents independently selected from ; - C(0)R ^20' and -S(0) ₂ R ^20' ; and C ₁ -C4 alkyl, preferably Ci alkyl, substituted with -NH ₂ , -N0 ₂ , or - NHC(0)CH ₃ ;

Z' is selected from -C ₁ -C ₁₂ alkyl-OH (preferably C ₂ -C ₇ alkyl-OH or C ₃ -C ₅ alkyl-OH), cyclohexyl, -cyclohexyl-OH, and piperidine;

Q' is -0-;

R ²⁰ is independently selected from methyl optionally substituted with -NH ₂ or tert- butoxycarbonyl-NH-; and pyrrolidine and piperidine, each of which is optionally substituted with tert-butoxycarbonyl; and

n is 0. [0231] In some embodiments, a compound or salt of Formula (III) is represented by:

any of which may be optionally substituted by one of more substituents independently selected at each occurrence from R ²² .

[0232] A compound or salt of any one of Formulas (III), (Ilia), (Illb), and (IIIc) may be covalently bound to a linker, -L ³ . In certain embodiments, the linker is selected from -X'-L ³ , wherein X' is independently selected at each occurrence from a bond, -0-, -S-, - H-, -Ci-C ₆ alkylene, -Ci-C ₆ alkylene- H-, -Ci-C ₆ alkylene-O-, and -Ci-C ₆ alkylene-S-. A linker may, for example, link a compound or salt of any one of Formulas (III), (Ilia), (Illb), and (IIIc) to a residue of an antibody construct.

[0233] In some embodiments, for a compound or salt of any one of Formulas (III), (Ilia), (Illb), and (IIIc), the linker L ³ is covalently bound to an atom in B or Z'or L ³ is covalently bound to a substituent on B or Z' . For example, a compound or salt of any one of Formulas (III), (Ilia), (Illb), and (IIIc) may be represented by:

[0234] Exemplary linkers for a compound or salt of any one of the compounds or salts in this section are described in the subsequent section entitled "Linkers".

[0235] Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds. The term "stable", as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).

[0236] Included in the present disclosure are salts, particularly pharmaceutically acceptable salts, of the compounds described herein. The compounds of the present disclosure that possess a sufficiently acidic, a sufficiently basic, or both functional groups, can react with any of a number of inorganic bases, and inorganic and organic acids, to form a salt. Alternatively, compounds that are inherently charged, such as those with a quaternary nitrogen, can form a salt with an appropriate counterion, e.g., a halide such as bromide, chloride, or fluoride, particularly bromide.

[0237] The compounds described herein may in some cases exist as diastereomers, enantiomers, or other stereoisomeric forms. The compounds presented herein include all diastereomeric, enantiomeric, and epimeric forms as well as the appropriate mixtures thereof. Separation of stereoisomers may be performed by chromatography or by forming diastereomers and separating by recrystallization, or chromatography, or any combination thereof. (Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and Resolutions", John Wiley And Sons, Inc., 1981, herein incorporated by reference for this disclosure). Stereoisomers may also be obtained by stereoselective synthesis.

[0238] The methods and compositions described herein include the use of amorphous forms as well as crystalline forms (also known as polymorphs). The compounds described herein may be in the form of pharmaceutically acceptable salts. In some embodiments, active metabolites of these compounds having the same type of activity are included in the scope of the present disclosure. In addition, the compounds described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the compounds presented herein are also considered to be disclosed herein.

[0239] In certain embodiments, compounds or salts of the compounds of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc) may be prodrugs, e.g., wherein a hydroxyl in the parent compound is presented as an ester or a carbonate, or carboxylic acid present in the parent compound is presented as an ester. The term "prodrug" is intended to encompass compounds which, under physiologic conditions, are converted into pharmaceutical agents of the present disclosure. One method for making a prodrug is to include one or more selected moieties which are hydrolyzed under physiologic conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by enzymatic activity of the host animal such as by specific target cells in the host animal. For example, esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids and esters of phosphonic acids) are preferred prodrugs of the present disclosure.

[0240] Prodrug forms of the herein described compounds, wherein the prodrug is metabolized in vivo to produce a compound of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc) as set forth herein are included within the scope of the claims. In some cases, some of the herein-described compounds may be a prodrug for another derivative or active compound.

[0241] Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent is not. Prodrugs may help enhance the cell permeability of a compound relative to the parent drug. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. Prodrugs may be designed as reversible drug derivatives, for use as modifiers to enhance drug transport to site-specific tissues or to increase drug residence inside of a cell.

[0242] In certain embodiments, the prodrug may be converted, e.g., enzymatically or chemically, to the parent compound under the conditions within a cell. In certain embodiments, the parent compound includes an acidic moiety, e.g., resulting from the hydrolysis of the prodrug, which may be charged under the conditions within the cell. In some embodiments, the prodrug is converted to the parent compound once it has passed through the cell membrane into a cell. In certain embodiments, the parent compound has diminished cell membrane permeability properties relative to the prodrug, such as decreased lipophilicity and increased hydrophilicity.

[0243] In certain embodiments, the parent compound with the acidic moiety is retained within a cell for a longer duration than the same compound without the acidic moiety.

[0244] The parent compound, with an acidic moiety, may be retained within the cell, i.e., drug residence, for 10% or longer, such as 15% or longer, such as 20% or longer, such as 25% or longer, such as 30% or longer, such as 35% or longer, such as 40% or longer, such as 45% or longer, such as 50% or longer, such as 55% or longer, such as 60% or longer, such as 65% or longer, such as 70% or longer, such as 75% or longer, such as 80% or longer, such as 85% or longer, or even 90% or longer relative to the same compound without an acidic moiety.

[0245] In some embodiments, the design of a prodrug increases the lipophilicity of the pharmaceutical agent. In some embodiments, the design of a prodrug increases the effective water solubility. See, e.g., Fedorak et al, Am. J. Physiol, 269:G210-218 (1995); McLoed et al, Gastroenterol, 106:405-413 (1994); Hochhaus et al., Biomed. Chrom., 6:283-286 (1992); J.

Larsen and H. Bundgaard, Int. J. Pharmaceutics, 37, 87 (1987); J. Larsen et al., Int. J.

Pharmaceutics, 47, 103 (1988); Sinkula et al., J. Pharm. Sci., 64: 181-210 (1975); T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series; and Edward B. Roche, Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, all incorporated herein for such disclosure). According to another embodiment, the present disclosure provides methods of producing the above-defined compounds. The compounds may be synthesized using conventional techniques.

Advantageously, these compounds are conveniently synthesized from readily available starting materials.

[0246] Synthetic chemistry transformations and methodologies useful in synthesizing the compounds described herein are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations (1989); T. W. Greene and P. G. M.

Wuts, Protective Groups in Organic Synthesis, 2d. Ed. (1991); L. Fieser and M. Fieser, Fieser and Fieser 's Reagents for Organic Synthesis (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis (1995).

Conjugates and Linkers

[0247] The compounds and salts described herein may be bound to a linker, e.g., a cleavable linker or a non-cleavable linker. In certain embodiments, the linker is also bound to an antibody construct and may be referred to as an antibody construct conjugate or conjugate. Linkers of the conjugates may not affect the binding of active portions of a conjugate, e.g., the antigen binding domains, Fc domains, target binding domains, antibodies, or the like, to a target, which can be a cognate binding partner such as an antigen. A conjugate can comprise multiple linkers, each having one or more compounds attached. These linkers can be the same linkers or different linkers.

[0248] A linker can be short, flexible, rigid, cleavable, non-cleavable, hydrophilic, or hydrophobic. A linker can contain segments that have different characteristics, such as segments of flexibility or segments of rigidity. The linker can be chemically stable to extracellular environments, for example, chemically stable in the blood stream, or may include linkages that are not stable or selectively stable. The linker can include linkages that are designed to cleave and/or immolate or otherwise breakdown specifically or non-specifically inside cells. A cleavable linker can be sensitive to enzymes. A cleavable linker can be cleaved by enzymes such as proteases. A cleavable linker may comprise a valine-citrulline (Val-Cit) dipeptide or a valine- alanine (Val-Ala) dipeptide. A valine-citrulline or valine-alanine containing linker can contain a pentafluorophenyl group. A valine-citrulline or valine-alanine containing linker can contain a succinimide group. A valine-citrulline or valine-alanine containing linker can contain a para- aminobenzyl alcohol (PABA) group or a para-ami nob enzyl carbamate (PABC) group. A valine- citrulline or valine-alanine containing linker can contain a PABA group and a pentafluorophenyl group. A valine-citrulline or valine-alanine containing linker can contain a PABA group and a succinimide group.

[0249] A cleavable linker can include a maleimido group, such as maleimidocaproyl, attached to a peptide. The peptide can be, for example, valine-citrulline, valine-lysine, valine-alanine, or the like.

[0250] A non-cleavable linker can be protease insensitive. A non-cleavable linker can be maleimidocaproyl linker. A maleimidocaproyl linker can comprise N- maleimidomethylcyclohexane-l-carboxylate. A maleimidocaproyl linker can contain a succinimide group. A maleimidocaproyl linker can contain pentafluorophenyl group. A linker can be a combination of a maleimidocaproyl group and one or more polyethylene glycol molecules. A linker can be a maleimide-PEG4 linker. A linker can be a combination of a maleimidocaproyl linker containing a succinimide group and one or more polyethylene glycol molecules. A linker can be a combination of a maleimidocaproyl linker containing a

pentafluorophenyl group and one or more polyethylene glycol molecules. A linker can contain maleimides linked to polyethylene glycol molecules in which the polyethylene glycol can allow for more linker flexibility or can be used lengthen the linker. A linker can be a

(maleimidocaproyl)-(valine-citrulline)-(para-aminobenzylo xycarbonyl)- linker. A linker can be a THIOMAB linker. A THIOMAB linker can be a (maleimidocaproyl)-(valine-citrulline)-(para- aminobenzyloxycarbonyl)- linker.

[0251] A linker can also comprise alkylene, alkenylene, alkynylene, polyether, polyester, polyamide group(s) and also, polyamino acids, polypeptides, cleavable peptides, or

aminobenzylcarbamates. A linker can contain a maleimide at one end and an N- hydroxysuccinimidyl ester at the other end. A linker can contain a lysine with an N-terminal amine acetylated, and a valine-citrulline cleavage site. A linker can be a link created by a microbial transglutaminase, wherein the link can be created between an amine-containing moiety and a moiety engineered to contain glutamine as a result of the enzyme catalyzing a bond formation between the acyl group of a glutamine side chain and the primary amine of a lysine chain. A linker can contain a reactive primary amine. A linker can be a Sortase A linker. A Sortase A linker can be created by a Sortase A enzyme fusing an LXPTG recognition motif to an N-terminal GGG motif to regenerate a native amide bond. The linker created can therefore link a moiety attached to the LXPTG recognition motif with a moiety attached to the N-terminal GGG motif.

[0252] In the conjugates, a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ha), (lib), (lie), (III), (Ilia), (Illb), and (IIIc) is linked to the antibody construct by way of a linker(s), also referred to herein as L ³ . L ³ , as used herein, may be selected from any of the linkers discussed herein. The linker linking the compound or salt to the antibody construct of a conjugate may be short, long, hydrophobic, hydrophilic, flexible or rigid, or may be composed of segments that each independently have one or more of the above-mentioned properties such that the linker may include segments having different properties. The linkers may be polyvalent such that they covalently link more than one compound or salt to a residue of an antibody construct, or monovalent such that covalently they link a single compound or salt to a a residue of an antibody construct.

[0253] As will be appreciated by skilled artisans, the linkers may link a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc) to the antibody construct by covalent linkages between the linker and the antibody construct and compound. As used herein, the term "linker" may be used to refer to (i) unconjugated forms of the linker that include a functional group capable of covalently linking the linker to a

compound(s) or salt(s) of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc) and a functional group capable of covalently linking the linker to an antibody construct; (ii) partially conjugated forms of the linker that include a functional group capable of covalently linking the linker to an antibody construct and that is covalently linked to a compound(s) or salt(s) of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc), or vice versa; and (iii) fully conjugated forms of the linker that are covalently linked to both a compound(s) or salt(s) of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc) and an antibody construct. One embodiment pertains to a conjugate formed by contacting an antibody construct that binds a cell surface receptor or tumor associated antigen expressed on a tumor cell with a linker-compound described herein under conditions in which the linker-compound covalently links to the antibody construct. One embodiment pertains to a method of making a conjugate formed by contacting a linker- compound described herein under conditions in which the linker-compound covalently links to the antibody construct. One embodiment pertains to a method of stimulating immune activity in a cell that expresses a target antigen, comprising contacting the cell with a conjugate that is capable of binding the cell, under conditions in which the conjugate binds the cell.

[0254] In certain embodiments, for a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc), L ³ is represented by the formula:

in which L ⁴ represents the C-terminus of the peptide and L ⁵ is selected from a bond, alkylene and heteroalkylene, wherein L ⁵ is optionally substituted with one or more groups independently selected from R ³² , RX is a reactive moiety; and R ³² is independently selected at each occurrence from halogen, -OH, -CN, -O-alkyl, -SH, =0, =S, - H ₂ , -N0 ₂ ; and Ci-i ₀ alkyl, C ₂ -io alkenyl, and C _2- io alkynyl, each of which is independently optionally substituted with one or more substituents selected from halogen, -OH, -CN, -O-alkyl, -SH, =0, =S, -NH ₂ , and -N0 ₂ .

[0255] In certain embodiments, for a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc), RX may include a leaving group or be a maleimide. In certain embodiments, L ³ is represented by the formula:

in which L ⁴ represents the C-terminal of the peptide and L ⁵ is selected from a bond, alkylene and heteroalkylene, wherein L ⁵ is optionally substituted with one or more groups independently selected from R ³² ; RX* is a bond, a succinimide moiety, or a hydrolyzed succinimide moiety bound to a residue of an antibody construct, wherein on RX* represents the point of attachment to the residue of the antibody construct; and R ³² is independently selected at each occurrence from halogen, -OH, -CN, -O-alkyl, -SH, =0, =S, -NH ₂ , -N0 ₂ ; and d-io alkyl, C ₂ . ₁₀ alkenyl, and C ₂ . ₁₀ alkynyl, each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -OH, -CN, -O-alkyl, -SH, =0, =S, -NH ₂ , and -N0 ₂ .

[0256] In certain embodiments, for a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc), the peptide comprises Cit-Val.

[0257] In certain embodiments, for a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc), L ³ is selected from:

[0258] In certain embodiments, for a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc), L ³ is represented by the formula:

in which RX comprises a reactive moiety. In certain embodiments, L is represented by the formula: in which RX* is a bond, a succinimide moiety, or a hydrolyzed succinimide moiety, wherein on RX* represents the point of attachment to the residue of the antibody construct. For example L ³ may be represented by the formula:

in which the wavy line indicates an attachment to a heterocyclic compound. [0259] In certain embodiments, for a compound or salt of any one of Formulas (I), (la), (lb),

(II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc), L ³ includes a hydrazone moiety. For example, L ³ may be selected from:

in which the wavy line indicates to the point of attachment to the compound. In some embodiments, M is selected from the group consisting of Ci-C ₆ alkyl, aryl, and -0-Ci-C ₆ alkyl.

[0260] In certain embodiments, for a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc), L ³ includes a self-stabilizing moiety. For example, L ³ m lected from:

in which the wavy line indicates to the point of attachment to the compound.

[0261] In certain embodiments, for a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc), L ³ includes a methylene carbamate unit.

[0262] A linker may be a polyvalent linker that may be used to link one or more compounds or salts of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc) to a residue of an antibody construct. For example, Fleximer® linker technology has the potential to enable high-DAR (drug-to-antibody ratio) conjugates with useful physicochemical properties. As shown below, the Fleximer® linker technology is based on incorporating drug molecules into a solubilizing poly-acetal backbone via a sequence of ester bonds:

O-Drug' O-Drug' O-Drug'

The methodology renders highly-loaded conjugates (DAR up to 20) while maintaining useful physicochemical properties. This methodology could be utilized with a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc).

[0263] In certain embodiments, to utilize the Fleximer® linker technology depicted above, an aliphatic alcohol can be present or introduced into a heterocyclic compound or salt thereof. The alcohol moiety is then conjugated to an alanine moiety, which is then synthetically incorporated into the Fleximer® linker. Liposomal processing of the conjugate in vitro releases the parent alcohol-containing drug.

[0264] By way of example and not limitation, some cleavable and noncleavable linkers that may be included in the conjugates are described below.

[0265] Cleavable linkers can be cleavable in vitro and in vivo. Cleavable linkers can include chemically or enzymatically unstable or degradable linkages. Cleavable linkers can rely on processes inside the cell to liberate a heterocyclic compound, such as reduction in the cytoplasm, exposure to acidic conditions in the lysosome, or cleavage by specific proteases or other enzymes within the cell. Cleavable linkers can incorporate one or more chemical bonds that are either chemically or enzymatically cleavable while the remainder of the linker can be noncleavable.

