HK1244235B - Quaternary amine compounds and antibody-drug conjugates thereof - Google Patents

Description

Quaternary ammonium compounds and antibody-drug conjugates thereof

Cross-reference to related applications

This nonprovisional application, filed under 37 CFR §1.53(b), claims the benefit of U.S. Provisional Application No. 62/087,127, filed on December 3, 2014, under 35 USC §119(e), which is incorporated herein by reference in its entirety.

Technical Field

The present application relates to novel quaternary ammonium compounds that can be used as linkers for antibody-drug conjugates (ADCs). The present application also relates to ADCs containing quaternary ammonium linkers. The present application also relates to methods for treating diseases in humans.

Background Art

The use of monoclonal antibodies (mABs) to deliver anticancer drugs directly to tumor cells has attracted considerable attention in recent years. Two new antibody-drug conjugates have been approved by the FDA for the treatment of cancer. Brentuximab vedotin is a CD30-directed antibody-drug conjugate (ADC) indicated for the treatment of relapsed or refractory Hodgkin's lymphoma and systemic anaplastic large cell lymphoma (ALCL). Ado-trastuzumab emtansine is a new therapy approved for patients with HER2-positive advanced (metastatic) breast cancer. In order to achieve both potent antitumor activity and an acceptable therapeutic index in an ADC, several aspects of the design can be optimized. Specifically, the chemical structure of the known linker can significantly affect both the efficacy and safety of the ADC (Ducry & Stump, Bioconjugate Chem, 2010, 21, 5-13). Selecting the appropriate linker has an impact on the proper delivery of the drug to the intended cellular compartment of the cancer cell. Traditionally, the selection of conjugated drugs (sometimes also referred to as toxins) containing tertiary amine or pyridine functional groups has been primarily limited by how to link these drugs to other functional groups in the molecule. Therefore, it is desirable to develop methods for linking amine- or pyridine-containing toxins by forming quaternary salts. This application provides different types of ADCs containing quaternary salts that can be cleaved in tumor cells.

Summary of the Invention

The present application relates to an antibody-drug conjugate represented by formula (I):

Ab—(L—D) _p

Ab is antibody;

p is 1-8;

L-D is a chemical moiety represented by the following formula:

—Str—(Pep)—Sp—D

Str is a stretching unit covalently linked to Ab;

Pep is a linker;

D is an antitumor agent represented by the following formula:

wherein R ²⁰ and R ³⁰ are each independently C ₁ -C ₆ alkyl, and

R ¹⁰ is a non-hydrogen substituent; or

R ³⁰ is C ₁ -C ₆ alkyl, and R ¹⁰ and R ²⁰ together with N form a substituted C ₃ -C ₇ heterocycloalkyl ring; or

R ³⁰ is absent, and R ¹⁰ and R ²⁰ together with N form a substituted heteroaryl ring;

Sp-D is a spacer moiety of the formula:

The present application also relates to methods for treating cancer, uses of the antibody-drug conjugates of formula (I) in therapy, and uses of the antibody-drug conjugates of formula (I) in the preparation of medicaments for treating cancer. The present application also relates to methods for preparing the antibody-drug conjugates of formula (I).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a comparison of the efficacy of various CD22 ADCs in SCID mice bearing WSU-DLCL2 human diffuse large B-cell lymphoma. Both CD22 ADC1-2 and ADC2-2 showed dose-dependent inhibition of tumor growth compared to the vehicle group, with ADC1-2 showing stronger anti-tumor activity than ADC2-2. The non-binding control NaPi2b ADC had minimal effect on tumor growth.

DETAILED DESCRIPTION

The present application relates to antibody-conjugates of formula (I).

In one embodiment, D is an anti-tumor moiety with a potency of ₁₀ nM or less.

In one embodiment, Pep is a peptidyl moiety.

In one embodiment, Pep is a Val-Cit moiety.

In one embodiment, Pep is a Phe-Lys moiety.

In one embodiment, Pep is a Val-Ala moiety.

In one embodiment, Pep is a non-peptide chemical moiety selected from:

W is -NH-heterocycloalkyl- or heterocycloalkyl;

Y is heteroaryl, aryl, -C(O)C ₁ -C ₆ alkylene, C ₂ -C ₆ alkenyl, C ₁ -C ₆ alkylene, or -C ₁ -C ₆ alkylene-NH-;

Each R ¹ is independently C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, (C ₁ -C ₁₀ alkyl)NHC(NH)NH ₂ or (C ₁ -C ₁₀ alkyl)NHC(O)NH ₂ ;

R ³ and R ² are each independently H, C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, arylalkyl or heteroarylalkyl, or R ³ and R ² together may form a C ₃ -C ₇ cycloalkyl group;

R ⁴ and R ⁵ are each independently C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, arylalkyl, heteroarylalkyl, (C ₁ -C ₁₀ alkyl)OCH ₂ -, or R ⁴ and R ⁵ may form a C ₃ -C ₇ cycloalkyl ring; and

p is an integer from 1 to 8.

The present application also relates to an antibody-conjugate of formula (I), which is represented by the following formula:

Ab—(L—D) _p

Wherein Ab is an antibody; L-D is a chemical moiety represented by the following formula:

R ¹ is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, (C ₁ -C ₆ alkyl)NHC(NH)NH ₂ or (C ₁ -C ₆ alkyl)NHC(O)NH ₂ ; and

R ³ and R ² are each independently H or C ₁ -C ₁₀ alkyl.

The present application also relates to an antibody-conjugate of formula (I), which is represented by the following formula:

Ab—(L—D) _p

Wherein Ab is an antibody; L-D is a chemical moiety of the formula:

R ¹ is C ₁ -C ₆ alkyl, (C ₁ -C ₆ alkyl)NHC(NH)NH ₂ or (C ₁ -C ₆ alkyl)NHC(O)NH ₂ ; and

R ⁴ and R ⁵ together form a C ₃ -C ₇ cycloalkyl ring.

The present application also relates to an antibody-conjugate of formula (I), which is represented by the following formula:

Ab—(L—D) _p

Where Ab is an antibody;

L-D is a chemical moiety represented by the following formula:

and

R ¹ is C ₁ -C ₆ alkyl, (C ₁ -C ₆ alkyl)NHC(NH)NH ₂ or (C ₁ -C ₆ alkyl)NHC(O)NH ₂ .

The present application also relates to an antibody-conjugate of formula (I) represented by the following formula:

in

Str is a chemical moiety represented by the formula:

wherein R ⁶ is selected from C ₁ -C ₁₀ alkylene and C ₁ -C ₁₀ alkylene-C(O)N(R ^a )-C ₂ -C ₆ alkylene, wherein each alkylene group may be substituted with 1 to 5 substituents selected from the group consisting of halogen, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamide, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthioaryl, C ₃ -C ₈ cycloalkyl, C ₄ -C ₇ heterocycloalkyl, heteroarylalkyl, and heteroaryl;

Each ^Ra is independently H or _C1 - _C6 alkyl; and

p is 1, 2, 3, or 4.

The present application also relates to an antibody-conjugate of formula (I) represented by the following formula:

in

Str is a chemical moiety represented by the formula:

wherein R ⁶ is selected from C ₁ -C ₁₀ alkylene and C ₁ -C ₁₀ alkylene-C(O)N(R ^a )-C ₂ -C ₆ alkylene, wherein each alkylene group may be substituted with 1 to 5 substituents selected from the group consisting of halogen, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamide, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthioaryl, C ₃ -C ₈ cycloalkyl, C ₄ -C ₇ heterocycloalkyl, heteroarylalkyl, and heteroaryl;

Each ^Ra is independently H or _C1 - _C6 alkyl; and

p is 1, 2, 3, or 4.

The present application also relates to an antibody-conjugate of formula (I) represented by the following formula:

in

Str is a chemical moiety represented by the formula:

wherein R ⁶ is selected from C ₁ -C ₁₀ alkylene and C ₁ -C ₁₀ alkylene-C(O)N(R ^a )-C ₂ -C ₆ alkylene, wherein each alkylene group may be substituted with 1 to 5 substituents selected from the group consisting of halogen, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamide, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthioaryl, C ₃ -C ₈ cycloalkyl, C ₄ -C ₇ heterocycloalkyl, heteroarylalkyl, and heteroaryl;

Each ^Ra is independently H or _C1 - _C6 alkyl; and

p is 1, 2, 3, or 4.

The present application also relates to an antibody-conjugate of formula (I) (A1) represented by the following formula:

in

R ¹ is C ₁ -C ₆ alkyl-NH ₂ , (C ₁ -C ₆ alkyl)NHC(NH)NH ₂ or (C ₁ -C ₆ alkyl)NHC(O)NH ₂ ; and

p is 1, 2, 3, or 4.

The present application also relates to an antibody-conjugate of formula (I) (B1) represented by the following formula:

in

p is 1, 2, 3, or 4;

R ¹ is C ₁ -C ₆ alkyl-NH ₂ , (C ₁ -C ₆ alkyl)NHC(NH)NH ₂ or (C ₁ -C ₆ alkyl)NHC(O)NH ₂ ; and

^R4 and ^R5 are each independently _C1 - _C6 alkyl, wherein the alkyl group is unsubstituted, or ^R4 and ^R5 may form a _C3 - _C7 cycloalkyl ring.

The present application also relates to an antibody-conjugate of formula (I) (C1) represented by the following formula:

in

R ¹ is C ₁ -C ₆ alkyl-NH ₂ , (C ₁ -C ₆ alkyl)NHC(NH)NH ₂ or (C ₁ -C ₆ alkyl)NHC(O)NH ₂ ; and

p is 1, 2, 3, or 4.

In one embodiment, Ab-(L-D) _p is a chemical moiety of formula (II):

In one embodiment, Ab-(L-D) _p is of formula (III):

The present application also relates to antibody-conjugates of formula (I), (I)(A1), (I)(A2), wherein Y is heteroaryl, aryl, or alkenyl; and R ⁶ is C ₁ -C ₁₀ alkylene.

The present application also relates to antibody-conjugates of formula (I), (I)(A1), (I)(A2), wherein Y is

The present application also relates to any of the above-mentioned antibody-conjugates, wherein Y is

The present application also relates to any of the above-mentioned antibody-conjugates, wherein Y is

The present application also relates to any of the above-mentioned antibody-conjugates, wherein

Str is a chemical moiety represented by the formula:

wherein ^R6 is selected from _C1 - _C10 alkylene, _C2 - _C10 alkenyl, _C3 - _C8 cycloalkyl, ( _C1 - _C8 alkylene)O- and _C1 - _C10 alkylene-C(O)N( ^Ra ) _-C2 - _C6 alkylene, wherein each alkylene group may be substituted with 1 to 5 substituents selected from the group consisting of halogen, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamide, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthioaryl, _C3 - _C8 cycloalkyl, _C4 - _C7 heterocycloalkyl, heteroarylalkyl and heteroaryl; and

Each ^Ra is independently H or _C1 - _C6 alkyl.

The present application also relates to any of the above-mentioned antibody-conjugates, wherein Str has the following formula:

wherein ^R7 is selected from _C1 - _C10 alkylene, _C2 - _C10 alkenyl, ( _C1 - _C10 alkylene)O-, N( ^Rc )-( _C2 - _C6 alkylene)-N( ^Rc ) and N( ^Rc )-( _C2 - _C6 alkylene); and

Each R ^c is independently H or C ₁ -C ₆ alkyl.

The present application also relates to any of the above-mentioned antibody-conjugates, wherein

Str is a chemical moiety represented by the formula:

and

R ⁶ is a C ₁ -C ₆ alkylene group.

Linker-Drug

The present application also relates to compounds of formula L-D, which are represented by the following formula:

—Str—(Pep)—Sp—D

Str is a stretching unit covalently linked to Ab;

Pep is a linker;

D is an antitumor antibiotic drug represented by the following formula:

wherein R ²⁰ and R ³⁰ are each independently C ₁ -C ₆ alkyl, and

R ¹⁰ is a non-hydrogen substituent; or

R ³⁰ is C ₁ -C ₆ alkyl, and R ¹⁰ and R ²⁰ together with N form a substituted C ₃ -C ₇ heterocycloalkyl ring; or

R ³⁰ is absent, and R ¹⁰ and R ²⁰ together with N form a substituted heteroaryl ring; and

Sp—D is the chemical moiety of the formula:

The present application also relates to non-peptide compounds of the following formula:

in

Str is a stretching unit that can be covalently linked to an antibody;

Y is heteroaryl, aryl, -C(O)C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkenyl, or -C ₂ -C ₆ alkenyl-NH-;

R ¹ is C ₁ -C ₁₀ alkyl, (C ₁ -C ₁₀ alkyl)NHC(NH)NH ₂ or (C ₁ -C ₁₀ alkyl)NHC(O)NH ₂ ;

^R3 and ^R2 are each independently H, _C1 - _C10 alkyl, arylalkyl or heteroarylalkyl, or ^R3 and ^R2 together can form a _C3 - _C7 cycloalkyl; and

D is for medicine.

The present application also relates to non-peptide compounds represented by the following formula:

wherein R ₆ is a C ₁ -C ₁₀ alkylene group.

The present application also relates to non-peptide compounds represented by the following formula:

The present application also relates to non-peptide compounds of the following formula:

in

Str is a stretching unit that can be covalently linked to an antibody;

R ¹ is C ₁ -C ₁₀ alkyl, (C ₁ -C ₁₀ alkyl)NHC(NH)NH ₂ or (C ₁ -C ₁₀ alkyl)NHC(O)NH ₂ ; and

R ⁴ and R ⁵ are each independently C ₁ -C ₁₀ alkyl, arylalkyl, heteroarylalkyl, (C ₁ -C ₁₀ alkyl)OCH ₂ —, or R ⁴ and R ⁵ may form a C ₃ -C ₇ cycloalkyl ring.

The present application also relates to non-peptide compounds represented by the following formula:

wherein R ₆ is a C ₁ -C ₁₀ alkylene group; and R ⁴ and R ⁵ together form a C ₃ -C ₇ cycloalkyl ring.

The present application also relates to non-peptide compounds represented by the following formula:

The present application also relates to compounds of the following formula:

wherein Het is optionally substituted heteroaryl.

The present application also relates to compounds of the following formula:

wherein q is 0-5; and R ¹¹ is NO ₂ or CN.

The present application also relates to compounds of the following formula:

The present application also relates to compounds of the following formula:

The present application also relates to any one of the compounds of formula (I)(A1)(LD), (I)(A2)(LD), (I)(A3)(LD), (I)(B1)(LD), (I)(B2)(LD), (I)(B3)(LD), (II)(A1)(LD) or (III)(A1)(LD) above, wherein Str has the following formula:

wherein R ⁶ is selected from C ₁ -C ₁₀ alkylene, C ₃ -C ₈ cycloalkyl, O-(C ₁ -C ₈ alkylene) and C ₁ -C ₁₀ alkylene-C(O)N(R ^a )-C ₂ -C ₆ alkylene, wherein each alkylene group may be substituted with 1 to 5 substituents selected from the group consisting of halogen, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamide, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthioaryl, C ₃ -C ₈ cycloalkyl, C ₄ -C ₇ heterocycloalkyl and heteroaryl;

Each ^Ra is independently H or _C1 - _C6 alkyl;

Sp is —Ar—R ^b —, wherein Ar is aryl or heteroaryl; and

R ^b is (C ₁ -C ₁₀ alkylene)O—.

The present application also relates to compounds of formula (I)(A1)(LD), (I)(A2)(LD), (I)(A3)(LD), (I)(B1)(LD), (I)(B2)(LD), (I)(B3)(LD), (II)(A1)(LD), (II)(A2)(LD), (II)(A3)(LD) and (IV)(A1)(LD), wherein R ₆ is C ₁ -C ₁₀ alkylene;

Sp is —Ar—R ^b —, wherein Ar is aryl; and

R ^b is a C ₁ -C ₆ alkylene group.

The present application also relates to any compound of formula (I)(A1)(LD), (I)(A2)(LD), (I)(A3)(LD), (I)(B1)(LD), (I)(B2)(LD), (I)(B3)(LD), (II)(A1)(LD) or (III)(A1)(LD) wherein _R6 is -( _CH2 ) _{and q} is 1-10.

The present application also relates to compounds of formula (I)(A1)(LD), (I)(A2)(LD), (I)(A3)(LD), (I)(B1)(LD), (I)(B2)(LD), (I)(B3)(LD), (II)(A1)(LD), (II)(A2)(LD), (II)(A3)(LD) and (IV)(A1)(LD), wherein

Str is a chemical moiety represented by the formula:

wherein ^R6 is selected from _C1 - _C10 alkylene, _C2 - _C10 alkenyl, _C3 - _C8 cycloalkyl, ( _C1 - _C8 alkylene)O- and _C1 - _C10 alkylene-C(O)N( ^Ra ) _-C2 - _C6 alkylene, wherein each alkylene group may be substituted with 1 to 5 substituents selected from the group consisting of halogen, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamide, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthioaryl, _C3 - _C8 cycloalkyl, _C4 - _C7 heterocycloalkyl, heteroarylalkyl and heteroaryl; and

Each ^Ra is independently H or _C1 - _C6 alkyl.

The present application also relates to compounds of formula (I)(A1)(LD), (I)(A2)(LD), (I)(A3)(LD), (I)(B1)(LD), (I)(B2)(LD), (I)(B3)(LD), (II)(A1)(LD), (II)(A2)(LD), (II)(A3)(LD) and (IV)(A1)(LD), wherein Str has the formula:

wherein ^R7 is selected from _C1 - _C10 alkylene, _C2 - _C10 alkenyl, ( _C1 - _C10 alkylene)O-, N( ^Rc )-( _C2 - _C6 alkylene)-N( ^Rc ) and N( ^Rc )-( _C2 - _C6 alkylene); and

Each R ^c is independently H or C ₁ -C ₆ alkyl.

The present application also relates to non-peptide compounds, wherein R ₆ is C ₁ -C ₁₀ alkylene.

The present application also relates to any one of the above-mentioned antibody-drug conjugates, wherein p is 1.

The present application also relates to any one of the above-mentioned antibody-drug conjugates, wherein p is 2.

The present application also relates to any of the above-mentioned antibody-drug conjugates, wherein the antibody is bound to one or more polypeptides selected from the group consisting of:

CLL1;BMPR1B;E16;STEAP1;0772P;MPF;NaPi2b;Sema 5b;PSCA hlg; ETBR; MSG783; STEAP2; TrpM4; CRIPTO; CD21; CD79b; FcRH2; HER2; NCA; MDP; IL20Rα; brevican; EphB2R; ASLG659; PSCA; GEDA; BAFF-R; CD22; CD79a; CXCR5; HLA-DOB; P2X5; CD72; LY64; FcRH1; IRTA2; TENB2; PMEL17; TMEFF1; GDNF-Ra1; Ly6E; TMEM46; Ly6G6D; LGR5; RET; LY6K; GPR19; GPR54; ASPHD1; tyrosinase; TMEM118; GPR172A; MUC16; and CD33.

The present application also relates to a method of treating a disease in a human in need thereof, comprising administering to said human an effective amount of an antibody-drug conjugate according to any one of the above embodiments.

The present application also relates to a pharmaceutical composition comprising the antibody-drug conjugate according to any one of the above embodiments and a pharmaceutically acceptable carrier thereof.

The present application also relates to any of the above-mentioned antibody-drug conjugates, wherein the antibody is bound to one or more polypeptides selected from the group consisting of:

CLL1; STEAP1; NaPi2b; STEAP2; TrpM4; CRIPTO; CD21; CD79b; FcRH2; HER2; CD22; CD79a; CD72; LY64; Ly6E; MUC16; and CD33.

The present application also relates to any of the above-mentioned antibody-drug conjugates, wherein the antibody binds to CD33.

The present application also relates to any one of the above-mentioned antibody-drug conjugates, wherein the antibody binds to CD33, and the anti-CD33 antibody comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 11, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 13, HVR-H1 comprising the amino acid sequence of SEQ ID NO: 14, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16.

The present application also relates to any one of the above-mentioned antibody-drug conjugates, wherein the antibody binds to CD33, and the anti-CD33 antibody comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 17 and a VH domain comprising the amino acid sequence of SEQ ID NO: 18.

In some embodiments, the antibody of the antibody-drug conjugate binds to CD33. In some embodiments, the antibody of the antibody-drug conjugate comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 19; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 21.

In some embodiments, the antibody comprises the VH of any one of the above embodiments and the VL of any one of the above embodiments. In one embodiment, the antibody comprises the corresponding VL and VH sequences in SEQ ID NO: 25 and SEQ ID NO: 26, including post-translational modifications of those sequences.

The present application also relates to any of the above-mentioned antibody-drug conjugates, wherein the antibody binds to NaPi2b.

The present application also relates to any one of the above-mentioned antibody-drug conjugates, wherein the antibody binds to NaPi2b, and the NaPi2b antibody comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3, HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6.

The present application also relates to any one of the above-mentioned antibody-drug conjugates, wherein the antibody binds to NaPi2b, and the NaPi2b antibody comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 7 and a VH domain comprising the amino acid sequence of SEQ ID NO: 8.