[0266] A linker can contain a chemically labile group such as hydrazone and/or disulfide groups. Linkers comprising chemically labile groups can exploit differential properties between the plasma and some cytoplasmic compartments. The intracellular conditions that can facilitate heterocyclic compound release for hydrazone containing linkers can be the acidic environment of endosomes and lysosomes, while the disulfide containing linkers can be reduced in the cytosol, which can contain high thiol concentrations, e.g., glutathione. The plasma stability of a linker containing a chemically labile group can be increased by introducing steric hindrance using substituents near the chemically labile group. [0267] Acid-labile groups, such as hydrazone, can remain intact during systemic circulation in the blood's neutral pH environment (pH 7.3-7.5) and can undergo hydrolysis and can release the heterocyclic compound once the conjugate (e.g, the antibody construct, the heterocyclic compound and the linker) is internalized into mildly acidic endosomal (pH 5.0-6.5) and lysosomal (pH 4.5-5.0) compartments of the cell. This pH dependent release mechanism can be associated with nonspecific release of the drug. To increase the stability of the hydrazone group of the linker, the linker can be varied by chemical modification, e.g., substitution, allowing tuning to achieve more efficient release in the lysosome with a minimized loss in circulation.

[0268] Hydrazone-containing linkers can contain additional cleavage sites, such as additional acid-labile cleavage sites and/or enzymatically labile cleavage sites. Exemplary hydrazone- containing linkers include for example, the following structures:

in which the wavy line represents the point of attachment to a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc). In some

embodiments, M may be selected from the group consisting of Ci-C ₆ alkyl, aryl, and -0-Ci-C ₆ alkyl. The point of attachment to a residue of an antibody construct may be at the opposite end of the linker. Certain linkers can include two or more cleavable groups such as a disulfide and a hydrazone moiety. For such linkers, effective release of the unmodified free heterocyclic compound can require acidic pH or disulfide reduction and acidic pH. Other linkers can be effective with a single hydrazone cleavage site.

[0269] Acid-labile linkers may also include silyl ethers. For example, in certain

embodiments, for a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (He), (III), (Ilia), (Illb), and (IIIc), L ³ is represented by the formula: ^ ^S '^o( ^{C Cs alkylene} )~RX

in which each R ³⁰ is independently selected from optionally substituted Ci- ₆ alkyl and optionally substituted phenyl; the wavy line indicates an attachment to the reaminder of the compound; and RX includes a reactive moiety. RX may include a leaving group or be a maleimide. In certain embodiments, L ³ is represented by the formula: wherein RX ^* is a bond, a succinimide moiety, or a hydrolyzed succinimide moiety bound to a residue of an antibody construct, wherein ^ on RX* represents the point of attachment to the residue of the antibody construct; and R ³⁰ is independently selected from optioinally substituted Ci-C ₆ alkyl and optionally substituted phenyl.

[0270] Other acid-labile groups that can be included in linkers include cz ^' s-aconityl-containing linkers, cz ^' s- Aconityl chemistry can use a carboxylic acid juxtaposed to an amide bond to accelerate amide hydrolysis under acidic conditions.

[0271] Cleavable linkers can also include a disulfide group. Disulfides can be

thermodynamically stable at physiological pH and can be designed to release the heterocyclic compound upon internalization inside cells, wherein the cytosol can provide a significantly more reducing environment compared to the extracellular environment. Scission of disulfide bonds can require the presence of a cytoplasmic thiol cofactor, such as (reduced) glutathione (GSH), such that disulfide-containing linkers can be reasonably stable in circulation, selectively releasing the heterocyclic compound in the cytosol. The intracellular enzyme protein disulfide isomerase, or similar enzymes capable of cleaving disulfide bonds, can also contribute to the preferential cleavage of disulfide bonds inside cells. GSH can be present in cells in the concentration range of 0.5-10 mM compared with a significantly lower concentration of GSH or cysteine, the most abundant low-molecular weight thiol, in circulation at approximately 5 μΜ. Tumor cells, where irregular blood flow can lead to a hypoxic state, can result in enhanced activity of reductive enzymes and therefore even higher glutathione concentrations. The in vivo stability of a disulfide-containing linker can be enhanced by chemical modification of the linker, e.g., use of steric hindrance adjacent to the disulfide bond.

[0272] Disulfide-containing linkers can include the following structures:

in which the wavy line represents the point of attachment to a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc) and R is independently selected at each occurrence from hydrogen or alkyl, for example. The point of attachment to a residue of an antibody construct may be at the opposite end of the linker (e.g., -SH or - H ₂ ). Increasing steric hindrance adjacent to the disulfide bond can increase the stability of the linker. Structures such as those shown above can show increased in vivo stability when one or more R groups is selected from a lower alkyl group such as methyl.

[0273] Another type of linker that can be used is a linker that is specifically cleaved by an enzyme. For example, the linker can be cleaved by a lysosomal enzyme. Such linkers can be peptide-based or can include peptidic regions that can act as substrates for enzymes. Peptide based linkers can be more stable in plasma and extracellular milieu than chemically labile linkers.

[0274] Peptide bonds can have good serum stability, as lysosomal proteolytic enzymes can have very low activity in blood due to endogenous inhibitors and the unfavorably high pH value of blood compared to lysosomes. Release of a heterocyclic compound from a residue of an antibody construct can occur due to the action of lysosomal proteases, e.g., cathepsin and plasmin. These proteases can be present at elevated levels in certain tumor tissues. The linker can be cleavable by a lysosomal enzyme. The lysosomal enzyme can be, for example, cathepsin B, cathepsin S, β-glucuronidase, or β-galactosidase.

[0275] The cleavable peptide can be selected from tetrapeptides such as Gly-Phe-Leu-Gly, Ala-Leu- Ala-Leu or dipeptides such as Val-Cit, Val-Ala, and Phe-Lys; and optionally with a maleimidyl caproyl group (mc). Dipeptides can have lower hydrophobicity compared to longer peptides. In an example, a compound or salt of Formula (I), having a linker L ³ attached thereto, may be represented by:

For example, a compound or salt of Formula (I) may be:

[0276] A variety of dipeptide-based cleavable linkers can be used in the conjugates.

[0277] Enzymatically cleavable linkers can include a self-immolative component to spatially separate the heterocyclic compound from the site of enzymatic cleavage. The direct attachment of a heterocyclic compound to a peptide linker can result in proteolytic release of the heterocyclic compound or of an amino acid adduct of the heterocyclic compound, thereby impairing its activity. The use of a self-immolative component can allow for the elimination of the fully active, chemically unmodified heterocyclic compound upon amide bond hydrolysis.

[0278] One self-immolative component (e.g., spacer) can be a bifunctional /?ara-aminobenzyl alcohol group, which can link to the peptide through the amino group, forming an amide bond, while amine-containing heterocyclic compounds can be attached through carbamate

functionalities to the benzylic hydroxyl group of the linker (to give a ^-amidobenzylcarbamate, PABC). The resulting pro-heterocyclic compound can be activated upon protease-mediated cleavage, leading to a 1,6-elimination reaction releasing the unmodified heterocyclic compound, carbon dioxide, and remnants of the linker. The following scheme depicts the fragmentation of p- amidobenzyl carbamate and release of the heterocyclic compound:

X-D

wherein X-D represents the unmodified heterocyclic compound. Heterocyclic variants of this self-immolative group have also been described. Another example of a self-immolative component includes a methylene carbamate unit. A self-immolative component may have the form:

in which the wavy line represents the point of attachment to additional linker components or a residue of an antibody construct; Y is an activatable self-immolative moiety; X-D is a heterocyclic compound or salt thereof, where X is, for example, a heteroatom such as oxygen; and R is independently selected at each occurrence from hydrogen, optionally substituted Ci _-6 alkylene, an optionally substituted C ₃ . ₁₂ carbocycle, and an optionally substituted 3- to 12- membered heterocycle, and a carbocycle or heterocycle interspersed with one or more alkylene groups.

[0279] The enzymatically cleavable linker can be a β-glucuronic acid-based linker. Facile release of the heterocyclic compound can be realized through cleavage of the β-glucuronide glycosidic bond by the lysosomal enzyme β-glucuronidase. This enzyme can be abundantly present within lysosomes and can be overexpressed in some tumor types, while the enzyme activity outside cells can be low. β-Glucuronic acid-based linkers can be used to circumvent the tendency of conjugate to undergo aggregation due to the hydrophilic nature of β-glucuronides. In certain embodiments, β-glucuronic acid-based linkers can link an antibody construct to a hydrophobic heterocyclic compound. The following scheme depicts the release of a heterocyclic compound (D) from an antibody construct-heterocyclic compound conjugate containing a β- lucuronic acid-based linker:

in which Ab indicates the antibody construct.

[0280] A variety of cleavable β-glucuronic acid-based linkers useful for linking drugs such as auristatins, camptothecin and doxorubicin analogues, CBI minor-groove binders, and psymberin to antibodies have been described. These β-glucuronic acid-based linkers may be used in the conjugates. In certain embodiments, the enzymatically cleavable linker is a β-galactoside-based linker. β-Galactoside is present abundantly within lysosomes, while the enzyme activity outside cells is low.

[0281] Additionally, heterocyclic compounds containing a phenol group can be covalently bonded to a linker through the phenolic oxygen. One such linker relies on a methodology in which a diamino-ethane "Space Link" is used in conjunction with traditional "PAB"-based self- immolative groups to deliver phenols.

[0282] Cleavable linkers can include non-cleavable portions or segments, and/or cleavable segments or portions can be included in an otherwise non-cleavable linker to render it cleavable. By way of example only, polyethylene glycol (PEG) and related polymers can include cleavable groups in the polymer backbone. For example, a polyethylene glycol or polymer linker can include one or more cleavable groups such as a disulfide, a hydrazone or a dipeptide.

[0283] Other degradable linkages that can be included in linkers can include ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a heterocyclic compound, wherein such ester groups can hydrolyze under

physiological conditions to release the heterocyclic compound. Hydrolytically degradable linkages can include, but are not limited to, carbonate linkages; imine linkages resulting from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a hydroxyl group of an oligonucleotide. [0284] A linker can contain an enzymatically cleavable peptide moiety. For example, a linker may comprise a moiety selected from:

or a salt thereof, in which "peptide" represents a peptide (illustrated in N→C orientation, in which peptide includes the amino and carboxy "termini") that is cleavable by a lysosomal enzyme; T represents a polymer including one or more ethylene glycol units or an alkylene chain, or combinations thereof; R ^a is selected from hydrogen, alkyl, sulfonate and methyl sulfonate; R ^y is hydrogen or C _1-4 alkyl-(0) _r -(Ci _-4 alkylene) _s -G ¹ or C _1-4 alkyl-(N)-[(Ci _-4 alkylene)- G ¹ ^; R ^z is C _M alkyl-(0) _r -(Ci _-4 alkylene) _s -G ² ; G ¹ is S0 ₃ H, C0 ₂ H, PEG 4-32, or a sugar moiety; G ² is S0 ₃ H, C0 ₂ H, or a PEG 4-32 moiety; r is 0 or 1; s is 0 or 1; p is an integer ranging from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1; the wavy line represents the point of attachment of the linker to a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ha), (lib), (lie), (III), (Ilia), (Illb), and (IIIc); and * represents the point of attachment to another portion of the linker or to a residue of an antibody construct.

[0285] In certain embodiments, the peptide can be selected from natural amino acids, unnatural amino acids or combinations thereof. In certain embodiments, the peptide can be selected from a tripeptide or a dipeptide. In certain embodiments, the dipeptide can include L- amino acids and be selected from: 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; He-Cit; Cit-He; Phe-Arg; Arg-Phe; Cit-Trp; and Trp-Cit, or salts thereof. [0286] Exemplary embodiments of linkers including an enzymatically cleavable peptide moiety are illustrated below (as illustrated, the linkers include a reactive group suitable for covalently linking the linker to an antibody construct):

in which the wavy line indicates an attachment site of a linker L ³ to a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc).

[0287] Exemplary embodiments of linkers including an enzymatically cleavable peptide moiety are illustrated below (as illustrated, the linkers can include a reactive group suitable for covalently linking the linker to an antibody construct):

(IVc.l),

(IVc.5), in which the wavy line indicates an attachment site to a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (nib), and (IIIc).

[0288] The linker can contain an enzymatically cleavable sugar moiety. For example, a linker ma include one of the following structures:

(Vd), and

or a salt thereof, wherein: q is 0 or 1 ; r is 0 or 1 ; X ¹ is CH ₂ , O or H; the wavy line represents the point of attachment of the linker L ³ to the compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc); and * represents the point of attachment to another portion of the linker or to a residue of an antibody construct.

[0289] Exemplary embodiments of linkers according to structural formula (Va) that may be included in the conjugates include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody construct):

118 8),

in which the wavy line represents the point of attachment of the linker L ³ to the compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc).

[0290] Exemplary embodiments of linkers according to structural formula (Vb) that may be included in the conjugates include the linkers illustrated below (as illustrated, the linkers include a roup suitable for covalently linking the linker to an antibody construct):

121

in which the wavy line represents the point of attachment of the linker L ³ to the compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc).

[0291] Exemplary embodiments of linkers according to structural formula (Vc) that may be included in the conjugates include the linkers illustrated below (as illustrated, the linkers include a roup suitable for covalently linking the linker to an antibody construct):



in which the wavy line represents the point of attachment of the linker L ³ to the compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc).

[0292] Exemplary embodiments of linkers according to structural formula (Vd) that may be included in the conjugates include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody construct):

(Vd.3),

(Vd.4),

in which the wavy line represents the point of attachment of the linker L ³ to the compound of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc). [0293] Exemplary embodiments of linkers according to structural formula (Ve) that may be included in the conjugates include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody construct):

in which the wavy line represents the point of attachment of the linker L ³ to the compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc).

[0294] Although cleavable linkers can provide certain advantages, the linkers need not be cleavable. For non-cleavable linkers, the heterocyclic compound release may not depend on the differential properties between the plasma and some cytoplasmic compartments. The release of the heterocyclic compound can occur after internalization of the conjugate via antigen-mediated endocytosis and delivery to lysosomal compartment, where the antibody construct or a portion thereof can be degraded to the level of amino acids through intracellular proteolytic degradation. This process can release a heterocyclic compound derivative (a metabolite of the conjugate containing a non-cleavable linker-heterocyclic compound), which is formed by the heterocyclic compound, the linker, and the amino acid residue or residues to which the linker was covalently attached. The heterocyclic compound derivative from conjugates with non-cleavable linkers can be more hydrophilic and less membrane permeable, which can lead to less bystander effects and less nonspecific toxicities compared to conjugates with a cleavable linker. Conjugates with non- cleavable linkers can have greater stability in circulation than conjugates with cleavable linkers. Non-cleavable linkers can include alkylene chains, or can be polymeric, such as, for example, based upon polyalkylene glycol polymers, amide polymers, or can include segments of alkylene chains, polyalkylene glycols and/or amide polymers. The linker can contain a polyethylene glycol segment having from 1 to 6 ethylene glycol units.

[0295] The linker can be non-cleavable in vivo. For example, a linker may include one of the

followin structures: (Via),

or salts thereof, which is selected from hydrogen, alkyl, sulfonate and methyl sulfonate; R ^x is a reactive moiety including a functional group capable of covalently linking the linker to an antibody construct; and the wavy line represents the point of attachment of the linker L ³ to the compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc).

[0296] Exemplary embodiments of linkers according to structural formula (Vla)-(VIe) that may be included in the conjugates include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody construct, and wavy lines represent the point of attachment of the linker L ³ to the compound or salt of any one of Formulas (I) (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc)):

O (VId. l),

in which the wavy line indicates an attachment to a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc).

[0297] Attachment groups that are used to attach the linkers to an antibody construct can be electrophilic in nature and include, for example, maleimide groups, activated disulfides, active esters such as NHS esters and HOBt esters, haloformates, acid halides, alkyl, and benzyl halides such as haloacetamides. There are also emerging technologies related to "self-stabilizing" maleimides and "bridging disulfide" that can be used in accordance with the disclosure.