The present application also relates to any one of the above-mentioned antibody-drug conjugates, wherein the antibody binds to NaPi2b, and the NaPi2b antibody comprises the amino acid sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10.

The present application also relates to any of the above-mentioned antibody-drug conjugates, wherein the antibody binds to NaPi2b.

The present application also relates to any of the above-mentioned antibody-drug conjugates, wherein the antibody binds to CD-22, and the anti-CD-22 antibody comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 41, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 42, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 43, HVR-H1 comprising the amino acid sequence of SEQ ID NO: 44, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 45, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 46.

The present application also relates to any of the above-mentioned antibody-drug conjugates, wherein the antibody binds to CD-22, and the CD-22 antibody comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 47 and a VH domain comprising the amino acid sequence of SEQ ID NO: 48.

The present application also relates to any one of the above-mentioned antibody-drug conjugates, wherein the antibody binds to CD-22, and the CD-22 antibody comprises the amino acid sequence of SEQ ID NO: 49 and the amino acid sequence of SEQ ID NO: 50.

definition

Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings: When a trade name is used herein, applicants intend to include independently the trade name product formulation, the generic name drug, and the active pharmaceutical ingredient of the trade name product.

As used herein, the term "peptide mimetic" or PM refers to a non-peptide chemical moiety. A peptide is a short chain of amino acid monomers connected by a peptide (amide) bond (i.e., a covalent chemical bond formed when the carboxyl group of one amino acid reacts with the amino group of another amino acid). The shortest peptide is a dipeptide consisting of two amino acids connected by a peptide bond, followed by tripeptides, tetrapeptides, and the like. A quaternary amine chemical moiety includes a non-amino acid chemical moiety. A quaternary amine chemical moiety may also include one or more amino acids separated by one or more non-amino acid chemical units. A quaternary amine chemical moiety does not contain two or more adjacent amino acids connected by a peptide bond in any part of its chemical structure.

As used herein, the term "amino acid" refers to glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, tyrosine, cysteine, methionine, lysine, arginine, histidine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, or citrulline.

The term "antibody" in this application is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170:4854-4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th edition, Garland Publishing, New York). The target antigen typically has multiple binding sites (also called epitopes) that are recognized by the CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, an antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules containing an antigen binding site that immunospecifically binds to a specific target or a portion thereof, and the above-mentioned targets include, but are not limited to, cells that are not limited to cancer cells or produce autoimmune antibodies associated with autoimmune diseases. Immunoglobulins disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, and IgA), categories (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclasses of immunoglobulin molecules. Immunoglobulins can be derived from any species. However, in one aspect, immunoglobulins are human, murine, or rabbit-derived.

The term "antibody fragment" as used herein comprises a portion of a full-length antibody, typically its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; diabodies; linear antibodies; miniantibodies (Olafsen et al. (2004) Protein Eng. Design & Sel. 17(4): 315-323), fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions), and epitope-binding fragments of any of the above that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The term "monoclonal antibody" used herein refers to an antibody obtained from a substantially homogeneous antibody group, i.e., each antibody constituting the group is identical, except for possible naturally occurring mutations that may exist in very small amounts. Monoclonal antibodies are highly specific for a single antigenic site. In addition, unlike polyclonal antibody preparations comprising different antibodies for different determinants (epitopes), each monoclonal antibody is for a single determinant on the antigen. Except for its specificity, monoclonal antibodies are advantageous because they can be synthesized without being contaminated by other antibodies. The modifier "monoclonal" represents that an antibody is obtained from a substantially homogeneous antibody group and is not interpreted as requiring antibody to be produced by any specific method. For example, the monoclonal antibody used herein can be prepared by the hybridoma method described in Kohler et al. (1975) Nature, 256:495 for the first time or can be prepared by a recombinant DNA method (see, for example, US4816567; US5807715). Monoclonal antibodies can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597.

The monoclonal antibodies in the present application specifically include "chimeric" antibodies and fragments of the above antibodies, wherein a portion of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, and the rest of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as long as they exhibit the desired biological activity (US4816567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81: 6851-6855). The chimeric antibodies of interest in the present application include "primatized" antibodies, which comprise variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

The term "intact antibody" as used herein refers to an antibody comprising VL and VH domains and a light chain constant domain (CL) and heavy chain constant domains CHI, _CH2 , and _CH3 . The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more "effector functions," which refer to those biological activities attributable to the Fc constant region (native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC) mediated by cells; phagocytosis; and downregulation of cell surface receptors such as B cell receptors and BCRs.

The term "Fc region" as used herein refers to the C-terminal region of at least a portion of an immunoglobulin heavy chain containing a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise specified herein, the numbering of the amino acid residues in the Fc region or constant region conforms to the EU numbering system (also referred to as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The term "framework" or "FR" as used herein refers to the variable domain residues excluding the hypervariable region (HVR) residues. The FR of the variable domain is generally composed of four FR domains, namely FR1, FR2, FR3 and FR4. Therefore, the HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4. Complete antibodies can be classified into different "classes" depending on the amino acid sequence of the constant domain of their heavy chains. There are five major classes of complete immunoglobulin antibodies, namely IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are called α, δ, ε, γ and μ, respectively. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are known. Ig forms include hinge-modified or hingeless forms (Roux et al. (1998) J. Immunol. 161:4083-4090; Lund et al. (2000) Eur. J. Biochem. 267:7246-7256; US 2005/0048572; US 2004/0229310).

The term "human antibody", as used herein, refers to antibodies that have an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or that are derived from non-human sources using human antibody libraries or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

As used herein, the term "human consensus framework" refers to a framework that represents the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, a human immunoglobulin VL or VH sequence is selected from a subset of variable domain sequences. Typically, the subset of sequences is the subset described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI described above by Kabat et al. In one embodiment, for VH, the subgroup is subgroup III described above by Kabat et al.

The term "humanized antibody" as used herein refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all variable domains (at least one and typically two), wherein all or substantially all HVRs (e.g., CDRs) correspond to those of non-human antibodies, and all or substantially all FRs correspond to those of human antibodies. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has been humanized.

The term "hypervariable region" or "HVR" as used herein refers to each of the following regions of an antibody variable domain that are highly variable in sequence and/or form structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs), which have the highest sequence variability and/or are involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, amino acid residues 50-56 of L2, amino acid residues 89-97 of L3, amino acid residues 31-35B of H1, amino acid residues 50-65 of H2, and amino acid residues 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, 1994). Health, Bethesda, MD (1991). With the exception of CDR1 in VH, CDRs typically contain amino acid residues that form hypervariable loops. CDRs also contain "specificity determining residues" or "SDRs," which are residues that contact the antigen. SDRs are contained in regions of the CDRs known as abbreviated CDRs or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at the amino acid position of L1. The amino acid residues of the HVR and other residues (e.g., FR residues) in the variable domain are numbered according to Kabat et al., supra. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008). Unless otherwise indicated, the HVR residues and other residues (e.g., FR residues) in the variable domain are numbered according to Kabat et al., supra.

The term "variable region" or "variable domain" as used herein refers to the domain of an antibody heavy chain or light chain that participates in binding of an antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. Additionally, antibodies that bind to a specific antigen can be isolated using VH or VL domains from antibodies that bind to the antigen to screen for libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

As used herein, the term "vector" refers to a nucleic acid molecule that is capable of propagating another nucleic acid to which it is linked. The term encompasses vectors that are self-replicating nucleic acid structures as well as vectors that are introduced into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

The term "free cysteine" as used herein refers to a cysteine residue that has been engineered into a parent antibody and has a thiol functional group (-SH) and is not paired into an intra- or intermolecular disulfide bond.

The term "linker," "linker unit," or "linker" as used herein refers to a chemical moiety comprising a chain of atoms that covalently links the drug moiety to the antibody. In various embodiments, the linker is a divalent group designated L.

As used herein, the term "drug moiety" refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioisotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleolytic enzymes; and various antitumor or anticancer agents described below.

As used herein, unless otherwise defined in the claims, the term "acyl" refers to the group -C(O)R', wherein R' is alkyl, _C3 - _C6 cycloalkyl, or heterocyclyl, each as defined herein.

Unless otherwise defined in the claims, the term "alkoxy" as used herein refers to the group -OR', wherein R' is a _C1 - _C4 alkyl or _C3 - _C6 cycloalkyl group as defined above. Examples of "alkoxy" include methoxy, ethoxy, isopropoxy, propoxy, butoxy, tert-butoxy, isobutoxy, cyclopropyloxy, and cyclobutyloxy, and halogenated forms thereof such as fluoromethoxy and difluoromethoxy.

Unless otherwise defined in the claims, the term "alkyl" as used herein refers to a linear or branched monovalent or divalent hydrocarbon chain group having 1 to 12 ( _C1 - _C12 ) carbon atoms, which may be unsubstituted or substituted with multiple degrees of substitution, for example, one, two, three, four, five or six, as included herein. Examples of substituents are selected from halogen, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamide, sulfoxide, hydroxyl, alkoxy, ester, carboxylic acid and alkylthio. As used herein, examples of “alkyl” include, but are not limited to, methyl (Me, —CH ₃ ), ethyl (Et, —CH ₂ CH ₃ ), 1-propyl (n-Pr, n-propyl, —CH ₂ CH ₂ CH ₃ ), 2-propyl (i-Pr, isopropyl, —CH(CH ₃ ) ₂ ), 1-butyl (n-Bu, n-butyl, —CH ₂ CH ₂ CH ₂ CH ₃ ), 2-methyl-1-propyl (i-Bu, isobutyl, —CH ₂ CH(CH ₃ ) ₂ ), 2-butyl (s-Bu, sec-butyl, —CH(CH ₃ )CH ₂ CH ₃ ), 2-methyl-2-propyl (t-Bu, tert-butyl, —C(CH ₃ ) ₃ ), 1-pentyl (n-pentyl, —CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ), 2-pentyl (—CH(CH ₃ )CH _{2 3-} Pentyl (-CH( _{CH2CH3) 2} ), 2-Methyl-2-butyl (-C( _CH3 ) _2CH2CH3 ), 3-Methyl- ₂ -butyl ( _-CH ( _CH3 )CH( _{CH3 ) 2 )} , 3-Methyl- ₁ -butyl ( _-CH2CH2CH ( _CH3 ) ₂ ), 2-Methyl-1-butyl ( _-CH2CH ( _{CH3 ) CH2CH3} ), 1 _{- hexyl} (-CH2CH2CH2CH2CH2CH3 ₎ , 2 _- hexyl ₍ -CH( _CH3 )CH2CH2CH2CH3), 3 _- hexyl ( _{-CH ( CH2CH3 ) ( CH2CH2CH3 )} ) _, 2-methyl _{- 2} -pentyl (-C( _{CH3 ) 2CH2CH2CH 3} ), 3-methyl-2-pentyl (—CH(CH ₃ )CH(CH ₃ )CH ₂ CH ₃ ), 4-methyl-2-pentyl (—CH(CH ₃ )CH ₂ CH(CH ₃ ) ₂ ), 3-methyl-3-pentyl (—C(CH ₃ )(CH ₂ CH ₃ ) ₂ ), 2-methyl-3-pentyl (—CH(CH ₂ CH ₃ )CH(CH ₃ ) ₂ ), 2,3-dimethyl-2-butyl (—C(CH ₃ ) ₂ CH(CH ₃ ) ₂ ), 3,3-dimethyl-2-butyl (—CH(CH ₃ )C(CH ₃ ) ₃ , and divalent (“alkylene”) and substituted versions thereof. Examples of substituted alkyl groups include, but are not limited to, hydroxymethyl, difluoromethyl, and trifluoromethyl.

Unless otherwise defined in the claims, the term "alkenyl" as used herein refers to any linear or branched monovalent or divalent hydrocarbon chain radical of 2 to 8 carbon atoms ( _C2 - _C10 ) in length having at least one site of unsaturation, i.e., a carbon-carbon ^sp2 double bond, wherein the alkenyl group may be optionally substituted independently with one or more substituents described above in the definition of "alkyl" and includes groups having "cis" and "trans" orientations or "E" and "Z" orientations. Examples of alkenyl groups include, but are not limited to, vinyl (—CH═CH ₂ ), prop-1-enyl (—CH═CHCH ₃ ), prop-2-enyl (—CH ₂ CH═CH ₂ ), 2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl, buta-1,3-dienyl, 2-methylbuta-1,3-dienyl, hex-1-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl, hexa-1,3-dienyl, and divalent (“alkenylene”) and substituted versions thereof.

As used herein, unless otherwise defined in the claims, the term "alkynyl" refers to any linear or branched monovalent or divalent hydrocarbon radical of 2 to 8 carbon atoms ( _C2 - _C10 ) in length, having at least one site of unsaturation, i.e., a carbon-carbon sp triple bond, wherein the alkynyl radical may be optionally substituted independently with one or more substituents as described above in the definition of alkyl. Examples of alkynyl radicals include, but are not limited to, ethynyl (-C≡CH), prop-1-ynyl ( _-C≡CCH3 ), prop-2-ynyl (propargyl, _-CH2C≡CH ), but-1-ynyl, but-2-ynyl, and but-3-ynyl, and divalent ("alkynylene") and substituted forms thereof.

Unless otherwise defined in the claims, the term "alkylamino" as used herein refers to the group -NR'R", wherein R' is H, C ₁ -C ₆ alkyl or C ₃ -C ₆ cycloalkyl, and R" is C ₁ -C ₆ alkyl or C ₃ -C ₆ cycloalkyl, examples of alkylamino include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, propylamino and cyclopropylamino.

Unless otherwise defined in the claims, the term "amide" as used herein refers to the group -C(O)NR'R", wherein R' and R" are each independently H, C ₁ -C ₆ alkyl, or C ₃ -C ₆ cycloalkyl; examples of amides include, but are not limited to, -C(O)NH ₂ , -C(O)NHCH ₃ , and -C(O)N(CH ₃ ) ₂ .

Unless otherwise defined in the claims, the term "aryl" as used herein refers to an aromatic hydrocarbon ring system. The ring system can be monocyclic or fused polycyclic (e.g., bicyclic, tricyclic, etc.) and substituted or unsubstituted. In various embodiments, the monocyclic aryl ring is _C5 - _C10 or _C5 - _C7 or _C5 - _C6 , where these carbon numbers refer to the number of carbon atoms forming the ring system. A _C6 ring system, i.e., a phenyl ring, is an aryl group. In various embodiments, the polycyclic ring is a bicyclic aryl group, where examples of bicyclic aryls include _C8 - _C12 or _C9 - _C10 . A naphthyl ring with 10 carbon atoms is a polycyclic aryl group. Examples of substituents for aryl are described below in the definition of "optionally substituted".

Unless otherwise defined in the claims, the term "cyano" as used herein refers to the group -CN.

Unless otherwise defined in the claims, "cycloalkyl" as used herein refers to a non-aromatic, substituted or unsubstituted, saturated or partially unsaturated hydrocarbon ring group. Examples of substituents are described in the definition of "optionally substituted". In one example, the cycloalkyl has 3-12 carbon atoms ( _C3 - _C12 ). In other examples, the cycloalkyl is _C3 - _C8 , _C3 - _C10 , or _C5 - _C10 . In other examples, the cycloalkyl as a monocycle is _C3 - _C8 , _C3 - _C6 , or _C5 - _C6 . In another example, the cycloalkyl as a bicycle is C7- _C12 . In another example, _the cycloalkyl as a spirocyclic system is _C5 - _C12 . Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, perdeuterated cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl. Exemplary arrangements of bicyclic cycloalkyls with 7-12 ring atoms include, but are not limited to, [4,4], [4,5], [5,5], [5,6], or [6,6] ring systems. Exemplary bridged bicyclic cycloalkyls include, but are not limited to, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and bicyclo[3.2.2]nonane. Examples of spirocyclic cycloalkyl groups include spiro[2.2]pentane, spiro[2.3]hexane, spiro[2.4]heptane, spiro[2.5]octane, and spiro[4.5]decane.

As used herein, unless otherwise defined in the claims, the term "ester" refers to the group -C(O)OR', wherein R' is C ₁ -C ₆ alkyl or C ₃ -C ₆ cycloalkyl.

Unless otherwise defined in the claims, the terms "heterocycle," "heterocycloalkyl," or "heterocyclyl" as used herein refer to unsubstituted and substituted monocyclic or polycyclic non-aromatic ring systems containing 2-12 ring carbon atoms and 1-3 ring heteroatoms. Polycyclic ring systems can be fused bicyclic or tricyclic, spirocyclic, or bridged. Examples of heteroatoms include N, O, and S, including N-oxides, sulfur oxides, and dioxides. In one embodiment, the ring is 3-8 members and is fully saturated or has one or more degrees of unsaturation. Multiple degrees of substitution are included in this definition. Examples of substituents are defined below. Examples of "heterocyclic" groups include, but are not limited to, tetrahydrofuranyl, pyranyl, 1,4-dioxanyl, 1,3-dioxanyl, oxolanyl, oxetanyl, 2-oxa-6-azaspiro[3.3]hept-6-yl, piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl, piperazinyl, pyrrolidonyl, piperazinonyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, and their various tautomers.

Unless otherwise defined in the claims, the term "heteroaryl" as used herein refers to an aromatic ring system containing 1-9 carbon atoms and at least one heteroatom. Examples of heteroatoms include N, O, and S. Heteroaryl groups may be monocyclic or polycyclic and substituted or unsubstituted. Monocyclic heteroaryl groups may have 2-6 ring carbon atoms and 1-3 ring heteroatoms in the ring, while polycyclic heteroaryl groups may contain 3-9 ring carbon atoms and 1-5 ring heteroatoms. Polycyclic heteroaryl rings may contain fused, spirocyclic, or bridged ring bonds, for example, bicyclic heteroaryl is a polycyclic heteroaryl group. Bicyclic heteroaryl rings may contain 8-12 member atoms. Monocyclic heteroaryl rings may contain 5-8 member atoms (carbon and heteroatoms). Exemplary heteroaryl groups include, but are not limited to, benzofuranyl, benzothiophenyl, furyl, imidazolyl, indolyl, azaindolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzimidazolyl, tetrazinyl, tetrazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinolyl, quinazolinyl, quinoxalinyl, triazinyl, triazolyl, thiazolyl and thienyl. Examples of substituents for heteroaryl are described below in the definition of "optionally substituted".

Unless otherwise defined in the claims, the term "arylalkyl" as used herein refers to the group (aryl)C ₁ -C ₃ alkyl.

Unless otherwise defined in the claims, the term "urea" as used herein refers to the group -NR'C(O)NR", wherein R' and R" are each independently H, C ₁ -C ₆ alkyl, or C ₃ -C ₆ cycloalkyl.

Unless otherwise defined in the claims, the term "optionally" as used herein means that the subsequently described event may or may not occur and includes both events that occur and events that do not occur.

Unless otherwise defined, the phrases "optionally substituted,""substituted," or variations thereof, as used herein, refer to optional substitution with one or more, for example, one, two, or three, substituents, including multiple degrees of substitution. The phrases should not be construed as a repetition of the substitutions described and depicted herein. Exemplary optional substituents include acyl, C ₁ -C ₆ alkyl, sulfonyl, amino, sulfonamide, sulfoxide, alkoxy, cyano, halogen, urea, ester, carboxylic acid, amide, hydroxy, oxo, and nitro.

Unless otherwise defined in the claims, the term "treating" as used herein means alleviating the condition, eliminating or reducing one or more symptoms of the condition, slowing down or eliminating the progression of the condition.

As used herein, unless otherwise defined in the claims, the term "effective amount" means that amount of a drug or pharmaceutical agent that will elicit the biological or medicinal response of a tissue, system, animal or human that is being sought, for example, by a researcher or clinician.

Unless otherwise defined in the claims, the term "therapeutically effective amount" as used herein refers to any amount that achieves treatment of a disease, condition, or side effect or reduces the rate of progression of a disease or condition compared to a corresponding subject that has not received the above amount. The term also includes within its scope amounts that effectively enhance normal physiological function. For use in therapy, a therapeutically effective amount of a compound of Formula I and a salt thereof can be administered in the form of a raw chemical. Additionally, the active ingredient can be provided in the form of a pharmaceutical composition.

This application also refers to any one of the examples in the experimental part.

The term "pharmaceutical salt" used herein refers to a pharmaceutical organic or inorganic salt of an antibody-drug conjugate (ADC) or a linker-drug moiety. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis(2-hydroxy-3-naphthoate)). Pharmaceutical salts may include another molecule such as acetate ion, succinate ion, or other counter ion. Counter ion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. When multiple charged atoms are part of a pharmaceutically acceptable salt, there may be multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

Other salts that are not pharmaceutically acceptable may be used to prepare the compounds of the present invention, and these salts should be considered to constitute another aspect of the present invention. Although these salts, such as oxalates or trifluoroacetates, are not pharmaceutically acceptable themselves, they may be used to prepare salts that are useful as intermediates for obtaining the compounds of the present invention and their pharmaceutically acceptable salts.