[0298] Maleimide groups are frequently used in the preparation of conjugates because of their specificity for reacting with thiol groups of, for example, cysteine groups of the antibody residue of a conjugate. The reaction between a thiol group of an antibody residue and a drug with a linker including a maleimide group proceeds according to the following scheme:

Antibod

[0299] The reverse reaction leading to maleimide elimination from a thio- substituted succinimide may also take place. This reverse reaction is undesirable as the maleimide group may subsequently react with another available thiol group such as other proteins in the body having available cysteines. Accordingly, the reverse reaction can undermine the specificity of a conjugate. One method of preventing the reverse reaction is to incorporate a basic group into the linker shown in the scheme above. Without wishing to be bound by theory, the presence of the basic group may increase the nucleophilicity of nearby water molecules to promote ring-opening hydrolysis of the succinimide group. The hydrolyzed form of the attachment group is resistant to deconjugation in the presence of plasma proteins. Such self-stabilizing linkers provide conjugates with im roved stability. A representative schematic is shown below:

[0300] The hydrolysis reaction schematically represented above may occur at either carbonyl group of the succinimide group. Accordingly, two possible isomers may result, as shown below:

[0301] The identity of the base as well as the distance between the base and the maleimide group can be modified to tune the rate of hydrolysis of the thio- substituted succinimide group and optimize the delivery of a conjugate to a target by, for example, improving the specificity and stability of the conjugate.

[0302] Bases suitable for inclusion in a linker L ³ described herein with a maleimide group prior to conjugation to an antibody construct may facilitate hydrolysis of a nearby succinimide group formed after conjugation of the antibody construct to the linker. Bases may include, for

26 27 26 2V

example, amines (e.g., -N(R )(R ), where R and R are independently selected from H and Ci _-6 alkyl), nitrogen-containing heterocycles (e.g., a 3- to 12-membered heterocycle including one or more nitrogen atoms and optionally one or more double bonds), ami dines, guanidines, and carbocycles or heterocycles substituted with one or more amine groups (e.g., a 3- to 12- membered aromatic or non-aromatic cycle optionally including a heteroatom such as a nitrogen

26 27 26 27 atom and substituted with one or more amines of the type -N(R )(R ), where R and R are independently selected from H or Ci _-6 alkyl). A basic unit may be separated from a maleimide group by, for example, an alkylene chain of the form -(CH ₂ ) _q -, where q is an integer from 0 to 10. An alkylene chain may be optionally substituted with other functional groups as described herein.

[0303] A linker L ³ with a maleimide group may include an electron withdrawing groups such as, but not limited to, -C(0)R, =0, -CN, -N0 ₂ , -CX ₃ , -X, -COOR, -CO R ₂ , -COR, -COX, - S0 ₂ R, -S0 ₂ OR, -S0 ₂ HR, -S0 ₂ NR ₂ , P0 ₃ R ₂ , -P(0)(CH ₃ ) HR, -NO, -NR ₃ ⁺ , -CR=CR ₂ , and - C≡CR, where each R is independently selected from H and Ci _-6 alkyl and each X is

independently selected from F, Br, CI, and I. Self-stabilizing linkers may also include aryl, e.g., phenyl, or heteroaryl, e.g., pyridine, groups optionally substituted with electron withdrawing groups such as those described herein.

[0304] Examples of self-stabilizing linkers are provided in, e.g., U.S. Patent Publication Number 2013/0309256, the linkers of which are incorporated by reference herein. It will be understood that a self-stabilizing linker useful in conjunction with the compounds of the present invention may be equivalently described as unsubstituted maleimide-including linkers, thio- substituted succinimide-including linkers, or hydrolyzed, ring-opened thio- substituted succinimide-including linkers.

[0305] In certain embodiments a linker L ³ includes a stabilizing linker selected from:

in which the wavy line indicates an attachment to a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc).

[0306] In the scheme provided above, the bottom structure may be referred to as (maleimido)- DPR-Val-Cit-PAB, where DPR refers to diaminopropinoic acid, Val refers to valine, Cit refers to citrulline, PAB refers to para-aminobenzylcarbonyl, and the wavy line represents the point of attachment to compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc). [0307] A method for bridging a pair of sulfhydryl groups derived from reduction of a native hinge disulfide bond has been disclosed and is depicted in the schematic below. An advantage of this methodology is the ability to synthesize homogenous DAR4 conjugates by full reduction of

IgGs (to give 4 pairs of sulfhydryls from interchain disulfides) followed by reaction with 4 equivalents of the alkylating agent. Conjugates containing bridged disulfides are also claimed to have increased stability.

"bridged disulfide"

[0308] Similarly, as depicted below, a maleimide derivative that is capable of bridging a pair of sulfhydryl groups has been develo ed:

A linker L can contain the followin structural formulas (Vila), (Vllb), or (VIIc):

or salts thereof, wherein: R ^q is H or-0-(CH ₂ CH ₂ 0)ii-CH ₃ ; x is 0 or 1; y is 0 or 1; G ² is- CH ₂ CH ₂ CH ₂ S0 ₃ H or -CH ₂ CH ₂ 0-(CH ₂ CH ₂ 0)n-CH ₃ ; R ^w is-0-CH ₂ CH ₂ S0 ₃ H or - H(CO)- CH ₂ CH ₂ 0-(CH ₂ CH ₂ 0)i ₂ -CH ₃ ; and * represents the point of attachment to another portion of the linker or a site of attachment to a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc).

[0309] Exemplary embodiments of linkers according to structural formula (Vila) and (Vllb) that can be included in the conjugates can include the linkers illustrated below (as illustrated, the linkers can include a group suitable for covalently linking the linker to a residue of an antibody construct):

ı33 (VIIb.3),

(VIIb.4),

OH (VIIb.6),

OH (VIIb.8), in which the wavy line represents the point of attachment of the linker L ³ to the compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc).

[0310] Exemplary embodiments of linkers according to structural formula (VIIc) that can be included in the conjugates can include the linkers illustrated below (as illustrated, the linkers can include a roup suitable for covalently linking the linker to an antibody construct):

OH (VIIc. l),

in which the wavy line represents the point of attachment of the linker L ³ to the compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc). [0311] As is known by skilled artisans, the linker selected for a particular conjugate may be influenced by a variety of factors including, but not limited to, the site of attachment to the residue of an antibody construct (e.g., Lys, Cys, or other amino acid residues), structural constraints of the drug pharmacophore (i.e., the heterocyclic compound), and the lipophilicity of the heterocyclic compound. The specific linker selected for a conjugate should seek to balance these different factors for the specific antibody construct-drug combination.

[0312] For example, heterocyclic compounds and conjugates including the same have been observed to effect killing of bystander antigen-negative cells present in the vicinity of the antigen-positive tumor cells. The mechanism of bystander cell killing by conjugates has indicated that metabolic products formed during intracellular processing of the conjugates may play a role. Neutral and hydrophobic cytotoxic metabolites generated by metabolism of the conjugates in antigen-positive cells appear to play a role in bystander cell killing while charged and hydrophilic metabolites may be prevented from diffusing across the membrane into the medium, or from the medium across the membrane, and therefore cannot affect bystander killing. In certain embodiments, the linker is selected to attenuate a bystander effect caused by cellular metabolites of the conjugate. In certain embodiments, the linker is selected to increase the bystander effect.

[0313] The properties of the linker, or linker-compound, may also impact aggregation of the conjugate under conditions of use and/or storage. Typically, conjugates reported in the literature contain no more than 3-4 drug molecules per antibody molecule. Attempts to obtain higher drug- to-antibody ratios ("DAR") often failed, particularly if both the drug and the linker were hydrophobic, due to aggregation of the conjugate. In many instances, DARs higher than 3-4 could be beneficial as a means of increasing potency. In instances where the heterocyclic compound is more hydrophobic in nature, it may be desirable to select linkers that are relatively hydrophilic as a means of reducing conjugate aggregation, especially in instances where DARs greater than 3-4 are desired. Thus, in certain embodiments, the linker incorporates chemical moieties that reduce aggregation of the conjugates during storage and/or use. A linker may incorporate polar or hydrophilic groups such as charged groups or groups that become charged under physiological pH to reduce the aggregation of the conjugates. For example, a linker may incorporate charged groups such as salts or groups that deprotonate, e.g., carboxylates, or protonate, e.g., amines, at physiological pH.

[0314] In some embodiments, the aggregation of the conjugates during storage or use is less than about 40% as determined by size-exclusion chromatography (SEC). In certain

embodiments, the aggregation of the conjugates during storage or use is less than 35%, such as less than about 30%>, such as less than about 25%, such as less than about 20%, such as less than about 15%, such as less than about 10%, such as less than about 5%, such as less than about 4%, or even less, as determined by size-exclusion chromatography (SEC).

Conjugates of PROTACS

[0315] In certain embodiments, a conjugate of a compound described herein can be designed to increase ubiquitin-mediated target protein destruction via the ubiquitin pathway. The process of attaching ubiquitin molecules to a protein target typically involves 3 enzymes and processes: 1) an El enzyme that can activate ubiquitin, 2) an E2 enzyme that can transfer activated ubiquitin, and 3) a multi-subunit E3 enzyme ligase that can receive the activated ubiquitin and catalyze a ubiquitin attachment to the target protein.

[0316] In some embodiments, a conjugate includes a proteolysis or protein targeting module (PTM; also referred to as a proteoly sis-targeting chimera or PROTAC). A PTM can comprise a small molecule that can bind to an E3 ubiquitin ligase subunit and a target binding moiety (a compound described herein) that binds a protein target. The E3 ubiquitin ligase binding small molecule is attached, directly or by a spacer (S), to the target binding moiety. In some embodiments, a PTM includes a protein targeting moiety, such as a compound or salt described herein (designated D), that is covalently attached to an E3 ubiquitin ligase binding moiety (ULM) through a spacer (S), and a linker (L) that is covalently attached to the PTM and to the antibody construct (Ab), as represented by the formula Ab-(L-PTM _n ) _z , where n is from 1-20 and z is from 1 to 20. In some embodiments, L is a cleavable linker. In some embodiments, L is a non-cleavable linker. A cleavable linker can be a peptide linker or other cleavable linker.

Linkers can be, for example, as described herein. In some embodiments, the Fc domain of the conjugate is an Fc null. In some embodiments, the Fc domain is a wild-type IgG that can bind to Fey receptors and to FcRn.

[0317] In some embodiments, an Fc domain or region of the antibody construct portion of a conjugate can exhibit increased binding affinity to one or more Fc receptors. In some embodiments, an Fc domain or region can exhibit increased binding affinity to one or more Fcgamma receptors. In some embodiments, an Fc domain or region can exhibit increased binding affinity to FcRn receptors. In some embodiments, an Fc domain or region can exhibit increased binding affinity to Fcgamma and FcRn receptors.

[0318] In some embodiments, an Fc domain or region of the antibody construct portion of a conjugate can exhibit reduced binding affinity to one or more Fc receptors. In some

embodiments, an Fc domain or region can exhibit reduced binding affinity to one or more Fcgamma receptors. In some embodiments, an Fc domain or region can exhibit reduced binding affinity to FcRn receptors. In some embodiments, an Fc domain or region can exhibit reduced binding affinity to Fcgamma and FcRn receptors. In some embodiments, an Fc domain is an Fc null domain or region. In some embodiments, an Fc domain or region can exhibit reduced binding affinity to FcRn receptors, but have the same or increased binding affinity to one or more Fcgamma receptors as compared to a wildtype IgG. In some embodiments, an Fc domain or region can exhibit increased binding affinity to FcRn receptors, but have the same or decreased binding affinity to one or more Fcgamma receptors.

[0319] In some embodiments, a protein targeting moiety, such as a compound or salt described herein (designated D), is covalently attached to an E3 ubiquitin ligase binding moiety (ULM) through a spacer (S) and a linker (L) (e.g., as described elsewhere herein) is covalently attached to the spacer (S), n is from 1-20 and z is from 1 to 20 as represented by Formula PI :

(PI)

In some embodiments, n is 1, and z may be from 1 to 20, 1 to 10, 1 to 9, 1 to 8, 2 to 8, 1 to 6 or 3 to 5. In certain embodiments, z is 4. In some embodiments, n is 1 and z may be from 1 to 10, 1 to 9, 1 to 8, 2 to 8, 1 to 6, 3 to 5, 2 or 4.

[0320] In some embodiments, a protein targeting moiety, such as a compound or salt described herein (designated D), is covalently attached to an E3 ubiquitin ligase binding moiety (ULM) through a spacer (S) and a linker (L) is covalently attached to the protein targeting moiety, n is from 1-20 and z is from 1 to 20 as represented by Formula P2:

(P2)

In some embodiments, n is 1, and z may be from 1 to 20, 1 to 10, 1 to 9, 1 to 8, 2 to 8, 1 to 6 or 3 to 5. In certain embodiments, z is 4. In some embodiments, n is 1 and z may be from 1 to 10, 1 to 9, 1 to 8, 2 to 8, 1 to 6, 3 to 5, 2 or 4.

[0321] In some embodiments, a protein targeting moiety, such as a compound or salt described herein (designated D), is covalently attached to an E3 ubiquitin ligase binding moiety (ULM) through a spacer (S) and linker (L) (e.g., as described elsewhere herein) is covalently attached to the ubiquitin E3 ligase moiety (ULM), n is from 1-20 and z is from 1 to 20 as represented by Formula P3 :

(P3)

In some embodiments, n is 1, and z may be from 1 to 20, 1 to 10, 1 to 9, 1 to 8, 2 to 8, 1 to 6 or 3 to 5. In certain embodiments, z is 4. In some embodiments, n is 1 and z may be from 1 to 10, 1 to 9, 1 to 8, 2 to 8, 1 to 6, 3 to 5, 2 or 4.

[0322] In certain embodiments, for any one of Formulas PI, P2, or P3, L is a cleavable linker. In some embodiments, L is a non-cleavable linker. The cleavable linker can be a peptide linker or other cleavable linker as described in the section on linkers. In some embodiments, the Fc domain of the conjugate is an Fc null. In some embodiments, the Fc domain is a wild-type IgG that can bind to Fey receptors.

[0323] In certain embodiments, for any one of Formulas PI, P2, or P3, an Fc domain or region of the antibody construct portion of a conjugate can exhibit increased binding affinity to one or more Fc receptors. In some embodiments, an Fc domain or region can exhibit increased binding affinity to one or more Fcgamma receptors. In some embodiments, an Fc domain or region can exhibit increased binding affinity to FcRn receptors. In some embodiments, an Fc domain or region can exhibit increased binding affinity to Fcgamma and FcRn receptors.

[0324] In certain embodiments, for any one of Formulas PI, P2, or P3, an Fc domain or region of the antibody construct portion of a conjugate can exhibit reduced binding affinity to one or more Fc receptors. In some embodiments, an Fc domain or region can exhibit reduced binding affinity to one or more Fcgamma receptors. In some embodiments, an Fc domain or region can exhibit reduced binding affinity to FcRn receptors. In some embodiments, an Fc domain or region can exhibit reduced binding affinity to Fcgamma and FcRn receptors. In some embodiments, an Fc domain is an Fc null domain or region. In some embodiments, an Fc domain or region can exhibit reduced binding affinity to FcRn receptors, but have the same or increased binding affinity to one or more Fcgamma receptors as compared to a wildtype IgG. In some embodiments, an Fc domain or region can exhibit increased binding affinity to FcRn receptors, but have the same or decreased binding affinity to one or more Fcgamma receptors.

[0325] In certain embodiments, for any one of Formulas PI, P2, or P3, the E3 ubiquitin ligase binding moiety is linked to a protein targeting moiety, such as a compound described herein, in the conjugate as described herein, via a spacer. In certain embodiments, the E3 ubiquitin ligase binding moiety can be linked to the protein targeting moiety via a spacer having a linear non- hydrogen atom number in the range of 1 to 25 or 1 to 20. In certain embodiments, the spacer has

5 to 20 or 5 to 15 linear non-hydrogen atoms. The spacer is typically non-cleavable.

[0326] In certain embodiments, for any one of Formulas PI, P2, or P3, the E3 ubiquitin ligase binding moiety can be linked to the spacer of the protein targeting moiety with a functional group such as an ether, amide, alkane, alkene, alkyne, ketone, hydroxyl, carboxylic acid, thioether, sulfoxide, and sulfone. The E3 ubiquitin ligase binding moiety can be linked to the spacer of the protein targeting moiety via a spacer comprising an aromatic, heteroaromatic, cyclic, bicyclic, and/or tricyclic moiety.

[0327] In certain embodiments, for any one of Formulas PI, P2, or P3, spacer length can be varied to optimize the activity of the protein targeting moiety for its target protein. In some embodiments, the spacer is non-cleavable and comprises segments of alkylene, alkenylene, alkynylene, -(CH ₂ 0)-, -CH ₂ CH ₂ 0)-, -(CH ₂ OCH ₂ )-, -C(O)-, - H-, and -0-, having a length of from 1-25, 1-20, 1-15, 5-25, 5-20, or 5-15 linear non-hydrogen atoms. A spacer may be optionally substituted with Ci-C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, -(CH ₂ 0) _n iH, - (CH ₂ CH ₂ 0)„iH, -(CH ₂ 0)„iCH ₃ , -C(0)OH or - H ₂ , wherein nl is from 1 to 8, and may further optionally comprise a reactive group, R ^x , to form a functional group, such as an ether, amide, alkane, alkene, alkyne, ketone, hydroxyl, carboxylic acid, thioether, sulfoxide, and sulfone, forming an attachment to a linker (L). In some embodiments, the spacer is not substituted. In some embodiments, the spacer is substituted with R ^x .