The compounds of the present invention may exist in solid or liquid form. In the solid state, they may exist in crystalline or amorphous form or as mixtures thereof. Those skilled in the art will recognize that pharmaceutically acceptable solvates may be formed for crystalline or amorphous compounds. In crystalline solvates, solvent molecules enter the crystal lattice during the crystallization process. Solvates may involve non-aqueous solvents such as, but not limited to, ethanol, isopropanol, DMSO, acetic acid, ethanolamine, or ethyl acetate, or may involve water as the solvent that enters the crystal lattice. Solvates in which water is the solvent that enters the crystal lattice are generally referred to as "hydrates." Hydrates include stoichiometric hydrates and compositions containing variable amounts of water. The present application encompasses all such solvates.

It will also be appreciated by those skilled in the art that certain compounds of the present invention, including their various solvates, that exist in crystalline form may exhibit polymorphism (i.e., the ability to appear in different crystalline structures). These different crystalline forms are generally referred to as "polymorphs." The present application includes all of the above-mentioned polymorphs. Polymorphs have the same chemical composition, but differ in terms of packing, geometric arrangement, and other descriptive properties of the crystalline solid state. Therefore, polymorphs may have different physical properties, such as shape, density, hardness, deformability, stability, and solubility. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffraction patterns, which can be used for identification. It will be appreciated by those skilled in the art that different polymorphs may be produced, for example, by changing or adjusting the reaction conditions or reagents used to prepare the compound. For example, changes in temperature, pressure, or solvent may result in polymorphs. In addition, a polymorph may spontaneously transform into another polymorph under certain conditions.

The compounds of the present application or their salts may exist in stereoisomeric forms (e.g., they contain one or more asymmetric carbon atoms). Individual stereoisomers (enantiomers and diastereomers) and mixtures thereof are included within the scope of the present application. Similarly, it should be understood that the compounds or salts of formula (I) may exist in tautomeric forms different from those shown in formula (I), and these are also included within the scope of the present application. It should be understood that the present application includes all combinations and subsets of the above-mentioned specific groups. The scope of the present application includes mixtures of stereoisomers and purified enantiomers or mixtures enriched in enantiomers/diastereomers. It should be understood that the present application includes all combinations and subsets of the above-mentioned specific groups.

The present application also includes isotopically labeled forms of the compounds of the present application, in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of isotopes that can be incorporated into the compounds of the present application and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, ³⁶ Cl, ¹²³ I, and ¹²⁵ I.

Compounds of the present invention containing the above-mentioned isotopes and/or other isotopes of other atoms and pharmaceutically acceptable salts of the compounds are within the scope of this application. Isotopically labeled compounds of the present invention, for example, those into which radioactive isotopes such as ³ H and ¹⁴ C have been introduced, can be used in drug and/or substrate tissue distribution assays. Tritium, i.e., ³ H, and carbon-14, i.e., ¹⁴ C, isotopes are frequently used due to their ease of preparation and detection. ¹¹ C and ¹⁸ F isotopes can be used for PET (positron emission tomography), and ¹²⁵ I isotopes can be used for SPECT (single photon emission computed tomography), both of which can be used for brain imaging. In addition, substitution with heavier isotopes, such as deuterium, i.e., ² H, can provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some cases. Isotopically labeled compounds of Formula I and the following compounds can generally be prepared by carrying out the procedures disclosed in the following schemes and/or examples by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

Pharmaceutical compositions of ADCs

The pharmaceutical preparations of the therapeutic antibody-drug conjugates (ADCs) of the present application are generally prepared with pharmaceutically acceptable parenteral vehicles for parenteral administration, i.e., bolus injection, intravenous injection, intratumoral injection, and in the form of unit dose injections. The antibody-drug conjugates (ADCs) of the desired purity are optionally mixed with pharmaceutically acceptable diluents, carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. ed.) into lyophilized preparations or aqueous solutions.

Cysteine-engineered antibodies

The present application compound includes antibody-drug conjugates, which include cysteine engineered antibodies, wherein one or more amino acids of the wild-type or parent antibody are replaced by cysteine. Any form of antibody can be so engineered, i.e., mutated. For example, the parent Fab antibody fragment can be engineered to form a cysteine engineered Fab, which is referred to as "ThioFab" in this application. Similarly, the parent monoclonal antibody can be engineered to form a "ThioMab." It should be noted that a single-site mutation produces one engineered cysteine residue in ThioFab, while a single-site mutation produces two engineered cysteine residues in ThioMab due to the dimer nature of the IgG antibody. The reactivity of the newly introduced engineered cysteine sulfhydryl groups is evaluated for mutants with cysteine (Cys) residues replaced ("engineered"). The sulfhydryl reactivity value is a relative value ranging from 0 to 1.0 and can be measured for any cysteine engineered antibody. The sulfhydryl reactivity value of the cysteine engineered antibodies of the present application is 0.6 to 1.0, 0.7 to 1.0 or 0.8 to 1.0. In order to prepare cysteine engineered antibodies by mutagenesis, DNA encoding amino acid sequence variants of the starting polypeptide is prepared by various methods known in the art. These methods include but are not limited to preparing by site-guided (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis and cassette mutagenesis of the previously prepared coded polypeptide DNA. Variants of the recombinant antibodies can also be constructed by restriction fragment manipulation or by overlapping extension PCR with synthetic oligonucleotides. Mutagenic primers encode cysteine codon replacement. Standard mutagenesis techniques can be used to generate DNA encoding the above-mentioned mutant cysteine engineered antibodies. For general guidance, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, N.Y., 1993.

Cysteine can be engineered at reactive sites of antibodies without forming intrachain or intermolecular disulfide bonds (Junutula et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; US7521541; US7723485; WO2009/052249, Shen et al. (2012) Nature Biotech., 30(2):184-191; Junutula et al. (2008) Jour of Immun. Methods 332:41-52). The engineered cysteine thiol group can react with a linker reagent or linker-drug intermediate of the present invention having a thiol-reactive electrophilic group such as maleimide or α-haloamide to form an ADC having a cysteine engineered antibody (ThioMabs) and a drug (D) moiety. Therefore, the location of the drug moiety can be designed, controlled, and known. Drug loading can be controlled because engineered cysteine thiols typically react with thiol-reactive linker reagents or linker-drug intermediates in high yield. Antibodies can be engineered to introduce a cysteine by substitution at a single site on the heavy or light chain, resulting in two new cysteines in a symmetrical antibody. Drug loadings approaching 2 can be achieved, and the conjugated ADCs are nearly homogeneous.

The present application's cysteine engineered antibodies preferably retain the antigen binding capacity of their wild-type parent antibody counterparts. Therefore, cysteine engineered antibodies are able to preferably specifically bind to antigens. The above-mentioned antigens include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, transmembrane proteins, signal transduction proteins, cell survival regulatory factors, cell proliferation regulatory factors, molecules related to tissue development or differentiation (e.g., known or suspected functionally promoting tissue development or differentiation), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules related to angiogenesis, and molecules related to angiogenesis (e.g., known or suspected functionally promoting angiogenesis). Tumor-associated antigens can be cluster differentiation factors (i.e., CD proteins). The antigen that cysteine engineered antibodies can bind to can be a member of a subset of one of the above-mentioned categories, wherein other subsets of the categories include other molecules/antigens with unique characteristics (with respect to the antigen being paid attention to).

Cysteine-engineered antibodies were prepared for conjugation to linker-drug intermediates by reduction and reoxidation of intrachain disulfide groups.

Tumor-associated antigens

The antibodies (including but not limited to cysteine engineered antibodies) that can be used for the antibody-drug conjugates of the present application in the treatment of cancer include but are not limited to antibodies against cell surface receptors and tumor-associated antigens (TAA). Certain tumor-associated antigens are known in the art and can be prepared using methods and information known in the art for the generation of antibodies. In order to find effective cell targets for cancer diagnosis and treatment, researchers have attempted to identify transmembrane tumor-associated polypeptides or other tumor-associated polypeptides that are specifically expressed on the surface of one or more specific types of cancer cells compared to one or more normal non-cancerous cells. Typically, the above-mentioned tumor-associated polypeptides are more abundantly expressed on the surface of cancer cells compared to the surface of non-cancerous cells. The identification of the above-mentioned tumor-associated cell surface antigen polypeptides has been able to be more specifically targeted to cancer cells for destruction by antibody-based therapy.

Examples of tumor-associated antigens TAAs include, but are not limited to, those listed below. For convenience, information related to these antigens is known in the art and follows the National Center for Biotechnology Information (NCBI) nucleic acid and protein sequence identification protocol, including names, alternative names, Genbank accession numbers, and primary references. Nucleic acid and protein sequences corresponding to the following TAAs are available in public databases such as GenBank. Tumor-associated antigens targeted by antibodies include all of the following amino acid sequence variants and isoforms, which have at least about 70%, 80%, 85%, 90% or 95% sequence identity with the sequences identified in the cited references and/or show substantially the same biological properties or characteristics as the TAAs having the sequences found in the cited references. For example, TAAs with variant sequences are generally capable of specifically binding to the following antibodies, which specifically bind to TAAs having the listed corresponding sequences. The sequences and contents of the references specifically described in this application are expressly incorporated into this application by reference.