[0328] In certain embodiments, for any one of Formulas PI, P2, or P3, a spacer may be a Ci- ₂₅ alkylene or optionally substituted Ci- ₂₅ heteroalkylene, wherein the heteroalkylene is a Ci-C ₂₄ alkylene chain interspersed with one or more groups independently selected from: -0-, -S-, - NH ₂ -, and -C(0) H-. The spacer may also be optionally substituted with a reactive group, R ^x , that can form a functional bond, such as an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond, or a thioether bond; such reactive groups can be, for example, amino groups; carboxyl groups; aldehyde groups; azide groups; alkyne and alkene groups;

ketones; carbonates; carbonyl functionalities bonded to leaving groups such as cyano and succinimidyl and hydroxyl groups. In some embodiments, R ^x can be -NH ₂ , -S or a maleimide. In some embodiments, R ^x is - H ₂ . The spacer may also be optionally substituted with Ci- C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, -(CH ₂ 0)„iH, -(CH ₂ CH ₂ 0)mH, -(CH ₂ 0)„iCH ₃ , -C(0)OH or - H ₂ , wherein nl is from 1 to 8. In some embodiments, the spacer is not substituted. In some embodiments, the spacer is substituted with R ^x .

[0329] In certain embodiments, for any one of Formulas PI, P2, or P3, the spacer (S) has the formula -C(O)N(R ¹⁰⁰ )R ¹⁰¹ C(O)N(R ¹⁰⁰ )-, -C(O)R ¹⁰¹ C(O)-, -C(O)R ¹⁰¹ N(Ri ₀₀ )-, -N(Ri ₀₀ )R ¹⁰¹ C(O)- , -N(R ¹⁰⁰ )C(O)R ¹⁰¹ C(O)-, -N(R ¹⁰⁰ )C(0)R ¹⁰¹ N(Rioo)-, -N(Ri ₀₀ )R ¹⁰¹ C(O)N(R100)-, - N(R ¹⁰⁰ )C(O)R ¹⁰¹ C(O)N(R ¹⁰⁰ )-, -N(R ¹⁰⁰ )C(0)R ¹⁰¹ N(Rioo)C(0)-, and -

C(0)N(Rioo)R ¹⁰¹ C(0)N(R100)-; wherein each R ¹⁰⁰ is independently selected from H or C1-C3 alkyl and R ¹⁰¹ is -Ci-C ₂₅ alkylene-, -Ci-C ₂₅ alkenylene-, -Ci-C ₂₅ alkynlene-, -Ci- Ci ₂ alkylene(CH ₂ 0) _n Ci-Ci ₅ alkylene-, -Ci-Ci ₂ alkylene((CH ₂ OCH ₂ ) _n Ci-Ci ₂ alkylene-, -C

Ci ₂ alkylene(CH ₂ CH ₂ 0) _n Ci-Ci ₂ alkylene-, -Ci-Ci ₂ alkenylene-((CH ₂ 0) _n Ci-Ci ₂ alkylene-, -C Ci ₂ alkenylene-(CH ₂ CH ₂ 0)„Ci-Ci ₂ alkylene-, -Ci-Ci ₂ alkenylene-((CH ₂ OCH ₂ ) _n Ci-Ci ₂ alkylene-, - Ci-Ci ₂ alkylene-(CH ₂ 0) _n Ci-Ci ₂ alkenylene-, -Ci-Ci ₂ alkylene-(CH ₂ CH ₂ 0) _n Ci-Ci ₂ alkenylene-, - Ci-Ci ₂ alkylene-(CH ₂ OCH ₂ ) _n Ci-Ci ₂ alkenylene-, -Ci-Ci ₂ alkynylene-(CH ₂ 0) _n Ci-Ci ₂ alkylene-, - Ci-Ci ₂ alkynylene-(CH ₂ CH ₂ 0) _n Ci-Ci ₂ alkylene-, -Ci-Ci ₂ alkynylene-(CH ₂ OCH ₂ ) _n Ci-Ci ₂ alkylene-, -Ci-Ci ₂ alkynylene-(CH ₂ 0) _n Ci-Ci ₂ alkenylene-, -C i-Ci ₂ alkynylene-(CH ₂ CH ₂ 0) _n Ci- Ci ₂ alkenylene-, -Ci-Ci ₂ alkynylene-(CH ₂ OCH ₂ ) _n Ci-Ci ₂ alkenylene-,-Ci-Ci ₂ alkynylene- (CH ₂ 0) _n Ci-Ci ₂ alkynylene-, -Ci-Ci ₂ alkynylene-(CH ₂ CH ₂ 0) _n Ci-Ci ₂ alkynylene-, -C

Ci ₂ alkynylene-(CH ₂ OCH ₂ ) _n Ci-Ci ₂ alkynylene-, in each case optionally substituted with a reactive moiety R ^x for attachment to the linker (L), and n is 0 to 8. R ^x can be a reactive group that can form an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond, or a thioether bond; such reactive groups can be, for example, amino groups; carboxyl groups;

aldehyde groups; azide groups; alkyne and alkene groups; ketones; carbonates; carbonyl functionalities bonded to leaving groups such as cyano and succinimidyl and hydroxyl groups. In some embodiments, R ^x can be - H ₂ , -S or a maleimide. In some embodiments, R ^x is - H ₂ .

[0330] In certain embodiments, for any one of Formulas PI, P2, or P3, the spacer (S) comprises glutamate, a glycine-glutamate dipeptide, glycine-PEGl-glutamate, glycine-PEG2- glutamate, glycine-PEG3 -glutamate, glycine-PEG4-glutamate or glycine-PEG5 -glutamate, wherein the E3 ubquitin ligase binding moiety and the protein targeting moiety are attached to the spacer via amide bonds.

[0331] In certain embodiments, for any one of Formulas PI, P2, or P3, an E3 ubiquitin ligase binding moiety can bind to an E3 ubiquitin ligase, such as Von Hippel-Lindaue E3 ubiquitin ligase (VHL), cereblon, mouse double minute 2 homolog (MDM2), AMFR, APC/Cdc20, APC/Cdhl, C6orfl 57, Cbl, CBLL1, CHFR, CHIP, DTL (Cdt2), E6-AP, HACE1, HECTD1, HECTD2, HECTD3, HECWl, HECW2, HERC2, HERC3, HERC4, HERC5, HUWEl, HYD, ITCH, LNXl, mahogunin, MARCH-I, MARCH-II, MARCH-III, MARCH-IV, MARCH- VI, MARCH- VII, MARCH- VIII, MARCH-X, MEKK1, MIB 1, MIB2, MycBP2, NEDD4,

NEDD4L, Parkin, PELIl , Pirh2, PJAl, PJA2, RFFL, RFWD2, Rictor, RNF5, RNF8, RNF 19, RNF190, RNF20, RNF34, RNF40, RNF125, RNF 128, RNF138, RNF 168, SCF/p-TrCP, SCF/FBW7, SCF/Skp2, SHPRH, SIAHl, SIAH2, SMURF1, SMURF2, TOPORS, TRAF6, TRAF7, TRFM63, UBE3B, UBE3C, UBR1, UBR2, UHRF2, WWP1, WWP2, or ZNRFl . [0332] In certain embodiments, for any one of Formulas PI, P2, or P3, an E3 ubiquitin ligase binding moiety can be selected from a molecule that binds an E3 ubiquitin ligase selected from von Rippel-Lindau (VHL), cereblon, XIAP, E3 A, MDM2, Anaphase-promoting complex (APC),

UBR5 (EDDI), SOCS/ BC-box/ eloBC/ CUL5/ RING, LNXp80, CBX4, CBLLI, HACEI,

HECTDI, HECTD2, HECTD3, HECWI, HECW2, HERCI, HERC2, HERC3, HERC4, HUWEI,

ITCH, NEDD4, NEDD4L, PPIL2, PRPFI9, PIASI, PIAS2, PIAS3, PIAS4, RANBP2, RNF4,

RBXI, SMURFI, SMURF2, STUB I, TOPORS, TRIPI2, UBE3A, UBE3B, UBE3C, UBE4A,

UBE4B, UBOX5, UBR5, WWPI, WWP2, Parkin, A20/TNFAIP3, AMFR/gp78, ARA54, beta-

TrCPI/BTRC, BRCAI, CBL, CHIP/STUB I, E6, E6AP/UBE3A, F-box protein I5/FBXOI5,

FBXW7/Cdc4, GRAIL/RNFI28, HOIP/RNF3 I, cIAP-I/HIAP-2, cIAP-2/HIAP-I, cIAP (pan),

ITCH/AIP4, KAPI, MARCH8, Mind Bomb I/MIBI, Mind Bomb 2/MIB2, MuRFI/TRFM63,

NDFIPI, NEDD4, NleL, Parkin, RNF2, RNF4, RNF8, RNFI68, RNF43, SARTI, Skp2,

SMURF2, TRAF-I, TRAF-2, TRAF-3, TRAF-4, TRAF-5, TRAF-6, TRIMS, TRFM2I, TRFM32,

UBR5, and ZNRF3.

[0333] In certain embodiments, for any one of Formulas PI, P2, or P3, an E3 ubiquitin ligase can be selected from the following types: HECT type, RING-type, PARKIN-finger type, RING- variant type, U-box type, A20-finger type, PIAS-finger type, PHD-finger type, Skpl-like type, Cullin-type, F-box type, SOCS-box type, BTB-type, DDBl-like type and APC/cyclosome type.

[0334] In certain embodiments, for any one of Formulas PI, P2, or P3, an E3 ubiquitin ligase binding moiety can be a VHL binding moiety such as (S)-2-amino-Nl-(4-(5-amino-6-((4- mo holinopyridin-3-yl)carbamoyl)pyrazin-2-yl)benzyl)-N5-(2-(3-( ((S)-l-((2S,4R)-4-hydroxy- 2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-l-yl )-3,3-dimethyl-l-oxobutan-2- yl)amino)-3-oxopropoxy)ethyl)pentanediamide or a cereblon binding moiety such as 3-amino-6- (4-(2-((2S)-2-amino-6-(2-((2-(2,6-dioxopiperidin-3-yl)-l,3-d ioxoisoindolin-4- yl)oxy)acetamido)hexanamido)ethyl)phenyl)-N-(4-mo holinopyridin-3-yl)pyrazine-2- carboxamide. Other compounds that bind VHL may be hydroxyproline compounds such as those disclosed in WO 2013/106643, and other compounds described in US 2016/0045607, WO 2014187777, US 2014/0356322, and U.S. 9,249, 153 (incorporated herein by reference). Other compounds that bind to cereblon include thalidomide, lenalidomide, pomalidomide and analogs thereof. Other small molecule compounds that bind to cereblon are also known, e.g., the compounds disclosed in US 2016/0058872 and US 2015/0291562 (incorporated herein by reference).

[0335] In certain embodiments, for any one of Formulas PI, P2, or P3, the linker (L) is attached to the PTM at a reactive site R ^x in the spacer. In certain embodiments, the linker (L) is attached to the PTM via an attachment site in the E3 ubiquitin ligase binding moiety. In certain embodiments, the linker (L) is attached to the PTM via an attachment site in the protein targeting moiety.

[0336] In certain embodiments, for any one of Formulas PI, P2, or P3, the linker (L) and/or covalent attachment site(s) of the linker (L) to the proteolysis targeting module can be cleavable or non-cleavable. In certain embodiments, the linker is cleavable, such as a peptide-cleavable linker. For example, a cleavable linker can be maleimidocaproyl-peptide-para-aminobenzyl alcohol (PABA) or a maleimidocaproyl-peptide-para-aminobenzyl carbonyl, wherein the peptide is valine-citrulline, valine-alanine or phenylalanine-lysine.

[0337] In certain embodiments, for any one of Formulas PI, P2, or P3, the linker is non- cleavable linker. In some embodiments, the linker is non-cleavable and is attached to the proteolysis targeting module at site wherein the protein targeting moiety can bind to its protein target, and, if active, does not lose activity, as determined by Kd measurement, by altered target protein function in a cell-based assay, or both. Linker length can be varied to optimize the activity of the protein targeting moiety for its target protein. Such linkers can be short, flexible, rigid, hydrophilic, or hydrophobic. The linker can contain segments that have different characteristics, such as segments of flexibility or segments of rigidity. The linker can be chemically stable to extracellular environments. Non-limiting examples can be

maleimidocaproyl linkers. A maleimidocaproyl linker can comprise N- maleimidomethylcyclohexane-l-carboxylate.

[0338] In certain embodiments, for any one of Formulas PI, P2, or P3, a linker (L) can be a combination of a maleimidocaproyl group and one or more polyethylene glycol molecules. A linker (L) may comprise from 5 to 100 linear non-hydrogen atoms, with optional substituions, that may be covalently attached to an antibody construct. In certain embodiments, the linker (L) is selected, for example, from linkers known in the art in addition to any of the linkers described herein, e.g., any L ³ linker described herein.

Pharmaceutical Formulations

[0339] The conjugates, and methods described herein can be considered useful as

pharmaceutical compositions for administration to a subject in need thereof. Pharmaceutical compositions can comprise at least one of the compounds, salts, or conjugates and one or more pharmaceutically acceptable carriers, diluents, excipients, stabilizers, dispersing agents, suspending agents, and/or thickening agents. The composition can comprise the conjugate having an antibody construct and a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (He), (III), (Ilia), (Illb), and (IIIc) connected via a linker, as described herein. The composition can comprise the conjugate having an antibody construct, a target binding domain, and a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ha), (lib), (He), (III), (Ilia),

(Illb), and (IIIc) connected via a linker, as described herein. A composition can comprise any conjugate described herein.

[0340] An antibody construct portion of the conjugate can recognize a tumor antigen. A tumor antigen can include CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH- 1, BCMA, CS-1, PD-L1, B7-H3, B7-DC (PD-L2), HLD-DR, carcinoembryonic antigen (CEA), TAG-72, EpCAM, MUC1, folate-binding protein, A33, G250, prostate-specific membrane antigen (PMSA), ferritin, GD2, GD3, GM2, Le ^y , CA-125, CA19-9 (MUC1 sLe(a)), epidermal growth factor, pl85HER2, IL-2 receptor, EGFRvIII (de2-7 EGFR), fibroblast activation protein, tenascin, a metalloproteinase, endosialin, vascular endothelial growth factor, avB3, WTl, LMP2, HPV E6. HPV E7, Her-2/neu, MAGE A3, p53 nonmutant, NY-ESO-1, Mel an A/MART 1 , Ras mutant, gplOO, p53 mutant, PR1, bcr-abl, tyronsinase, survivin, PSA, hTERT, a Sarcoma translocation breakpoint protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin (MSLN), PSCA, MAGE Al, sLe(a), CYPIBI, PLAV1, GM3, BORIS, Tn, GloboH, ETV6- AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TESl, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, , Legumain, Tie 3, Page4, VEGFR2, MAD- CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAIL 1, MUC16, MAGE A4, MAGE C2, GAGE, EGFR, CMET, HER3, MUC1, MUC15, CA6, NAPI2B, TROP2, CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, or Fos-related antigen 1.

[0341] In certain embodiments, the tumor antigen is selected from MUC16, UPK1B, VTCN1, TMPRSS3, TMEM238, Clorfl86, TMPRSS4, CLDN6, CLDN8 and STRA6. In certain embodiments, the tumor antigen is selected from MUC16, UPK1B, VTCN1, TMPRSS3, TMEM238, Clorfl86 and TMPRSS4. In certain embodiments, the tumor antigen is MUC16. In certain embodiments, the tumor antigen is UPK1B. In certain embodiments, the tumor antigen is VTCN1. In certain embodiments, the tumor antigen is TMPRSS3. In certain embodiments, the tumor antigen is TMEM238. In certain embodiments, the tumor antigen is Clorfl86. In certain embodiments, the tumor antigen is TMPRSS4. In certain embodiments, the tumor antigen is CLDN6. In certain embodiments, the tumor antigen is CLDN8. In certain embodiments, the tumor antigen is STRA6.