(1) BMPR1B (bone morphogenetic protein receptor type IB, Genbank accession number NM_001203)

ten Dijke, P. et al., Science 264(5155):101-104 (1994), Oncogene 14(11):1377-1382 (1997); WO2004063362 (claim 2); WO2003042661 (claim 12); US2003134790A1 (pp. 38-39); WO2002102235 (claim 13; p. 296); WO2003055443 (pp. 91-92); WO200299122 (Example 2; pp. 528-530); ); WO2003029421 (claim 6); WO2003024392 (claim 2; FIG. 112 ); WO200298358 (claim 1; page 183); WO200254940 (pages 100-101); WO200259377 (pages 349-350); WO200230268 (claim 27; page 376); WO200148204 (Example; FIG. 4 )

NP_001194 bone morphogenetic protein receptor type IB/pid=NP_001194.1-

Cross Ref:MIM:603248;NP_001194.1;AY065994

(2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486)

Biochem.Biophys.Res.Commun.255(2),283-288(1999),Nature 395(6699):288-291(1998),Gaugitsch,H.W.et al.(1992)J.Biol.Chem.267(16):11267-11273;WO2004048938(Example 2);WO2004032842(Example IV);WO2003042661(Claim 12);WO2003016475(Claim 1);WO200278524(Example 2);WO2002 99074 (claim 19; pp. 127-129); WO200286443 (claim 27; pp. 222, 393); WO2003003906 (claim 10; p. 293); WO200264798 (claim 33; pp. 93-95); WO200014228 (claim 5; pp. 133-136); US2003224454 (Fig. 3); WO2003025138 (claim 12; p. 150);

NP_003477 Solute carrier family 7 (cationic amino acid transporter, y+ system), member 5/pid=NP_003477.3 - Homo sapiens

Cross-reference: MIM:600182; NP_003477.3; NM_015923; NM_003486_1

(3) STEAP1 (prostate transmembrane epithelial antigen 6, Genbank accession number NM_012449)

Cancer Res. 61(15), 5857-5860(2001), Hubert, R.S. et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96(25): 14523-14528; WO2004065577 (claim 6); WO2004027049 (Figure 1L); EP1394274 (Example 11); WO2004016225 (Claim 2); WO2003042661 (Claim 12); US2003157089 (Example 5); US2003185830 (Example 5); US2003064397 (Figure 2); WO200289747 (Example 5; pages 618-619); WO2003022995 (Example 9; Figure 13A, Example 53; page 173, Example 2; Figure 2A);

NP_036581 Prostate six-transmembrane epithelial antigen

Cross-reference: MIM:604415; NP_036581.1; NM_012449_1

(4) 0772P (CA125, MUC16, Genbank accession number AF361486)

J. Biol. Chem. 276(29):27371-27375 (2001); WO2004045553 (claim 14); WO200292836 (claim 6; FIG. 12); WO200283866 (claim 15; pages 116-121); US2003124140 (Example 16); Cross-references: GI:34501467; AAK74120.3; AF361486_1

(5) MPF (MPF, MSLN, SMR, megakaryocyte enhancer factor, mesothelin, Genbank accession number NM_005823) Yamaguchi, N. et al., Biol. Chem. 269(2), 805-808(1994), Proc. Natl. Acad. Sci. U.S.A. 96(20): 11531-11536(1999), Proc. Natl. Acad. Sci. U.S.A. 93(1): 136-140(1996), J.Biol. Ol.Chem.270(37):21984-21990(1995); WO2003101283(claim 14); (WO2002102235(claim 13; pages 287-288); WO2002101075(claim 4; pages 308-309); WO200271928(pages 320-321); WO9410312(pages 52-57); Cross-reference: MIM:601051; NP_005814.2; NM_005823_1

(6) NaPi2b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number NM_006424)

J.Biol.Chem.277(22):19665-19672(2002),Genomics62(2):281-284(1999), Feild, J.A. et al. (1999)Biochem.Biophys.Res.Commun.258(3):578-582); WO2004022778 (claim 2); EP1394274 (Example 11); WO2002102235 (claim 13; page 326); EP875569 (claim 1; pages 17-19); WO200157188 (claim 20; page 329); WO2004032842 (Example IV); WO200175177 (claim 24; pages 139-140);

Cross-reference: MIM:604217; NP_006415.1; NM_006424_1

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, 7 thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession number AB040878)

Nagase T. et al. (2000) DNA Res. 7(2):143-150; WO2004000997 (claim 1); WO2003003984 (claim 1); WO200206339 (claim 1; page 50); WO200188133 (claim 1; pages 41-43, 48-58); WO2003054152 (claim 20); WO2003101400 (claim 11);

Accession:Q9P283;EMBL;AB040878;BAA95969.1.Genew;HGNC:10737;

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628); Ross et al. (2002) Cancer Res.62:2546-2553; US2003129192 (claim 2); US2004044180 (claim 12); US2004044179 (claim 11); US2003096961 (claim 11); US2003232056 (Example 5); WO2003105758 (claim 12); US2003206918 (Example 5); EP1347046 (claim 1); WO2003025148 (claim 20);

Cross-reference: GI:37182378;AAQ88991.1;AY358628_1

(9) ETBR (endothelin type B receptor, Genbank accession number AY275463);

Nakamuta M. et al., Biochem. Biophys. Res. Commun. 177, 34-39, 1991; Ogawa Y. et al., Biochem. Biophys. Res. Commun. 178, 248-255, 1991; Arai H. et al., Jpn. Circ. J. 56, 1303-1307, 1992; Arai H. et al., J. Biol. Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M. et al., Biochem. Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N.A. et al., J. Biol. Chem. 268, 3873-3879, 1993; Haendler B. et al., J. Cardiovasc. Pharmacol. 20, s1-S4, 1992; Tsutsumi M. et al., Gene 228, 43-49, 1999; Strausberg R.L. et al., Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903, 2002; Bourgeois C. et al., J. Clin. Endocrinol. Metab. 82, 3116-3123, 1997; Okamoto Y. et al., Biol. Chem. 272, 21589-21596, 1997; Verheij J.B. et al., Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R.M.W. et al., Eur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E.G. et al., Cell 79, 1257-1266, 1994; Attie T. et al., Hum. Mol. Genet. 4, 2407-2409, 1995; Auricchio A. et al., Hum. Mol. Genet. 5: 351-354, 1996; Amiel J. et al., Hum. Mol. Genet. 5, 355-357, 1996; Hofstra R.M.W. et al., Nat. Genet. 12, 445-447, 1996; Svensson P.J. et al., Hum. Genet. 103, 145-148, 1998; Fuchs S. et al., Mol. Med. 7, 115-124, 2001; Pingault V. et al. (2002) Hum. Genet. 111, 198-206; WO2004045516 (claim 1); WO2004048938 (Example 2); WO2004040000 (claim 151); WO2003087768 (claim 1); WO2003016475 (claim 1); WO2003016475 (claim 1); WO200261087 (Figure 1); WO2003016 494 (FIG. 6); WO2003025138 (claim 12; page 144); WO200198351 (claim 1; pages 124-125); EP522868 (claim 8; FIG. 2); WO200177172 (claim 1; pages 297-299); US2003109676; US6518404 (FIG. 3); US5773223 (claim 1a; columns 31-34); WO2004001004;

(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM_017763);

WO2003104275 (claim 1); WO2004046342 (Example 2); WO2003042661 (claim 12); WO2003083074 (claim 14; page 61); WO2003018621 (claim 1); WO2003024392 (claim 2; FIG. 93); WO200166689 (Example 6);

Cross Ref: LocusID:54894;NP_060233.2;NM_017763_1

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, prostate 6-transmembrane epithelial antigen, 6-transmembrane prostate protein, Genbank accession number AF455138)

Lab. Invest. 82(11):1573-1582(2002); WO2003087306; US2003064397 (claim 1; FIG1); WO200272596 (claim 13; pp. 54-55); WO200172962 (claim 1; FIG4B); WO2003104270 (claim 11); WO2003104270 (claim 16); US2004005598 (claim 22); WO2003042661 (claim 12); US2003060612 (claim 12; FIG10); WO200226822 (claim 23; FIG2); WO200216429 (claim 12; FIG10);

Cross-reference: GI:22655488;AAN04080.1;AF455138_1

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM_017636)

Xu, X.Z. et al., Proc. Natl. Acad. Sci. U.S.A. 98(19):10692-10697 (2001), Cell 109(3):397-407 (2002), J. Biol. Chem. 278(33):30813-30820 (2003); US2003143557 (claim 4); WO200040614 (claim 14; pages 100-103); WO200210382 (claim 1; FIG. 9A ); WO2003042661 (claim 12); WO200230268 (claim 27; page 391); US2003219806 (claim 4); WO200162794 (claim 14; FIG. 1A-D );

Cross-reference: MIM:606936; NP_060106.2; NM_017636_1

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession number NP_003203 or NM_003212)

Ciccodicola, A. et al., EMBO J. 8(7):1987-1991 (1989), Am. J. Hum. Genet. 49(3):555-565 (1991)); US2003224411 (claim 1); WO2003083041 (Example 1); WO2003034984 (claim 12); WO200288170 (claim 2; pages 52-53); WO2003024392 (claim 2; Figure 58); WO200216413 (claim 1; pages 94-95, 105); WO200222808 (claim 2; Figure 1); US5854399 (Example 2; columns 17-18); US5792616 (Figure 2);

Cross Ref:MIM:187395;NP_003203.1;NM_003212_1

(14) CD21 (CR2 (complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792, Genbank accession number M26004)

Fujisaku et al. (1989) J. Biol. Chem. 264(4):2118-2125; Weis J. J. et al., J. Exp. Med. 167, 1047-1066, 1988; Moore M. et al., Proc. Natl. Acad. Sci. U.S.A. 84, 9194-9198, 1987; Barel M. et al., Mol. Immunol. 35, 1025-1031, 1998; Weis J. J. et al., Proc. Natl. Acad. Sci. U.S.A. 83, 5639-5643, 1986; Sinha S.K. et al. (1993) J. Immunol. 150, 5311-5320; WO2004045520 (Example 4); US2004005538 (Example 1); WO2003062401 (Claim 9); WO2004045520 (Example 4); WO9102536 (Figs. 9.1-9.9); WO2004020595 (Claim 1);

Accession: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1

(15) CD79b (CD79B, CD79b, IGb (immunoglobulin-related beta), B29, Genbank accession number NM_000626 or 11038674)

Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (7): 4126-4131, Blood (2002) 100 (9): 3068-3076, Muller et al. (1992) Eur. J. Immunol. 22 (6): 1621-1625); WO2004016225 (claim 2, FIG. 140); WO2003087768; US2004101874 (claim 1, 102); WO2003062401 (claim 9); WO200278524 (Example 2); US2002150573 (claim 5, page 15); US5644033; WO2003048202 (claim 1, pages 306 and 309); WO99/558658; US6534482 (claim 13, Figure 17A/B); WO200055351 (claim 11, pages 1145-1146);

Cross Ref:MIM:147245;NP_000617.1;NM_000626_1

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (phosphatase anchoring protein containing SH2 domain 1a), SPAP1B, SPAP1C, Genbank accession number NM_030764, AY358130)

Genome Res. 13(10): 2265-2270 (2003), Immunogenetics 54(2): 87-95 (2002), Blood 99(8): 2662-2669 (2002), Proc. Natl. Acad. Sci. U.S.A. 98(17): 9772-9777 (2001), Xu, M.J. et al. (2001) Biochem. Biophys. Res. Commun. 280(3): 768-775; WO2004016225 (claim 2); WO2003077836; WO200138490 (claim 5; Figures 18D-1-18D-2); WO2003097803 (claim 12); WO2003089624 (claim 25);

Cross-reference: MIM:606509; NP_110391.2; NM_030764_1

(17) HER2 (ErbB2, Genbank accession number M11730)