[0342] In certain embodiments, the tumor antigen is selected from ACSL5, AP1M2, AREG, CDH1, CDH17, CEACAM5, CEACAM6, CEACAM7, CLCA1, CLDN3, DPEP1, ERBB3, GPA33, GPRC5A, ITGA6, KRTCAP3, LSR, MUC13, NOX1, PLOD3, PLPP2, SLC12A2, SLC44A4, SLC52A2, SMFM22, ST14, TFRC, TMPRSS4, sLE(x), and TSPAN6. In certain embodiments, the tumor antigen is selected from CLDN4, CLDN7, EPCAM, PIGR, TMEM141, TMEM54, TSPANl, LRG5, and TSPAN8. In certain embodiments, the tumor antigen is selected from ACSL5, AP1M2, AREG, CDH1, CDH17, CEACAM5, CEACAM6, CEACAM7, CLCA1,

CLDN3, DPEP1, ERBB3, GPA33, GPRC5A, ITGA6, KRTCAP3, LSR, MUC13, NOX1,

PLOD3, PLPP2, SLC12A2, SLC44A4, SLC52A2, SMIM22, ST14, TFRC, TMPRSS4, sLE(x),

TSPAN6, CLDN4, CLDN7, EPCAM, PIGR, TMEM141, TMEM54, TSPANl, LRG5, and

TSPAN8. In certain embodiments, the tumor antigen is any one of ACSL5, AP1M2, AREG,

CDH1, CDH17, CEACAM5, CEACAM6, CEACAM7, CLCA1, CLDN3, DPEP1, ERBB3,

GPA33, GPRC5A, ITGA6, KRTCAP3, LSR, MUC13, NOX1, PLOD3, PLPP2, SLC12A2,

SLC44A4, SLC52A2, SMIM22, ST14, TFRC, TMPRSS4, sLE(x), TSPAN6, CLDN4, CLDN7,

EPCAM, PIGR, TMEM141, TMEM54, TSPANl, LRG5, and TSPAN8.

[0343] The antibody construct may be an anti-CEA antibody. A conjugate can comprise an anti-CEA antibody and a heterocyclic compound. A pharmaceutical composition can comprise at least the compounds, salts or conjugates described herein and one or more of buffers, antibiotics, steroids, carbohydrates, drugs (e.g., chemotherapy drugs), radiation, polypeptides, chelators, adjuvants and/or preservatives.

[0344] Pharmaceutical compositions can be formulated using one or more physiologically- acceptable carriers comprising excipients and auxiliaries. Formulation can be modified depending upon the route of administration chosen. Pharmaceutical compositions comprising a compound, salt or conjugate as described herein can be manufactured, for example, by lyophilizing the compound, salt or conjugate, mixing, dissolving, emulsifying, encapsulating or entrapping the conjugate. The pharmaceutical compositions can also include the compounds, salts, or conjugates described herein in a free-base form or pharmaceutically-acceptable salt form.

[0345] Methods for formulation of the conjugates can include formulating any of the compounds, salts, or conjugates with one or more inert, pharmaceutically-acceptable excipients or carriers to form a solid, semi-solid, or liquid composition. Solid compositions can include, for example, powders, tablets, dispersible granules, and capsules, and, in some aspects, the solid compositions further contain nontoxic, auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically-acceptable additives. Alternatively, the compounds, salts, or conjugates can be lyophilized or in powder form for re-constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0346] Pharmaceutical compositions can comprise at least one active ingredient (e.g., a compound, salt, or conjugate). The active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug-delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.

[0347] Pharmaceutical compositions often further can comprise more than one active compound (e.g., a compound, salt, or conjugate and other agents) as necessary for the particular indication being treated. The active compounds can have complementary activities that do not adversely affect each other. For example, the composition can also comprise a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth-inhibitory agent, an anti-hormonal agent, an anti- angiogenic agent, and/or a cardioprotectant. Such molecules can be present in combination in amounts that are effective for the purpose intended.

[0348] The compositions and formulations can be sterilized. Sterilization can be

accomplished by, for example, filtration (e.g., sterile filtration).

[0349] The compositions can be formulated for administration as an injection. Non-limiting examples of formulations for injection can include a sterile suspension, solution, or emulsion in oily or aqueous vehicles. Suitable oily vehicles can include, but are not limited to, lipophilic solvents or vehicles such as fatty oils or synthetic fatty acid esters, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension. The suspension can also contain suitable stabilizers. Injections can be formulated for bolus injection or continuous infusion. Alternatively, the compositions can be lyophilized or in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0350] For parenteral administration, the compounds, salts or conjugates can be formulated in a unit dosage injectable form (e.g., a solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles can be inherently non-toxic, and non-therapeutic. Vehicles can be water, saline, Ringer' s solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as fixed oils and ethyl oleate can also be used. Liposomes can be used as carriers. The vehicle can contain minor amounts of additives such as substances that enhance isotonicity and chemical stability (e.g., buffers and

preservatives).

[0351] Sustained-release preparations can also be prepared. Examples of sustained-release preparations can include semipermeable matrices of solid hydrophobic polymers that can contain the compound, salt, or conjugate, and these matrices can be in the form of shaped articles (e.g., films or microcapsules). Examples of sustained-release matrices can include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly (vinyl alcohol)), polylactides, copolymers of L-glutamic acid and γ ethyl -L-glutamate, non-degradable ethyl ene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPO™ (i.e., injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly- D-( -)-3-hydroxybutyric acid.

[0352] Pharmaceutical formulations can be prepared for storage by mixing a compound, salt, or conjugate with a pharmaceutically acceptable carrier, excipient, and/or a stabilizer. This formulation can be a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients, and/or stabilizers can be nontoxic to recipients at the dosages and concentrations used. Acceptable carriers, excipients, and/or stabilizers can include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine;

preservatives, polypeptides; proteins, such as serum albumin or gelatin; hydrophilic polymers; amino acids; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes; and/or non- ionic surfactants or polyethylene glycol.

[0353] Pharmaceutical formulations of the conjugates may have an average drug-antibody contruct ratio selected from about 1 to about 10, wherein the drug is a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), and (IIIc). In certain embodiments, the average DAR of the formulation is from about 2 to about 8, such as from about 3 to about 8, such as from about 3 to about 7, such as from about 3 to about 5, or such as from about 1 to about 3. In certain embodiments, a pharmaceutical formulation has an average DAR of about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, or about 6.6.

Therapeutic Applications

[0354] The compositions, conjugates and methods of the present disclosure can be useful for a plurality of different subjects including, but are not limited to, a mammal, human, non-human mammal, domesticated animal (e.g., laboratory animals, household pets, or livestock), non- domesticated animal (e.g., wildlife), dog, cat, rodent, mouse, hamster, cow, bird, chicken, fish, pig, horse, goat, sheep, rabbit, and any combination thereof.

[0355] The compositions, conjugates and methods can be useful as a therapeutic, for example, a treatment that can be administered to a subject in need thereof. A therapeutic effect of the present disclosure can be obtained in a subject by reduction, suppression, remission, or eradication of a disease state including, but not limited to, a symptom thereof. A therapeutic effect in a subject having a disease or condition, or pre-disposed to have or beginning to have the disease or condition, can be obtained by a reduction, a suppression, a prevention, a remission, or an eradication of the condition or disease, or pre-condition or pre-disease state. [0356] In practicing the methods described herein, therapeutically-effective amounts of the compositions and conjugates can be administered to a subject in need thereof, often for treating and/or preventing a condition or progression thereof. A pharmaceutical composition can affect the physiology of the subject, such as the immune system, an inflammatory response, or other physiologic affect. A therapeutically-effective amount can vary depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors.

[0357] Treat and/or treating refers to any indicia of success in the treatment or amelioration of the disease or condition. Treating can include, for example, reducing, delaying or alleviating the severity of one or more symptoms of the disease or condition, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient. "Treat" can be used herein to refer to a method that results in some level of treatment or amelioration of the disease or condition, and can contemplate a range of results directed to that end.

[0358] "Prevent," "preventing," and the like refer to the prevention of the disease or condition, e.g., tumor formation, in the patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present disclosure and does not later develop the tumor or other form of cancer, then the disease has been prevented, at least over a period of time, in that individual. Preventing can also refer to preventing re-occurrence of a disease or condition in a patient that has previously been treated for the disease or condition, e.g., by preventing relapse.

[0359] A therapeutically effective amount can be the amount of a composition or an active component thereof sufficient to provide a beneficial effect or to otherwise reduce a detrimental non-beneficial event to the individual to whom the composition is administered. A

therapeutically effective dose can be a dose that produces one or more desired or desirable (e.g., beneficial) effects for which it is administered, such administration occurring one or more times over a given period of time. An exact dose can depend on the purpose of the treatment, and can be ascertainable by one skilled in the art using known techniques.

[0360] The conjugates that can be used in therapy can be formulated and dosages established in a fashion consistent with good medical practice taking into account the disease or condition to be treated, the condition of the individual patient, the site of delivery of the composition, the method of administration, and other factors known to practitioners. The compositions can be prepared according to the description of preparation described herein.

[0361] Pharmaceutical compositions can be used in the methods described herein and can be administered to a subject in need thereof using a technique known to one of ordinary skill in the art which can be suitable as a therapy for the disease or condition affecting the subject. One of ordinary skill in the art would understand that the amount, duration and frequency of

administration of a pharmaceutical composition to a subject in need thereof depends on several factors including, for example, but not limited to, the health of the subject, the specific disease or condition of the patient, the grade or level of a specific disease or condition of the patient, the additional therapeutics the subject is being or has been administered, and the like.

[0362] The methods and compositions can be for administration to a subject in need thereof. Often, administration of the compositions can include routes of administration, non-limiting examples of administration routes include intravenous, intraarterial, subcutaneous, subdural, intramuscular, intracranial, intrasternal, intratumoral, or intraperitoneal. Additionally, a pharmaceutical composition can be administered to a subject by additional routes of

administration, for example, by inhalation, oral, dermal, intranasal, or intrathecal administration.

[0363] Compositions of the present disclosure can be administered to a subject in need thereof in a first administration, and in one or more additional administrations. The one or more additional administrations can be administered to the subject in need thereof minutes, hours, days, weeks or months following the first administration. Any one of the additional

administrations can be administered to the subject in need thereof less than 21 days, or less than 14 days, less than 10 days, less than 7 days, less than 4 days, or less than 1 day after the first administration. The one or more administrations can occur more than once per day, more than once per week, or more than once per month. The administrations can be weekly, biweekly (every two weeks), every three weeks, monthly, or bimonthly.

[0364] The compositions and methods provided herein can be useful for the treatment of a plurality of diseases, conditions, preventing relapse of a disease or a condition in a subject or other therapeutic applications for subjects in need thereof. Often the compositions and methods provided herein can be useful for treatment of hyperplastic conditions, including but not limited to, neoplasms, cancers, tumors and the like. The compositions and methods provided herein can be useful for specifically targeting TNIK. In one embodiment, the compounds of the present disclosure serve as TNIK inhibitors and activate an immune response. A condition, such as a cancer, can be associated with the presence or expression of an antigen on the cancer cells. Often, the an antigen can include an extracellular portion capable of recognition by the antibody construct of the conjugate. An antigen expressed by the cancer cells can be a tumor antigen. An antibody construct portion of the conjugate can recognize a tumor antigen. A tumor antigen can include CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC (PD-L2), HLD-DR, carcinoembryonic antigen (CEA), TAG-72, EpCAM, MUC1, folate-binding protein, A33, G250, prostate-specific membrane antigen (PMSA), ferritin, GD2, GD3, GM2, Le ^y , CA-125, CA19-9 (MUCl sLe(a)), epidermal growth factor, pl85HER2, IL-2 receptor, (EGFRvIII (de2-7 EGFR), fibroblast activation protein, tenascin, a metalloproteinase, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6, HPV E7, Her-2/neu, MAGE A3, p53 nonmutant, NY-ESO-1, Mel an A/MART 1 , Ras mutant, gplOO, p53 mutant, PR1, bcr-abl, tyronsinase, survivin, PSA, hTERT, a Sarcoma translocation breakpoint protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin (MSLN), PSCA, MAGE Al, sLe(a), CYPIBI, PLAV1, GM3, BORIS, Tn, GloboH, ETV6- AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TESl, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, Legumain, Tie 3, Page4, VEGFR2, MAD-CT- 1, PDGFR-B, MAD-CT-2, ROR2, TRAIL 1, MUCl 6, MAGE A4, MAGE C2, GAGE, EGFR, CMET, HER3, MUCl, MUCl 5, CA6, NAPI2B, TROP2, CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, or Fos-related antigen 1.

[0365] In certain embodiments, the tumor antigen is selected from MUC16, UPKIB, VTCN1, TMPRSS3, TMEM238, Clorfl86, TMPRSS4, CLDN6, CLDN8, and STRA6. In certain embodiments, the tumor antigen is selected from MUCl 6, UPKIB, VTCN1, TMPRSS3, TMEM238, Clorfl86, and TMPRSS4. In certain embodiments, the tumor antigen is MUCl 6. In certain embodiments, the tumor antigen is UPKIB. In certain embodiments, the tumor antigen is VTCN1. In certain embodiments, the tumor antigen is TMPRSS3. In certain embodiments, the tumor antigen is TMEM238. In certain embodiments, the tumor antigen is Clorfl86. In certain embodiments, the tumor antigen is TMPRSS4. In certain embodiments, the tumor antigen is CLDN6. In certain embodiments, the tumor antigen is CLDN8. In certain embodiments, the tumor antigen is STRA6.

[0366] In certain embodiments, the tumor antigen is selected from ACSL5, AP1M2, AREG, CDH1, CDH17, CEACAM5, CEACAM6, CEACAM7, CLCA1, CLDN3, DPEP1, ERBB3, GPA33, GPRC5A, ITGA6, KRTCAP3, LSR, MUC13, NOX1, PLOD3, PLPP2, SLC12A2, SLC44A4, SLC52A2, SMFM22, ST14, TFRC, TMPRSS4, sLE(x), and TSPAN6. In certain embodiments, the tumor antigen is selected from CLDN4, CLDN7, EPCAM, PIGR, TMEM141, TMEM54, TSPANl, LRG5, and TSPAN8. In certain embodiments, the tumor antigen is selected from ACSL5, AP1M2, AREG, CDH1, CDH17, CEACAM5, CEACAM6, CEACAM7, CLCA1, CLDN3, DPEP1, ERBB3, GPA33, GPRC5A, ITGA6, KRTCAP3, LSR, MUC13, NOX1, PLOD3, PLPP2, SLC12A2, SLC44A4, SLC52A2, SMFM22, ST14, TFRC, TMPRSS4, sLE(x), TSPAN6, CLDN4, CLDN7, EPCAM, PIGR, TMEM141, TMEM54, TSPANl, LRG5, and TSPAN8. In certain embodiments, the tumor antigen is any one of ACSL5, AP1M2, AREG, CDH1, CDH17, CEACAM5, CEACAM6, CEACAM7, CLCA1, CLDN3, DPEP1, ERBB3, GPA33, GPRC5A, ITGA6, KRTCAP3, LSR, MUC13, NOX1, PLOD3, PLPP2, SLC12A2,

SLC44A4, SLC52A2, SMFM22, ST14, TFRC, TMPRSS4, sLE(x), TSPAN6, CLDN4, CLDN7, EPCAM, PIGR, TMEM141, TMEM54, TSPAN1, LRG5, and TSPAN8.

[0367] An antigen binding domain portion of the conjugate may be configured to recognize an antigen expressed by a cancer cell, such as for example, a disease antigen, tumor antigen or a cancer antigen. Often such antigens are known to those of ordinary skill in the art, or newly found to be associated with such a condition, to be commonly associated with, and/or, specific to, such conditions. For example, a disease antigen, tumor antigen, or a cancer antigen is, but is not limited to, CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC (PD-L2), HLD-DR, carcinoembryonic antigen (CEA), TAG-72, EpCAM, MUCl, folate-binding protein, A33, G250, prostate-specific membrane antigen

(PSMA), ferritin, GD2, GD3, GM2, Le ^y , CA-125, CA19-9 (MUCl sLe(a)), epidermal growth factor, pl85HER2, IL-2 receptor, EGFRvIII (de2-7 EGFR), fibroblast activation protein, tenascin, a metalloproteinase, endosialin, vascular endothelial growth factor, avB3, WTl, LMP2, HPV E6, HPV E7, Her-2/neu, MAGE A3, p53 nonmutant, NY-ESO-1, Mel an A/MART 1 , Ras mutant, gplOO, p53 mutant, PR1, bcr-abl, tyronsinase, survivin, PSA, hTERT, a Sarcoma translocation breakpoint protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin (MSLN), PSCA, MAGE Al, sLe(a), CYPIBI, PLAV1, GM3, BORIS, Tn, GloboH, ETV6- AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TESl, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, Legumain, Tie 3, Page4, VEGFR2, MAD-CT- 1, PDGFR-B, MAD-CT-2, ROR2, TRAIL 1, MUCl 6, MAGE A4, MAGE C2, GAGE, EGFR, CMET, HER3, MUCl, MUCl 5, CA6, NAPI2B, TROP2, CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, or Fos-related antigen 1.

[0368] In certain embodiments, the tumor antigen is selected from MUC16, UPKIB, VTCN1, TMPRSS3, TMEM238, Clorfl86, TMPRSS4, CLDN6, CLDN8, and STRA6. In certain embodiments, the tumor antigen is selected from MUCl 6, UPKIB, VTCN1, TMPRSS3, TMEM238, Clorfl86, and TMPRSS4. In certain embodiments, the tumor antigen is MUCl 6. In certain embodiments, the tumor antigen is UPKIB. In certain embodiments, the tumor antigen is VTCN1. In certain embodiments, the tumor antigen is TMPRSS3. In certain embodiments, the tumor antigen is TMEM238. In certain embodiments, the tumor antigen is Clorfl86. In certain embodiments, the tumor antigen is TMPRSS4. In certain embodiments, the tumor antigen is CLDN6. In certain embodiments, the tumor antigen is CLDN8. In certain embodiments, the tumor antigen is STRA6. [0369] In certain embodiments, the tumor antigen is selected from ACSL5, AP1M2, AREG,

CDH1, CDH17, CEACAM5, CEACAM6, CEACAM7, CLCA1, CLDN3, DPEP1, ERBB3,

GPA33, GPRC5A, ITGA6, KRTCAP3, LSR, MUC13, NOX1, PLOD3, PLPP2, SLC12A2,

SLC44A4, SLC52A2, SMIM22, ST14, TFRC, TMPRSS4, sLE(x), and TSPAN6. In certain embodiments, the tumor antigen is selected from CLDN4, CLDN7, EPCAM, PIGR, TMEM141,

TMEM54, TSPAN1, LRG5, and TSPAN8. In certain embodiments, the tumor antigen is selected from ACSL5, AP1M2, AREG, CDH1, CDH17, CEACAM5, CEACAM6, CEACAM7,

CLCA1, CLDN3, DPEP1, ERBB3, GPA33, GPRC5A, ITGA6, KRTCAP3, LSR, MUC13,

NOX1, PLOD3, PLPP2, SLC12A2, SLC44A4, SLC52A2, SMIM22, ST14, TFRC, TMPRSS4, sLE(x), TSPAN6, CLDN4, CLDN7, EPCAM, PIGR, TMEM141, TMEM54, TSPAN1, LRG5, and TSPAN8. In certain embodiments, the tumor antigen is any one of ACSL5, AP1M2, AREG,

CDH1, CDH17, CEACAM5, CEACAM6, CEACAM7, CLCA1, CLDN3, DPEP1, ERBB3,

GPA33, GPRC5A, ITGA6, KRTCAP3, LSR, MUC13, NOX1, PLOD3, PLPP2, SLC12A2,

SLC44A4, SLC52A2, SMFM22, ST14, TFRC, TMPRSS4, sLE(x), TSPAN6, CLDN4, CLDN7,

EPCAM, PIGR, TMEM141, TMEM54, TSPAN1, LRG5, and TSPAN8.

[0370] Additionally, such tumor antigens can be derived from the following specific conditions and/or families of conditions, including but not limited to, cancers such as brain cancers, skin cancers, lymphomas, sarcomas, lung cancer, liver cancer, leukemias, uterine cancer, breast cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, hemangiosarcomas, bone cancers, blood cancers, testicular cancer, prostate cancer, stomach cancer, intestinal cancers, pancreatic cancer, and other types of cancers as well as pre-cancerous conditions such as hyperplasia or the like.

[0371] Non-limiting examples of cancers can include Acute lymphoblastic leukemia (ALL); Acute myeloid leukemia; Adrenocortical carcinoma; Astrocytoma, childhood cerebellar or cerebral; Basal -cell carcinoma; Bladder cancer; Bone tumor, osteosarcoma/malignant fibrous histiocytoma; Brain cancer; Brain tumors, such as, cerebellar astrocytoma, malignant glioma, ependymoma, medulloblastoma, visual pathway and hypothalamic glioma; Brainstem glioma; Breast cancer; Bronchial adenomas/carcinoids; Burkitt's lymphoma; Cerebellar astrocytoma; Cervical cancer; Cholangiocarcinoma; Chondrosarcoma; Chronic lymphocytic leukemia;

Chronic myelogenous leukemia; Chronic myeloproliferative disorders; Colon cancer; Cutaneous T-cell lymphoma; Endometrial cancer; Ependymoma; Esophageal cancer; Eye cancers, such as, intraocular melanoma and retinoblastoma; Gallbladder cancer; Glioma; Hairy cell leukemia; Head and neck cancer; Heart cancer; Hepatocellular (liver) cancer; Hodgkin lymphoma;

Hypopharyngeal cancer; Islet cell carcinoma (endocrine pancreas); Kaposi sarcoma; Kidney cancer (renal cell cancer); Laryngeal cancer; Leukemia, such as, acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myelogenous and, hairy cell; Lip and oral cavity cancer;

Liposarcoma; Lung cancer, such as, non-small cell and small cell; Lymphoma, such as, AIDS- related, Burkitt; Lymphoma, cutaneous T-Cell, Hodgkin and Non-Hodgkin, Macroglobulinemia, Malignant fibrous histiocytoma of bone/osteosarcoma; Melanoma; Merkel cell cancer;

Mesothelioma; Multiple myeloma/plasma cell neoplasm; Mycosis fungoides; Myelodysplastic syndromes; Myelodysplastic/myeloproliferative diseases; Myeloproliferative disorders, chronic; Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma; Neuroblastoma;

Oligodendroglioma; Oropharyngeal cancer; Osteosarcoma/malignant fibrous histiocytoma of bone; Ovarian cancer; Pancreatic cancer; Parathyroid cancer; Pharyngeal cancer;

Pheochromocytoma; Pituitary adenoma; Plasma cell neoplasia; Pleuropulmonary blastoma; Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal pelvis and ureter, transitional cell cancer; Rhabdomyosarcoma; Salivary gland cancer; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, soft tissue; Sarcoma, uterine; Sezary syndrome; Skin cancer (non-melanoma); Skin carcinoma; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma; Squamous neck cancer with occult primary, metastatic; Stomach cancer; Testicular cancer; Throat cancer; Thymoma and thymic carcinoma; Thymoma; Thyroid cancer; Thyroid cancer, childhood; Uterine cancer; Vaginal cancer; Waldenstrom macroglobulinemia; Wilms tumor; and any combination thereof.

[0372] The invention provides any therapeutic compound or conjugate disclosed herein for use in a method of treatment of the human or animal body by therapy. Therapy may be by any mechanism disclosed herein, such as by stimulation of the immune system. The invention provides any therapeutic compound or conjugate disclosed herein for use in stimulation of the immune system, vaccination or immunotherapy, including for example enhancing an immune response. The invention further provides any therapeutic compound or conjugate disclosed herein for prevention or treatment of any condition disclosed herein, for example cancer, autoimmune disease, inflammation, sepsis, allergy, asthma, graft rejection, graft- versus-host disease, immunodeficiency or infectious disease (typically caused by an infectious pathogen). The invention also provides any therapeutic compound or conjugate disclosed herein for obtaining any clinical outcome disclosed herein for any condition disclosed herein, such as reducing tumour cells in vivo. The invention also provides use of any therapeutic compound or conjugate disclosed herein in the manufacture of a medicament for preventing or treating any condition disclosed herein. General Synthetic Schemes and Examples

[0373] The following synthetic schemes are provided for purposes of illustration, not limitation. The following examples illustrate the various methods of making compounds described herein. It is understood that one skilled in the art may be able to make these compounds by similar methods or by combining other methods known to one skilled in the art. It is also understood that one skilled in the art would be able to make, in a similar manner as described below by using the appropriate starting materials and modifying the synthetic route as needed. In general, starting materials and reagents can be obtained from commercial vendors or synthesized according to sources known to those skilled in the art or prepared as described herein.

Preparation of cyclic boronate ester TNIK inhibitors

Scheme 1

nthesis of benzoxaboroles

[0374] 5-N-Boc-aminosalicylaldehyde (iii) can be prepared using a two-step procedure starting from 3-aminophenol by protection of the amino group followed by ortho formylation using para formaldehyde and magnesium chloride-triethylamine according to the method described by Hofslokken and Skattebol (Acta Chemica Scandinavica, 53, 1999, 258-262). The phenol can be converted to the corresponding triflate ester (iv) using triflic anhydride and a tertiary amine base such as triethylamine. Aryl triflates (iv) can then be converted to boronate ester analogs (v) using a Miyaura borylation reaction with bis(pinacolato)diboron and a palladium catalyst such as PdCl ₂ dppf. A Henry reaction between aldehydes (v) and

nitromethane provides cyclic boronate esters (vi) which can be reduced with nickel in ammonium hydroxide to afford alkylamines (vii). The amine group of (vii) can be protected using trifluoroacetic anhydride and a tertiary amine base such as triethylamine to provide trifluoroacetamide analogs (viii). The boc group can be deprotected using a strong acid such as HC1 in dioxane to provide arylamines (ix) which can subsequently undergo nucleophilic aromatic substitution reactions with appropriately substituted quinazolines to give compounds (x) which can be converted to the amine analogs (xi) upon treatment with a base such as sodium hydroxide.

Scheme 2

of benzoxaborinols and benzoxaborapinols

xvi

[0375] Aryl bromides (xii) can be converted to the pinacolboronate esters (xiii) using bis(pinacolato) diboron and a palladium catalyst such as PdCl ₂ dppf. Reduction of the carboxylic ester provides the desired 3,4-dihydro-lH-benzo[c][l,2]oxaborinin-l-ols (m=l) or 4,5- dihydrobenzo[c][l,2]oxaborepin-l(3H)-ols (m=2) which can subsequently be nitrated using nitric acid to provide cyclic boronates (xv). The nitro group can be reduced to the target aryl amines (xvi) using a method such as hydrogenation using a metal catalyst such as palladium on carbon. Amine intermediates (xvi) can undergo a nucleophilic aromatic substitution reaction with an appropriately substituted quinazoline analog in the presence of catalytic toluenesulfonic acid to afford target molecules (xvii).

Scheme 3 Synthesis of diazaborinols

[0376] Aldehyde (v) can be deprotected using a strong acid such as HC1 in dioxane to afford aniline (xxiii). Aniline (xviii) can then be heated with a chloroquinazoline analog in the presence of an acid such as p-toluenesulfonic acid to provide intermediate boronates (xix) which can then undergo cyclization to the target diazaborines (xx) upon heating with an appropriately substituted hydrazine analog in the presence of a base such as ammonium hydroxide in ethanol.

Scheme 4

Synthesis of diazaborinols

xxii

[0377] Aldehyde (v) can be reacted with hydroxylamine hydrochloride in alcohol to provide benzoxazines (xxi) which can be deprotected using a strong acid such as HC1 in dioxane to afford aniline (xxii). Aniline (xxii) can then be heated with a chloroquinazoline analog in the presence of an acid such as p-toluenesulfonic acid to provide the desired targets (xxiii).

Preparation of macrocyclic TNIK inhibitors Scheme 5

Synthesis ofMacrocycles via Ring Closing Metathesis

xxx xxxi

[0378] Substituted 2-chloro-8-hydroxyquinazolines (xiv) can be alkylated to intermediates (xv) using either an co-haloalkene and a carbonate base such as potassium carbonate in DMF to provide intermediates (xv). 6-nitroquinazolin-4-ol can be converted to its olefinic ether analog (xvii) using an co-haloalkene and a carbonate base such as cesium carbonate in DMF at an elevated temperature. Reduction of the nitro group can be effected using iron in acetic acid to provide compounds (xviii) which can be reacted with 2-chloroquinazolines (xv) to afford S _N Ar adducts (xix). Ring closing metathesis reaction of bis-olefin (xix) using a ruthenium catalyst such as Grubbs 2 ^nd generation catalyst in a dilute solvent such as dichloroethane provides macrocyclic alkenes (xxx) which can be converted to alkanes (xxxi) using standard

hydrogenation conditions.

Scheme 6

Synthesis ofMacrocycles via Intramolecular Mitsunobu Reaction

χχχν xxxvi xxxvii

[0379] Dichloroquinazolines (xxxii) can be heated with amino alcohols to provide C-l adducts (xxxiii) which can be reacted with a second amine group in the presence of a base scavenger such as cesium carbonate in a polar aprotic solvent such as DMF elevated temperatures to afford C-3 adducts (xxxiv). The nitro group can be reduced to the target aryl amines (xxxv) using standard hydrogenation methodology such as hydrogen gas and catalytic palladium on carbon. Amine intermediates (xxxv) can undergo a nucleophilic aromatic substitution reaction with an appropriately substituted quinazoline analog in the presence of catalytic toluenesulfonic acid to afford target molecules (xxxvi). Macrocyclic ring formation using triphenylphosphine and diisopropyl azodicarboxylate can provide the target compounds (xxxvii)

EXAMPLES

Example 1

Synthesis of 3-(aminomethyl)-6-((8-((5-hydroxy-3,3-dimethylpentyl)oxy)qui nazolin-2- yl)amino)benzo[c][l,2]oxaborol-l(3H)-ol (Compound 1.1)

Int 1.1a

[0380] 2~ibrmylpher3ylboronic acid (6.0 g, 40.0 mmol) was added to solution of sodium hydroxide (1.70 g) in 20 niL of water at 10°C. To this suspension was added nitromethane (2.2 fflL) and the reaction was wanned to room temperature with stirring. After 30 minutes, the reaction was cooied in an ice bath and acidified with 3M HQ. The resulting white precipitate that formed was filtered and air dried to provide 6.0 g of 3-(nitromethyl)benzo[c][l,2]oxaborol-

I (3H)-oi (Int 1.1a) as an off-white solid. ^f H NMR (DMSO~d ₆ ) δ 9.48 (s, I H), 7.71-7.74 (d. i 6.9 Hz, Π ! }. 7.47-7.54 (m, 2H) 7.39 (t, .) 7.65 Hz, 1H) 5.73-7.78 (dd, i 2.7, j 9.0 Hz, 1H), 5.30-5.35 (dd, J=3.0, J=13.5 Hz, 1H), 4.52-4.59 (dd, J=13.5, J=9.3 Hz, 1H).

Step B: Preparation o Int Lib

Int 1.1a Int 1.1b

[0381] Int 1. la (5.7 g, 30 mmol) was added in small portions with stirring over 2 h to fuming nitric acid (200 mL) at 45°C. The cold reaction mixture was then poured into crushed ice and allowed to warm to room temperature. The aqueous phase was extracted with EtOAc and the organic phase was concentrated in vacuo. The residue was poured into crushed ice and the precipitate was filtered and washed with water. The solid was dissolved in EtOAc, dried (Na ₂ S0 ₄ ), and concentrated in vacuo to yield 4.0 g of Int 2.1b. 1H NMR (DMSO-d ⁶ ) δ 9.87 (bs, 1H), 8.57 (s, 1H), 8.40 (dd, J=8.4, 2.4 Hz, 1H), 7.85 (d, J=8.0 Hz, 1H), 5.92 (dd, J=8.0, 2.8 Hz, 1H), 5.42 (dd, J=13.6, 2.8 Hz, 1H), 4.82 (dd, J=13.6, 8.0 Hz, 1H).

[0382] A solution containing 2.4 g (10.0 mmol) of Int 1. lb and 500 mg of 10% Pd on carbon in 100 mL of EtOAc was degassed with N ₂ then stirred under a balloon of hydrogen for 5h. The reaction mixture was filtered through Celite then concentrated to provide compound Int 1.1c as a solid. 1H MR (DMSO-d ⁶ ) δ 9.25 (s, 1H), 7.13 (d, J=8.0 Hz, 1H), 6.85 (d, J=1.6 Hz, 1H), 6.68

(dd, J=8.0, 1.6 Hz, 1H), 5.58 (dd, J=9.2, 2.4 Hz, 1H), 5.21-5.16 (m, 3H), 4.36 (dd, J=13.2, 9.6

Hz, 1H). LCMS (M+H) = 309.

Step D: Preparation of Int Lid

Int 1.1d

[0383] A solution containing 2.0 g (7.74 mmol) of triphenylphosphine in 30 mLwas treated drop wise with 1.5 mL (7.74 mmol) of diisopropyl azodicarboxylate and the reaction was stirred for 10 minutes. A solution of 3.8 g (15.5 mmol) 5-((tert-butyldimethylsilyl)oxy)-3,3- dimethylpentan-l-ol in 5 mL of THF was added and the resulting reaction was stirred for 5 minutes before the addition of 929 mg (5.16 mmol) 2-chloro-8-hydroxyquinazoline. The reaction was stirred over 16h then the solvents were evaporated. The residue was then chromatographed (40 gram silica column; 0% to 100% EtOAc / Hexanes) to provide the desired compound Int 2. Id which was used directly in the next reaction. LCMS (M+H) = 409.