Coussens L. et al., Science (1985) 230(4730):1132-1139); Yamamoto T. et al., Nature 319, 230-234, 1986; Semba K. et al., Proc. Natl. Acad. Sci. U.S.A. 82, 6497-6501, 1985; Swiercz J.M. et al., J. Cell Biol. 165, 869-880, 2004; Kuhns J.J. et al., J. Biol. Chem. 274, 36422-36427, 1999; Cho H.-S. et al., Nature 421, 756-760, 2003; Ehsani A. et al. (1993) Genomics 15,426-429; WO2004048938 (Example 2); WO2004027049 (Figure 1I); WO2004009622; WO2003081210; WO2003089904 (claim 9); WO2003016475 (claim 1); US2003118592; WO2003008537 (claim 1); WO2003055439 (claim 29; Figures 1A-B); WO2003025228 (claim 37; Figure 5C); WO200222636 (Example 13; pages 95-107); WO200212341 (claim 68; Figure 7); WO 200213847 (pp. 71-74); WO200214503 (pp. 114-117); WO200153463 (claim 2; pp. 41-46); WO200141787 (p. 15); WO200044899 (claim 52; FIG. 7); WO200020579 (claim 3; FIG. 2); US5869445 (claim 3; columns 31-38); WO9630514 (claim 2; pp. 56-61); EP1439393 (claim 7); WO2004043361 (claim 7); WO2004022709; WO200100244 (Example 3; FIG. 4);

Accession: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1

(18) NCA (CEACAM6, Genbank accession number M18728);

Barnett T. et al., Genomics 3, 59-66, 1988; Tawaragi Y. et al., Biochem. Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R.L. et al., Proc. Natl. Acad. Sci. U.S.A. 99: 16899-16903, 2002; WO2004063709; EP1439393 (claim 7); WO2004044178 (Example 4); WO2004031238; WO2003042661 (claim 12); WO200278524 (Example 2); WO200286443 (claim 27; page 427); WO200260317 (claim 2);

Accession: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL; M18728;

(19) MDP (DPEP1, Genbank accession number BC017023)

Proc. Natl. Acad. Sci. U.S.A. 99(26):16899-16903(2002)); WO2003016475 (claim 1); WO200264798 (claim 33; pages 85-87); JP05003790 (Figures 6-8); WO9946284 (Figure 9);

Cross-reference: MIM:179780;AAH17023.1;BC017023_1

(20) IL20Rα (IL20Rα, ZCYTOR7, Genbank accession number AF184971);

Clark H.F. et al., Genome Res. 13, 2265-2270, 2003; Mungall A.J. et al., Nature 425, 805-811, 2003; Blumberg H. et al., Cell 104, 9-19, 2001; Dumoutier L. et al., J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J. et al., J. Biol. Chem. 277, 47517-47523, 2002; Pletnev S. et al. (2003) Biochemistry 42: 12617-12624; Sheikh F. et al. (2004) J. Immunol. 172, 2006-2010; EP1394274 (Example 11); US2004005320 (Example 5); WO2003029262 (pp. 74-75); WO2003002717 (claim 2; page 63); WO200222153 (pp. 45-47); US2002042366 (pp. 20-21); WO200146261 (pp. 57-59); WO200146232 (pp. 63-65); WO9837193 (claim 1; pages 55-59);

Accession:Q9UHF4;Q6UWA9;Q96SH8;EMBL;AF184971;AAF01320.1

(21) Brachyproteoglycan (BCAN, BEHAB, Genbank accession number AF229053)

Gary S.C. et al., Gene 256, 139-147, 2000; Clark H.F. et al., Genome Res. 13, 2265-2270, 2003; Strausberg R.L. et al., Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903, 2002; US2003186372 (claim 11); US2003186373 (claim 11); US2003119131 (claim 1; FIG52); US2003119122 (claim 1; FIG52); US2003119126 (Claim 1); US2003119121 (Claim 1; FIG. 52); US2003119129 (Claim 1); US2003119130 (Claim 1); US2003119128 (Claim 1; FIG. 52); US2003119125 (Claim 1); WO2003016475 (Claim 1); WO200202634 (Claim 1);

(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession number NM_004442)

Chan, J. and Watt, V.M., Oncogene 6(6), 1057-1061 (1991), Oncogene 10(5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998), Int. Rev. Cytol. 196:177-244 (2000); WO2003042661 (claim 12); WO200053216 (claim 1; page 41); WO2004065576 (claim 1); WO2004020583 (claim 9); WO2003004529 (pages 128-132); WO200053216 (claim 1; page 42);

Cross-reference: MIM:600997; NP_004433.2; NM_004442_1

(23)ASLG659 (B7h, Genbank accession number AX092328)

US20040101899 (claim 2); WO2003104399 (claim 11); WO2004000221 (Figure 3); US2003165504 (claim 1); US2003124140 (Example 2); US2003065143 (Figure 60); WO2002102235 (claim 13; page 299); US2003091580 (Example 2); WO200210187 (claim 6; Figure 10); WO200194641 (claim 12; Figure 7b); WO200202624 (claim 13; Figures 1A-1B); US20020347 49 (claim 54; pages 45-46); WO200206317 (Example 2; pages 320-321, claim 34; pages 321-322); WO200271928 (pages 468-469); WO200202587 (Example 1; Figure 1); WO200140269 (Example 3; pages 190-192); WO200036107 (Example 2; pages 205-207); WO2004053079 (claim 12); WO2003004989 (claim 1); WO200271928 (pages 233-234, 452-453); WO0116318;

(24) PSCA (prostate stem cell antigen precursor, Genbank accession number AJ297436)

Reiter R.E. et al., Proc. Natl. Acad. Sci. U.S.A. 95, 1735-1740, 1998; Gu Z. et al., Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res. Commun. (2000) 275(3):783-788; WO2004022709; EP1394274 (Example 11); US2004018553 (Claim 17); WO2003008537 (Claim 1); WO200281646 (Claim 1; page 164); WO2003003906 (claim 10; page 288); WO200140309 (Example 1; Figure 17); US2001055751 (Example 1; Figure 1b); WO200032752 (claim 18; Figure 1); WO9851805 (claim 17; page 97); WO9851824 (claim 10; page 94); WO9840403 (claim 2; Figure 1B);

Accession: O43653; EMBL; AF043498; AAC39607.1

(25) GEDA (Genbank accession number AY260763);

AAP14954 lipoma HMGIC fusion partner-like protein/pid=AAP14954.1 - Homo sapiens

Species: Homo sapiens (human)

WO2003054152 (claim 20); WO2003000842 (claim 1); WO2003023013 (Example 3, claim 20); US2003194704 (claim 45);

Cross Ref:GI:30102449;AAP14954.1;AY260763_1

(26) BAFF-R (B cell activating factor receptor, BLyS receptor 3, BR3, Genbank accession number AF116456); BAFF receptor/pid=NP_443177.1 - Homo sapiens

Thompson, J.S. et al., Science 293(5537), 2108-2111 (2001); WO2004058309; WO2004011611; WO2003045422 (Example; pp. 32-33); WO2003014294 (claim 35; FIG. 6B); WO2003035846 (claim 70; pp. 615-616); WO200294852 (cols. 136-137); WO200238766 (claim 3; p. 133); WO200224909 (Example 3; FIG. 3);

Cross Ref:MIM:606269;NP_443177.1;NM_052945_1;AF132600

(27) CD22 (B cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession number AK026467);

Wilson et al. (1991) J. Exp. Med. 173: 137-146; WO2003072036 (claim 1; Figure 1);

Cross-reference: MIM:107266; NP_001762.1; NM_001771_1

(28) CD79a (CD79A, CD79α, immunoglobulin-associated α, B cell-specific protein (CD79B) that covalently interacts with Igβ and forms a complex with IgM molecules on the surface to transduce signals involved in B cell differentiation), pI: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: 19q13.2, Genbank accession number NP_001774.10)

WO2003088808; US20030228319; WO2003062401 (claim 9); US2002150573 (claim 4, pages 13-14); WO9958658 (claim 13, FIG16); WO9207574 (FIG1); US5644033; Ha et al. (1992) J. Immunol. 148(5):1526-1531; Mueller et al. (1992) Eur. J. Biochem. 22:1621-1625; Hashimoto et al. (1994) Immunogenetics 40(4):287-295; Preud’homme et al. (1992) Clin. Exp. Immunol. 90(1):141-146; Yu et al. (1992) J. Immunol. 148(2):633-637; Sakaguchi et al. (1988) EMBO J. 7(11):3457-3464;

(29) CXCR5 (Burkitt lymphoma receptor 1, a G protein-coupled receptor activated by the CXCL13 chemokine that plays a role in lymphocyte migration and humoral defense, plays a role in HIV-2 infection, and may play a role in the development of AIDS, lymphoma, myeloma, and leukemia); 372 aa, pI: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3, Genbank accession number NP_001707.1)

WO2004040000; WO2004015426; US2003105292 (Example 2); US6555339 (Example 2); WO200261087 (Figure 1); WO200157188 (Claim 20, Page 269); WO200172830 (Pages 12-13); WO200022129 (Example 1, Pages 152-153, Example 2, Pages 254-256) ); WO9928468 (claim 1, page 38); US5440021 (Example 2, columns 49-52); WO9428931 (pages 56-58); WO9217497 (claim 7, Figure 5); Dobner et al. (1992) Eur. J. Immunol. 22:2795-2799; Barella et al. (1995) Biochem. J. 309:773-779;

(30) HLA-DOB (β subunit of MHC class II molecules (Ia antigens) that binds to peptides and presents them to CD4+ T lymphocytes); 273 aa, pI: 6.56 MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession number NP_002111.1)

Tonnelle et al. (1985) EMBO J. 4(11):2839-2847; Jonsson et al. (1989) Immunogenetics 29(6):411-413; Beck et al. (1992) J. Mol. Biol. 228:433-441; Strausberg et al. (2002) Proc. Natl. Acad. Sci USA 99:16899-16903; Servenius et al. (1987) J. Biol. Chem. 262:8759-8766; Beck et al. (1996) J. Mol. Biol. 255:1-13; Naruse et al. (2002) Tissue Antigens 59:512-519; WO9958658 (claim 13, FIG. 15); US6153408 (columns 35-38); US5976551 (columns 168-170); US6011146 (columns 145-146); Kasahara et al. (1989) Immunogenetics 30(1):66-68; Larhammar et al. (1985) J. Biol. Chem. 260(26):14111-14119;

(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, and defects may contribute to the pathophysiology of idiopathic detrusor instability); 422 aa), pI: 7.63, MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3, Genbank accession number NP_002552.2)

Le et al. (1997) FEBS Lett. 418(1-2):195-199; WO2004047749; WO2003072035 (claim 10); Touchman et al. (2000) Genome Res. 10:165-173; WO200222660 (claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1); WO2003029277 (page 82);