Step E: Pre aration of Int Lie

Int 1.1d Int 1.1 e

[0384] A mixture containing 800 mg (2.0 mmol) of Int 1. Id in 20 mL of toluene was added 620 mg (2.0 mmol) of Int 1.1c and a catalytic amount of p-toluenesulfonic acid. The reaction mixture was heated to 100°C for 16h then cooled and evaporated. The residue was chromatographed by preparative HPLC to afford the desired compound Int L ie. 1H NMR (DMSO-d ⁶ ) δ 9.24 (s, IH), 8.25-8.21 (m, 2H), 7.47-7.43 (m, 2H), 7.33-7.29 (m, 2H), 5.75 (dd, J=2.4, 8.4 Hz, IH), 5.25 (dd, J= 2.8, 8.4 Hz, IH), 4.53 (m, IH), 4.17 (t, J=6.4 Hz, 2H), 3.48 (t, J=6.4 Hz, 2H), 1.87 (t, J=7.2 Hz, 2H), 1.47 (t, J=7.2 Hz, 2H), 0.96 (s, 6H). LCMS (M+H) = 467.2.

Step F: Pre aration of Compound 1.1

Int 1.1 e Compound 1.1

[0385] A mixture of Int 2.1e (233 mg, 0.50 mmol), Raney nickel (100 mg), concentrated NH ₄ OH (1.0 mL), H ₂ 0 (10 mL), and MeOH (5 mL) was shaken in a Parr apparatus under an atmosphere of hydrogen (50 psi) at room temperature over 16 h. The mixture was filtered through Celite and the filtrate was concentrated. The residue was purified by preparative HPLC to afford the title compound. ¹ H MR (DMSO-d ⁶ ) δ 9.21 (s, IH), 8.25-8.21 (m, 2H), 7.45-7.39 (m, 2H), 7.30 (m, 2H), 5.29 (d, J=8.0Hz, IH), 4.16 (t, J=6.4 Hz, 2H), 3.49 (t, J=6.4 Hz, 2H), 2.74 (t, J=13.2 Hz, IH), 1.82 (t, J=7.2 Hz, 2H), 1.49 (t, J=7.2 Hz, 2H), 0.95 (s, 6H). LCMS (M+H) = 419.2.

[0386] Other heterocyclic compounds can be prepared in a manner similar to that described in Example 1 above. Structures and associated spectroscopic data are included in Table 1 below.

Table 1. Additional heterocyclic compounds.

Compound Structure Spectroscopic Data

1H NMR (DMSO-d6) δ 10.1 (s, 1H), 9.28 (s, 1H), 8.96 (s, 1H), 8.03 (s, 1H), 7.79 (s, 1H), 7.49 (d, J=8.0Hz, 1H), 7.36 (d,

1.13 J=7.6Hz, 1H), 7.36-7.28

(m, 2H), 4.75 (s, 1H),

1 -(1 -hydroxy-7-((8-(((1 s,4s)-4-hydroxycyclohexyl) 4.56 (s, 1H), 3.61 (m, oxy)quinazolin-2-yl)amino)benzo[d][1 ,2,3]

diazaborinin-2(1 H)-yl)ethan-1 -one 1H), 2.50 (s, 3H), 1.96- 1.65 (m, 8H). M+H = 428.2.

1H NMR (DMSO-d6) δ 10.0 (s, 1H), 8.96 (m, 1H), 8.03 (s, 1H), 7.83 (d, J=8.8Hz, 1H), 7.49 (m, 1H), 7.36-7.31 (m, 2H),

1.14

4.72 (s, 1H), 4.42 (s, 1H), 3.61 (m, 1H), 2.07-1.93 ferf-butyl (2-(1 -hydroxy-7-((8-(((1 s,4s)-4-hydroxycyclohexyl)

oxy)quinazolin-2-yl)amino)benzo[d][1 ,2,3] (m, 3H), 1.75-1.63 (m, diazaborinin-2(1 H)-yl)-2-oxoethyl)carbamate

5H). 1.50 (s, 9H). M+H = 543.3.

1H NMR (DMSO-d6) δ

NH ₂ 10.1 (s, 1H), 9.27 (s, 1H),

8.86 (d, J=8.8Hz, 1H), 7.66 (s, 1H), 7.49-7.28 (m, 5H), 6.67 (m, 2H),

1.15

4.73 (s, 1H), 4.63 (s, 1H),

2-amino-1 -(1 -hydroxy-7-((8-(((1 s,4s)-4- 3.65 (m, 2H), 3.12 (m, hydroxycyclohexyl)oxy)quinazolin-2- yl)amino)benzo[d][1 ,2,3]diazaborinin-2(1 H)- 1H), 1.99-1.93 (m, 2H), yl)ethan-1-one 1.75-1.63 (m, 6H). M+H

= 443.3. Compound Structure Spectroscopic Data

1H NMR (DMSO-d6) δ 9.24 (s, 1H), 9.80 (m, 1H), 8.43 (m, 1H), 8.05

Boc o OH _N ^k _N (s, 1H), 7.78 (d, J=9.6Hz,

1H), 7.48 (m, 1H), 7.32

1.16

(m, 2H), 5.45 (m, 1H), ferf-butyl 2-(1 -hydroxy-7-((8-(((1 s,4s)-4- 4.70 (m, 1H), 3.63 (m, hydroxycyclohexyl)oxy)quinazolin-2-yl)amino)- 1 ,2-dihydrobenzo[d][1 ,2,3]diazaborinine-2- 1H), 3.42 (m, 2H), 1.99- carbonyl)pyrrolidine-1-carboxylate 1.53 (m, 12H), 1.33-1.22

(m, 9H). M+H = 583.3.

1H NMR (DMSO-d6) δ 10.0 (s, 1H), 9.28 (s, 1H), 8.83 (m, 1H), 7.72-7.29 (m, 7H), 4.74 (m, 1H),

1.17

4.64 (m, 1H), 4.37 (m, 1H), 3.65 (m, 2H), 1.78-

1 -hydroxy-7-((8-(((1 s,4s)-4- hydroxycyclohexyl)oxy)quinazolin-2-yl)amino)-2- 1.69 (m, 8H). M+H = prolyl-1 ,2-dihydrobenzo[ ][1 ,2,3]diazaborinine

483.3.

1H NMR (DMSO-d6) δ 10.1 (s, 1H), 9.28 (s, 1H), 9.02 (m, 1H), 8.06 (s, 1H), 7.72 (s, 1H), 7.55 (d, J=7.8Hz, 1H), 7.36 (d,

° ^0H N^N

J=7.6Hz, 1H), 7.36-7.28

1.18

(m, 3H), 4.74 (m, 1H), 4.55 (m, 1H), 3.90 (m, fert-butyl 4-(1 -hydroxy-7-((8-(((1 s,4s)-4-hydroxycyclohexyl)

oxy)quinazolin-2-yl)amino)-1 ,2-dihydrobenzo[d][1 ,2,3] 2H), 3.80-3.61 (m, 2H), diazaborinine-2-carbonyl)piperidine-1 -carboxylate

2.81 (m, 2H), 1.97-1.65 (m, 9H), 1.31 (s, 9H). M+H = 597.4.

Example 2.1

Synthesis of 4, 13-dioxa-2-aza-l(2,8),3(6,4)-diquinazolinacyclotridecaphane TFA salt

(Compound 2.1)

Step A: Preparation of Int 2. la

1. Cs C0 ₃

Int 2. 1a

[0387] A mixture containing 955 mg (5.00 mmol) 6-nitroquinazolin-4(3H)-one in 25 mL of DMF was treated with 3.25 g (10.0 mmol) of Cs ₂ C0 ₃ and 1.49 g (10.0 mmol) of 5-bromo-l- pentene and the reaction was heated to 70°C for 16h. The reaction was cooled and quenched with water then extracted three times with EtOAc. The combined organic extracts were dried and concentrated. Silica gel chromatography (12g ISCO gold; 0% to 100% EtOAc / Hexanes) provided 857 mg of the alkylated intermediate which was immediately covered with 45 mL of HO Ac and 5 mL of water. 1.8 g (33.0 mmol) of iron powder was added and the mixture was heated at 50°C for 4h. The reaction was cooled and filtered through Celite then evaporated to a brown solid. The solid was covered with 2N Na ₂ C0 ₃ and extracted with EtOAc three times. The combined organic extracts were washed with NaHC0 ₃ then brine. The organic phase was separated and dried over Na ₂ S0 ₄ . Silica gel chromatography (12g ISCO gold; 0% to 100% EtOAc / Hexanes) provided Int 2.1a which was used directly. LCMS (M+H) = 230.2

Step B : Preparation o Int 2.1b

Int 2.1b

[0388] A mixture containing 500 mg (2.79 mmol) 2-chloro-8-hydroxyquinazoline in 8 mL of DMF was treated with 771 mg (5.58 mmol) of K ₂ C0 ₃ , 623 mg (4.18 mmol) of 5-bromo-l- pentene and 72 mg (0.20 mmol) of tetrabutyl ammonium iodide and the reaction was heated to 60°C for 16h. The reaction was cooled and quenched with water then extracted three times with EtOAc. The combined organic extracts were dried and concentrated. Silica gel chromatography (12g ISCO gold; 0% to 100% EtOAc / Hexanes) provided the desired compound Int 2.1b as a white solid. Step C: Preparation of Int 2.1c

[0389] A solution containing 132 mg (0.61 mmol) of Int 2.1a and 150 mg (0.61 mmol) of Int 2. lb in 4 mL of isopropanol was heated to 100°C in a sealed tube for 16h. The reaction mixture was cooled and filtered washing with cold IPA to afford 200 mg of the HC1 salt of the desired compound which was used in the next reaction without additional purification. LCMS (M+H) = 442.2.

Ste D : Preparation of Int 2. Id

Int 2.1c Int 2.1d

[0390] A solution containing 44 mg (0.1 mmol) of Int 2. lc in 20 mL of dichloroethane was degassed three times with nitrogen gas then treated with 9 mg (0.01 mmol) of benzylidene- bis(tricyclohexylphosphino)dichlororuthenium and the reaction was heated at 40°C for 16 h. The reaction mixture was cooled and concentrated then chromatographed (4g ISCO; 0% to 100% EtOAc / Hexanes) provided the desired compound Int 2. Id as a brown solid consisting of a mixture of cis and trans isomers. LCMS (M+H) = 414.5.

[0391] To a solution containing 44 mg (0.10 mmol) of Int 2. Id in 2 mL of EtOH was added

10 mg of 5% Pd on carbon. The resulting mixture was stirred under an atmosphere of H ₂ for 16h then filtered and evaporated. Reverse phase HPLC provided 29 mg of the TFA salt of the desired Compound 2.1 as a white solid. 1H MR (CDC1 ₃ ) δ 9.85 (s, 1H), 9.39 (bs, 1H), 9.09 (s,

1H), 7.81 (d, 9.0 Hz, 1H), 7.56 (dd, J=2.0, 8.5 Hz, 1H), 7.41 (m, 2H), 7.28 (m, 1H), 4.28 (t,

J=5.5 Hz, 2H), 4.20 (t, J=5.5, 2H), 2.13-2.10 (m, 2H), 1.87 (bs, 2H), 1.60 (m, 4H), 1.48-1.45 (m,

2H), 1.29-1.25 (m, 6H). LCMS (M+H) = 416.3.

[0392] Table 2 below depicts other compounds that can be prepared in a manner similar to that described in Example 2.

Table 2. Additional heterocyclic compounds.

Compound Structure Spectroscopic Data

2.8 CDC1 ₃ δ 9.53 (s, 1H), 9.07 (s,

HN A N XJ 1H), 8.95 (s, 1H), 8.21 (d,

J=8.4Hz, 1H), 7.62 (d, J=6.4Hz, 1H), 7.34-7.21 (m,

N ^N I / 3H), 5.65-5.61 (m, 1H), 5.44-

(Z)-4, 14-dioxa-2-aza-1 (2,8),3(6,4)- 5.41 (m, 1H), 4.91 (t, J=5.2Hz, diquinazolinacyclotetradecaphan-7-ene

2H), 4.32 (t, J=6.0Hz, 2H), 2.62 (m, 2H), 2.05 (m, 2H), 1.81 (m, 2H), 1.41 (m, 2H). M+H = 428.2.

2.9 CDCI3 δ 9.73 (s, 1H), 9.15 (s,

1H), 8.95 (s, 1H), 8.21 (d, J=8.4Hz, 1H), 7.94 (bs, 1H), 7.62 (d, J=8.0Hz, 1H), 7.41- 7.27 (m, 2H), 5.69-5.53 (m,

(£)-4, 14-dioxa-2-aza-1 (2,8),3(6,4)- diquinazolinacyclotetradecaphan-7-ene 2H), 4.83 (t, J=8.0Hz, 2H),

4.34 (t, J=8.0Hz, 2H), 2.82 (m, 2H), 2.29 (m, 4H), 1.61 (m, 2H). M+H = 428.2.

2.10 DMSO-d ⁶ δ 9.54 (s, 1H), 9.20

(s, 1H), 8.57 (d, J=8.0Hz, 2H), 7.94 (d, J=8.0Hz, 1H), 7.81 (d, J=8.0Hz, 1H), 7.62 (d, cis or trans (4-(4,12-dioxa-2-aza-1 (2,8),3(6,4)- J=8.0Hz, 2H), 7.47-7.34 (m, diquinazolinacyclododecaphan-7-en-3 ² - yl)phenyl)methanamine 3H), 6.00 (m, 1H), 5.70 (m,

1H), 5.06 (t, J=5.2Hz, 2H), 4.55 (t, J=8.0Hz, 2H), 4.50 (q, 2H), 4.25-4.20 (m, 4H), 2.62 (m, 2H), 2.25 (m, 4H), 1.98 (m, 2H). M+H = 505.1. Compound Structure Spectroscopic Data

2.11 CD ₃ OD δ 9.46 (s, 1H), 9.21 (s,

1H), 7.78 (d, J=7.2Hz, 1H), 7.70 (d, J=7.2Hz, 1H), 7.47 (d, J=6.4Hz, 1H), 7.38-7.31 (m,

(Z)-3 ² -(piperidin-4-yloxy)-4, 12-dioxa- 2H), 5.94 (m, 1H), 5.71 (m,

2-aza-1 (2,8),3(6,4)- 1H), 5.55 (m, 1H), 4.95 (t, diquinazolinacyclododecaphan-7-ene

J=5.2Hz, 2H), 4.54 (t, J=5.2Hz, 2H), 3.49-3.45 (m, 2H), 3.34 m, 2H), 2.55 (m, 2H), 2.35-2.22 (m, 6H), 1.99 (m, 2H), 0.90 (m, 2H). M+H = 499.1.

2.12 DMSO-d ⁶ δ 9.91 (s, 1H), 9.29

(s, 1H), 8.89 (s, 1H), 8.35 (s, 1H), 7.77-7.74 (m, 1H), 7.47

12-oxa-2,4-diaza-1 (2,8),3(6,4)- (m, 2H), 7.29 (m, 1H), 7.27 (d, diquinazolinacyclododecaphane

J=8.0Hz, 2H), 4.11 (t, J=5.6Hz, 2H), 3.60 (m, 3H), 1.75-1.72 (m, 4H), 1.54-1.49 (m, 7H). M+H = 401.2.

2.13 CDCI ₃ δ 9.52 (s, 1H), 9.16 (s,

1H), 8.76 (s, 1H), 8.25 (d, J=8.4Hz, 1H), 8.00 (bs, 1H),

(Z)-12-oxa-2,4-diaza-1 (2, 8), 3(6,4)- 7.45-7.27 (m, 4H), 5.85 (m, diquinazolinacyclododecaphan-7-ene

1H), 5.69 (m, 1H), 4.58 (t, J=5.6Hz, 2H), 4.05 (m, 2H), 2.48 (m, 2H), 2.28 (m, 2H), 2.08 (m, 2H). M+H = 399.2. Compound Structure Spectroscopic Data

2.14 DMSO-d ⁶ δ 10.4 (s, 1H), 9.39

(s, 1H), 9.29 (s, 1H), 8.42 (d,

J=7.6Hz, 2H), 8.30 (m, 3H),

7.88 (m, 2H), 7.70 (d,

J=8.4Hz, 2H), 7.58 (d,

(£)-(4-(12-oxa-2,4-diaza-1 (2, 8), 3(6,4)- diquinazolinacyclododecaphan-7-en-3 ² - J=7.6Hz, 1H), 7.41-7.27 (m, yl)phenyl)methanamine

2H), 5.67-5.46 (m, 2H), 4.37 (t, J=8.0Hz, 2H), 4.19 (t, J=8.0Hz, 2H), 3.87 (m, 2H), 2.53 (s, 2H), 2.12 (m, 2H), 1.81 (m, 2H). M+H = 504.3.

Example 2.2

Synthesis of (2S)-2-amino-Nl-(3-(2-((2-(2,6-dioxopiperidin-3-yl)-l,3-diox oisoindolin-4- yl)oxy)acetamido)propyl)-N5-((l-hydroxy-6-((8-(((ls,4s)-4-hy droxycyclohexyl)oxy)quinazolin- 2-yl)amino)-l,3-dihydrobenzo[c][l,2]oxaborol-3-yl)methyl)pen tanediamide (Compound 2.15)

Step A: Preparation of Int 2.2a

Int 2.2a

[0393] To a solution containing 116 mg (0.276 mmol) of compound 1.8 in 2 mL of DMF was added 93 mg (0.276 mmol) of Boc-Glu(OBzl)-OH, 105 mg (0.276 mmol) of HATU and 0.06 mL of N-methylmorpholine. The resulting mixture was stirred over 16 hours. The reaction mixture was quenched with NaHC0 ₃ and washed with EtOAc three times. The organic extracts were dried and evaporated. The residue was chromatographed on silica gel (0% to 20% Methanol / dichloromethane) to afford Int 2.2a as a white solid. M+H = 739.3.

Step B: Preparation of Int 2.2b

Int 2.2a Int 2.2b

[0394] To a solution containing 175 mg (0.269 mmol) of compound Int 2.2a in 10 mL of THF and 5 mL of methanol was added 1 mL of 2N LiOH. The resulting mixture was stirred for 3 hours. The reaction mixture was evaporated and purified by reverse phase chromatography to afford Int 2.2b as a white solid after lyophilization. M+H = 649.3.

Step C: Preparation of Compound 2.2

111

[0395] To a solution containing 75 mg (0.115 mmol) of compound Int 2.2a in 2 mL of DMF was added 57 mg (0.115 mmol) ofN-(3-aminopropyl)-2-[[2-(2,6-dioxo-3-piperidinyl)-2,3- dihydro-l,3-dioxo-lH-isoindol-4-yl]oxy]acetamide TFA salt (CAS No 2022182-58-5), 44 mg (0.115 mmol) of HATU and 2.0 equivalents of N-methylmorpholine. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with NaHC0 ₃ and washed with EtOAc three times. The organic extracts were dried and evaporated then chromatographed by reverse phase HPLC to afford 30 mg of the Boc protected product as a white solid (M+H = 1020.5). This material was redissolved in 5 mL of EtOAc and treated with 1 mL of 4N HCl in dioxane then stirred for 4 hours. The solvent was evapoarated and the residue was triturated in toluene then dried under high vacuum to afford 8 mg of Compound 2.15 as a white solid. 1H NMR (DMSO-d ⁶ ) δ 11.1 (s, 1H), 9.94 (s, 1H), 9.25 (s, 2H), 8.66 (m, 1H), 8.43 (t, J=8.0Hz, 1H), 8.30 (m, 1H), 8.25 (m, 2H), 8.13 (t, J=7.8Hz, 1H), 7.89 (s, 1H), 7.77 (t, J=7.8 Hz, 1H), 7.49 (m, 2H), 7.47 (d, J=7.6Hz, 2H), 7.38-7.26 (m, 2H), 5.12-5.08 (m, 2H), 4.77 (s, 2H), 4.72 (m, 1H), 3.72 (m, 1H), 3.61 (m, 2H), 3.11-2.92 (m, 7H), 1.97 (m, 3H), 1.91 (m, 8H), 1.68-1.63 (m, 8H). M+H = 921.3.

[0396] The following compounds can be prepared in a manner similar to that used for the synthesis of Compound 2.2.

oxohexane- 1 , 5-diyl)dicarbamate

dioxoisoindolin-4-yl)oxy)acetamido)-l-(((l-hydroxy-6-((8-

1 ,3 -dihydrobenzo[c] [ 1 ,2]oxaborol-3 -yl)methyl)hexanamide

Example 3

Preparation of Linker Compounds

Preparation of 4-((R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hex anamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl ((l-hydroxy-6-((8-(((ls,4s)-4- hydroxycyclohexyl)oxy)quinazolin-2-yl)amino)-l,3-dihydrobenz o[c][l,2]oxaborol-3- yl)methyl)carbamate (Compound 3.1)

[0397] To a stirred solution of Compound 1.8 (46 mg, 0.109 mmol) and Hunig's base (0.057 mL, 0.326 mmol) in DMF (3.4 mL) under nitrogen cooled in an ice-water bath was added dropwise a solution of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hex anamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (80 mg, 0.109 mmol) in DMF (2 mL). The resulting mixture was stirred overnight while the cooling bath expired. The mixture was then concentrated and the residue neutralized with saturated NaHC0 ₃ and purified by reverse phase column. Fractions were pooled and concentrated to afford

Compound 3.1 as an off-yellow solid. 1H MR (DMSO-d ⁶ + D ₂ 0) δ 9.22 (s, 1H), 8.58 (d, J=9.2Hz, 1H), 7.89 (d, J=2.5Hz, 1H), 7.85 (d, J=8.0Hz, 1H), 7.55-7.25 (m, 10H), 5.11 (m, 1H), 4.94 (s, 2H), 4.70 (m, 1H), 4.32 (m, 1H), 4.12 (m, 1H), 3.51-3.40 (m, 4H), 3.06-2.93 (m, 4H), 2.16-2.09 (m, 3H), 1.94-1.84 (m, 3H), 1.75-1.22 (m, 18H), 1.18 (m, 2H), 0.80 (t, J=7.5Hz, 6H). LCMS (M+H) = 1019.5.

[0398] The following compounds were prepared in a manner similar to that described for the preparation of Compound 3.1.

4-((R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hex anamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl (4-((Z)-4,12-dioxa-2-aza- l(2,8),3(6,4)-diquinazolinacyclododecaphan-7-en-32-yl)benzyl )carbamate

Example 4

IC ₅₀ Determination for TNIK Inhibitors

[0399] TNIK enzyme inhibition assays were performed by Reaction Biology Corp (Malvern, PA) using the protocol described below.

[0400] 20 uM peptide subtrate [RLGRDKYKTLRQIRQ] and 10 uM ATP were prepared in a mixture of fresh reaction buffer consisting of 20 mM Hepes (pH 7.5), 10 mM MgCl ₂ , 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na ₃ V0 ₄ , 2 mM DTT, 1% DMSO. The kinase was delivered into the substrate solution which was gently mixed. Compounds in 100% DMSO were added to the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range) and the mixture was incubated for 20 min at room temperature. ³³ P-ATP (Specific activity 10 uCi/uL) was added into the reaction mixture to initiate the reaction and the whole was incubated for 2 hours at room temperature. Radioactivity was detected by filter-binding method and kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions. IC ₅₀ values and curve fits were obtained using Prism (GraphPad Software). Compounds having an IC ₅₀ value between 0.1 nM and 10 nM are denoted as ++++, 10 nM and 100 nM as +++, 100 nM and 1000 nM as ++, and 1000 nM to 10,000 nM as +. Table 3 includes IC ₅₀ values for selected compounds.

Table 3. IC ₅₀ values for selected compounds.

1.19 ++++

1.20 +++

Int 2.1c >10,000 nM

Int 2.1d +++

2.1 ++++

2.2 ++++

2.3 ++++

2.4 +++

2.5 ++++

2.6 ++++

2.7 +++

2.8 ++

2.9 ++

2.10 +++

2.11 ++

2.12 +++

2.13 +++

2.14 ++++

FIG. 1 shows activity for selected compounds.

Example 5

Selectivity of TNIK Inhibitors

[0401] The selectivity of a selected compound for TNIK determined by Reaction Biology Corp. (Malvern, PA) using a panel of kinase assays of 371 wildtype kinases. Compound 2.2 was tested in single dose duplicate mode at a concentration of 0.1 μΜ and the results are shown below.

CK2a 85.61 RSK2 95.37 ZAK/MLTK 100.20 GRK5 113.51

MST4 85.62 CAMKla 95.44 TNKl 100.21 MYLK3 113.99

IRAK4 85.87 PIM3 95.47 EPHA4 100.31 STK32C/Y ANK3 114.79

GSK3a 85.88 DCAMKL1 95.49 PKCg 100.40 PKG2 PRKG2 115.67 c-Kit 85.95 FAK/PTK2 95.50 PRKX 100.48 BRSK2 116.03

NEK 11 86.24 NEK6 95.52 CHK1 100.49 PKCiota 116.67

LYN 86.59 IGF1R 95.54 PAK2 100.49 TAOK1 117.19

BTK 86.70 NEK7 95.60 WEE1 100.50 SNRK 126.44

PDGFRb 87.27 ERBB4/HER4 95.62 JNK2 100.51 TGFBR2 129.20

CKlepsilon 87.29 ROCK1 95.74 TAOK3/JIK 100.63 TLK1 137.03

STK39/STLK3 87.29 FES/FPS 95.74 EPHA3 100.98 ASK1 MAP3K5 137.67

SNARK/NUAK2 87.37 WNK3 95.80 EPHA1 101.02

c-Src 87.44 ULK3 95.81 SBK1 101.02

HIPK4 87.63 CDK7/cyclin H 95.87 FGFR3 101.11

GSK3b 87.75 PHKg2 95.89 NEK2 101.17

FYN 87.75 CAMK2d 95.90 PLK2 101.39

ULK1 87.81 LATS2 95.91 PLK3 101.58

EPHB4 87.82 c-MER 95.99 STK38/NDR1 101.60

ERK7 MAPK15 87.88 MEK3 96.00 MAPKAPK2 101.72

CDK3/cyclin E 87.90 MSSK1/STK23 96.01 PKN1 PRK1 101.76

TYK1 LTK 88.04 MELK 96.17 CKld 101.83

STK33 88.38 ERK5/MAPK7 96.17 TIE2/TEK 102.01

NEK8 88.53 PKD2 PRKD2 96.20 KSR2 102.18

CKlg3 88.65 MKK6 96.20 TESK2 102.18

c-MET 88.74 PKCnu PRKD3 96.21 DRAK1/STK17A 102.31

STK22D/TSSK1 89.18 SGK2 96.28 CHK2 102.32

HCK 89.20 PKCzeta 96.31 DMPK 102.38

PKCa 89.34 FGR 96.32 RIPK5 102.42

FLT4/VEGFR3 89.60 p70S6K/RPS6KBl 96.34 PKAcb 102.66

PIM2 89.60 TSSK3/STK22C 96.35 IR 102.67

MAPKAPK5 PR

89.65 CAMKlb 96.51 VRK1 102.74

AK

ALK6/BMPR1B 89.71 OSR1/OXSR1 96.57 GRK4 102.76

YES/YES 1 89.96 MARK2/P AR- 1 B a 96.62 PKCtheta 102.77

TXK 89.97 ULK2 96.65 ERN2/IRE2 102.89

SLK/STK2 90.19 JNKl 96.67 GRK6 102.98

SIK2 90.62 LY B 96.87 EGFR 103.04

LIMK1 90.65 PAK6 96.88 PKCeta 103.25

PKA 90.74 CAMK2b 96.98 GRK2 103.29

ERK1 90.91 ROCK2 96.98 KKa CHUK 103.36

GCK/MAP4K2 90.94 VRK2 97.03 Aurora B 103.54

Example 6

Lysine-based Bioconjugation

[0402] An antibody construct can be conjugated to a linker via lysine-based bioconjugation. An antibody construct can be exchanged into an appropriate buffer, for example, phosphate, borate, PBS, Tris-Acetate, Tris-Glycine, HEPES, MOPS, MES, EPS, HEPPS, Histidine, or HEPBS at a concentration of about 2 mg/mL to about 10 mg/mL. An appropriate number of equivalents of a heterocyclic compound, e.g., a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), or (IIIc) described herein, can be added as a solution with stirring. Dependent on the physical properties of the heterocyclic compound-linker construct, a co-solvent can be introduced prior to the addition of the heterocyclic compound- linker construct to facilitate solubility. The reaction can be stirred at room temperature for 2 hours to about 12 hours depending on the observed reactivity. The progression of the reaction can be monitored by LC-MS. Once the reaction is deemed complete, the remaining heterocyclic compound-linker constructs can be removed by applicable methods and the antibody construct- heterocyclic compound conjugate can be exchanged into the desired formulation buffer. Lysine- linked conjugates can be synthesized starting with antibody (mAb) or bispecific antibody (bsAb) and equivalents (e.g., 10 equivalents) of the heterocyclic compound-linker construct following Scheme A below (Conjugate = antibody construct-heterocyclic compound conjugate). Monomer content and heterocyclic compound-antibody construct ratios (molar ratios) can be determined by methods described herein.

Scheme A:

heterocyclic compound- mAb + Conjugate

linker construct

Example 7

Cysteine-based Bioconjugation

[0403] An antibody construct can be conjugated to a linker via cysteine-based bioconjugation. An antibody construct can be exchanged into an appropriate buffer, for example, phosphate, borate, PBS, Tris-Acetate, Tris-Glycine, HEPES, MOPS, MES, EPS, HEPPS, HEPBS or Histidine at a concentration of about 2 mg/mL to about 10 mg/mL with an appropriate number of equivalents of a reducing agent, for example, dithiothreitol or tris(2-carboxyethyl)phosphine. The resultant solution can be stirred for an appropriate amount of time and temperature to effect the desired reduction. A heterocyclic compound, e.g., a compound or salt of any one of Formulas (I), (la), (lb), (II), (Ila), (lib), (lie), (III), (Ilia), (Illb), or (IIIc) described herein, can be added as a solution with stirring. Dependent on the physical properties of the heterocyclic compound- linker construct, a co-solvent can be introduced prior to the addition of the heterocyclic compound-linker construct to facilitate solubility. The reaction can be stirred at room

temperature for about 1 hour to about 12 hours depending on the observed reactivity. The progression of the reaction can be monitored by liquid chromatography-mass spectrometry (LC- MS). Once the reaction is deemed complete, the remaining free heterocyclic compound-linker construct can be removed by applicable methods and the antibody construct-heterocyclic compound conjugate can be exchanged into the desired formulation buffer. Such cysteine-based conjugates can be synthesized starting with antibody (mAb) and equivalents (e.g., 7 equivalents) of heterocyclic compound-linker construct using the conditions described in Scheme B below (Conjugate = antibody construct-heterocyclic compound conjugate). Monomer content and drug-antibody ratios can be determined by methods described herein.

Scheme B:

1. reducing agent

mAb „. Conjugate

2. heterocyclic compound-linker construct Example 8

General Procedure for the Determination of the Drug-Antibody-Ratios

Hydrophobic Interaction Chromatography

[0404] 10 μΙ_, of a 6 mg/mL solution of a conjugate is injected into an HPLC system set-up with a TOSOH TSKgel Butyl-NPR TM hydrophobic interaction chromatography (HIC) column (2.5 μΜ particle size, 4.6 mm x 35 mm) attached. Then, over the course of 18 minutes, a method is run in which the mobile phase gradient runs from 100% mobile phase A to 100% mobile phase B over the course of several minutes, followed by a several-minute re-equilibration at 100% mobile phase A. The flow rate may be 0.8 mL/min and the detector may be set at 280 nM.

Mobile phase A may be 1.5 M ammonium sulfate, 25 mM sodium phosphate (pH 7). Mobile phase B may be 25% isopropanol in 25 mM sodium phosphate (pH 7). Post-run, the

chromatogram may be integrated and the molar ratio determined by summing the weighted peak area.

Mass Spectrometry

[0405] One microgram of conjugate is injected into an LC/MS such as an Agilent 6550 iFunnel Q-TOF equipped with an Agilent Dual Jet Stream ESI source coupled with Agilent 1290 Infinity UHPLC system. Raw data is obtained and is deconvoluted with software such as Agilent MassHunter Qualitative Analysis Software with BioConfirm using the Maximum Entropy deconvolution algorithm. The average mass of intact conjugates is calculated by the software, which may use top peak height at 25% for the calculation. This data is then imported into another program to calculate the molar ratio of the conjugate such as Agilent molar ratio calculator.

[0406] The procedures outlined in the preceding examples were used to prepare constructs comprising compounds disclosed herein and the anti-Her2 antibody pertuzumab. The percentage of monomers was calculated based on size exclusion chromatography (SEC). Drug antibody ratios were calculated as described above.

Construct % Monomer (SEC) DAR

Anti-Her2 : Compound 3.1 99% 3.70

Anti-Her2 : Compound 3.2 99% 2.94

Anti-Her2 : Compound 3.3 99% 3.50

Anti-Her2 : Compound 3.4 98% 3.25