(32) CD72 (B cell differentiation antigen CD72, Lyb-2) PROTEIN SEQUENCE Full size...tafrfpd (1..359; 359aa), pI: 8.66, MW: 40225TM: 1 [P] Gene Chromosome: 9p13.3, Genbank accession number NP_001773.1)

WO2004042346 (claim 65); WO2003026493 (pp. 51-52, 57-58); WO200075655 (pp. 105-106); Von Hoegen et al. (1990) J. Immunol. 144(12):4870-4877; Strausberg et al. (2002) Proc. Natl. Acad. Sci USA 99:16899-16903;

(33) LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family, regulates B cell activation and apoptosis, and loss of function is associated with increased disease activity in patients with systemic lupus erythematosus); 661aa, pI: 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12, Genbank accession number NP_005573.1)

US2002193567; WO9707198 (claim 11, pages 39-42); Miura et al. (1996) Genomics 38(3):299-304; Miura et al. (1998) Blood 92:2815-2822; WO2003083047; WO9744452 (claim 8, pages 57-61); WO200012130 (pages 24-26);

(34) FcRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain containing C2-type Ig-like and ITAM domains, which may play a role in B lymphocyte differentiation); 429aa, pI: 5.28, MW: 46925TM: 1[P] Gene Chromosome: 1q21-1q22, Genbank accession number NP_443170.1)

WO2003077836; WO200138490 (claim 6, Figures 18E-1-18-E-2); Davis et al. (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; WO2003089624 (claim 8); EP1347046 (claim 1); WO2003089624 (claim 7);

(35) IRTA2 (immunoglobulin superfamily receptor translocation-associated 2, a putative immune receptor that may play a role in B-cell development and lymphomagenesis; gene dysregulation via translocation occurs in some B-cell malignancies); 977aa, pI: 6.88MW: 106468TM: 1[P]Gene Chromosome: 1q21, Genbank accession numbers Human: AF343662, AF343663, AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL834187, AY358085; Mouse: AK089756, AY158090, AY506558; NP_112571.1

WO2003024392 (claim 2, FIG. 97 ); Nakayama et al. (2000) Biochem. Biophys. Res. Commun. 277(1):124-127; WO2003077836; WO200138490 (claim 3, FIG. 18B-1-18B-2);

(36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan related to growth factors of the EGF/nerve growth factor family and follistatin); 374 aa, NCBI accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; Genbank accession number AF179274; AY358907, CAF85723, CQ782436

WO2004074320 (SEQ ID NO 810); JP2004113151 (SEQ ID NO 2, 4, 8); WO2003042661 (SEQ ID NO 580); WO2003009814 (SEQ ID NO 411); EP1295944 (pages 69-70); WO200230268 (page 329); WO200190304 (SEQ ID NO 2706); US2004249130; US2004022727; WO2004063355; US2004197325; US2003232350; US2004005563; US2003124579; Horie et al. (2000) Genomics 67:146-152; Uchida et al. (1999) Biochem. Biophys. Res. Commun. 266:593-602; Liang et al. (2000) Cancer Res. 60:4907-12; Glynne-Jones et al. (2001) Int J Cancer. Oct 15; 94(2):178-84;

(37) PMEL17 (silver homologue; SILV; D12S53E; PMEL17; (SI); (SIL); ME20; gp100) BC001414; BT007202; M32295; M77348; NM_006928; McGlinchey, R.P. et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106(33), 13731-13736; Kummer, M.P. et al. (2009) J. Biol. Chem. 284(4), 2296-2306;

(38) TMEFF1 (transmembrane protein with EGF-like and two follistatin-like domains 1; Tomoregulin-1; H7365; C9orf2; C9ORF2; U19878; X83961) NM_080655; NM_003692; Harms, P.W. (2003) Genes Dev. 17(21), 2624-2629; Gery, S. et al. (2003) Oncogene 22(18): 2723-2727;

(39) GDNF-Ra1 (GDNF family receptor α1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-α1; GFR-A-1; U95847; BC014962; NM_145793) NM_005264; Kim, M.H. et al. (2009) Mol. Cell. Biol. 29(8), 2264-2277; Treanor, J.J. et al. (1996) Nature 382(6586):80-83;

(40) Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1) NP_002337.1; NM_002346.2; de Nooij-van Dalen, A.G. et al. (2003) Int. J. Cancer 103(6), 768-774; Zammit, D.J. et al. (2002) Mol. Cell. Biol. 22(3): 946-952;

(41) TMEM46 (shisa homolog 2 (Xenopus); SHISA2) NP_001007539.1; NM_001007538.1; Furushima, K. et al. (2007) Dev. Biol. 306(2), 480-492; Clark, H.F. et al. (2003) Genome Res. 13(10): 2265-2270;

(42) Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1) NP_067079.2; NM_021246.2; Mallya, M. et al. (2002) Genomics 80(1):113-123; Ribas, G. et al. (1999) J. Immunol. 163(1):278-287;

(43) LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR67) NP_003658.1; NM_003667.2; Salanti, G. et al. (2009) Am. J. Epidemiol. 170(5):537-545; Yamamoto, Y. et al. (2003) Hepatology 37(3):528-533;

(44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; (PTC); CDHF12; Hs.168114; RET51; RET-ELE1) NP_066124.1; NM_020975.4; Tsukamoto, H. et al. (2009) Cancer Sci. 100(10):1895-1901; Narita, N. et al. (2009) Oncogene 28(34):3058-3068;

(45) LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226) NP_059997.3; NM_017527.3; Ishikawa, N. et al. (2007) Cancer Res. 67(24):11601-11611; de Nooij-van Dalen, A.G. et al. (2003) Int. J. Cancer 103(6):768-774;

(46) GPR19 (G protein-coupled receptor 19; Mm. 4787) NP_006134.1; NM_006143.2; Montpetit, A. and Sinnett, D. (1999) Hum. Genet. 105(1-2):162-164; O’Dowd, B.F. et al. (1996) FEBSLett. 394(3):325-329;

(47) GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12) NP_115940.2; NM_032551.4; Navenot, J.M. et al. (2009) Mol. Pharmacol. 75(6):1300-1306; Hata, K. et al. (2009) Anticancer Res. 29(2):617-623;

(48) ASPHD1 (aspartate beta-hydroxylase domain-containing 1; LOC253982) NP_859069.2; NM_181718.3; Gerhard, D.S. et al. (2004) Genome Res. 14(10B):2121-2127;

(49) Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3) NP_000363.1; NM_000372.4; Bishop, D.T. et al. (2009) Nat. Genet. 41(8):920-925; Nan, H. et al. (2009) Int. J. Cancer 125(4):909-917;

(50) TMEM118 (RING finger protein, transmembrane 2; RNFT2; FLJ14627) NP_001103373.1; NM_001109903.1; Clark, H.F. et al. (2003) Genome Res. 13(10):2265-2270; Scherer, S.E. et al. (2006) Nature 440(7082):346-351

(51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e) NP_078807.1; NM_024531.3; Ericsson, T.A. et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100(11):6759-6764; Takeda, S. et al. (2002) FEBS Lett. 520(1-3):97-101.

In one embodiment, the antibody binds to one or more of the following polypeptides: BMPR1B, E16, STEAP1, 0772P, MPF, NaPi2b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, HER2, NCA, MDP, IL20Rα, brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, PMEL17, TMEFF1, GDNF-Ra1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, LY6K, GPR19, GPR54, ASPHD1, tyrosinase, TMEM118, GPR172A, or CD33.

In one embodiment, the antibody binds to BMPR1B.

In one embodiment, the antibody binds to E16.

In one embodiment, the antibody binds to STEAP1.

In one embodiment, the antibody binds to 0772P.

In one embodiment, the antibody binds to MPF.

In one embodiment, the antibody binds to NaPi2b.

In one embodiment, the antibody binds to Sema 5b.

In one embodiment, the antibody binds to PSCA h1g.

In one embodiment, the antibody binds to ETBR.

In one embodiment, the antibody binds to MSG783.

In one embodiment, the antibody binds to STEAP2.

In one embodiment, the antibody binds to TrpM4.

In one embodiment, the antibody binds to CRIPTO.

In one embodiment, the antibody binds to CD21.

In one embodiment, the antibody binds to CD79b.

In one embodiment, the antibody binds to FcRH2.

In one embodiment, the antibody binds to HER2.

In one embodiment, the antibody binds to NCA.

In one embodiment, the antibody binds to MDP.

In one embodiment, the antibody binds to IL20Rα.

In one embodiment, the antibody binds to brevican.

In one embodiment, the antibody binds to EphB2R.

In one embodiment, the antibody binds to ASLG659.

In one embodiment, the antibody binds to PSCA.

In one embodiment, the antibody binds to GEDA.

In one embodiment, the antibody binds to BAFF-R.

In one embodiment, the antibody binds to CD22.

In one embodiment, the antibody binds to CD79a.

In one embodiment, the antibody binds to CXCR5.

In one embodiment, the antibody binds to HLA-DOB.

In one embodiment, the antibody binds to P2X5.

In one embodiment, the antibody binds to CD72.

In one embodiment, the antibody binds to LY64.

In one embodiment, the antibody binds to FcRH1.

In one embodiment, the antibody binds to IRTA2.

In one embodiment, the antibody binds to TENB2.

In one embodiment, the antibody binds to PMEL17.

In one embodiment, the antibody binds to TMEFF1.

In one embodiment, the antibody binds to GDNF-Ra1.

In one embodiment, the antibody binds to Ly6E.

In one embodiment, the antibody binds to TMEM46.

In one embodiment, the antibody binds to Ly6G6D.

In one embodiment, the antibody binds to LGR5.

In one embodiment, the antibody binds to RET.

In one embodiment, the antibody binds to LY6K.

In one embodiment, the antibody binds to GPR19.

In one embodiment, the antibody binds to GPR54.

In one embodiment, the antibody binds to ASPHD1.

In one embodiment, the antibody binds to tyrosinase.

In one embodiment, the antibody binds to TMEM118.

In one embodiment, the antibody binds to GPR172A.

In one embodiment, the antibody binds to CD33.

The parent antibody may also be a fusion protein comprising an albumin binding peptide (ABP) sequence (Dennis et al. (2002) "Albumin Binding As A General Strategy for Improving The Pharmacokinetics Of Proteins" J Biol Chem. 277:35035-35043; WO01/45746). The antibodies of the present application include fusion proteins having the ABP sequences taught by: (i) Dennis et al. (2002) J Biol Chem. 277:35035-35043, Tables III and IV, page 35038; (ii) US20040001827, [0076]; and (iii) WO01/45746, pages 12-13, all of which are incorporated herein by reference.

Antibodies can be prepared using recombinant methods and compositions, for example as described in US4816567 and known in the art. In some embodiments, the antibodies are produced in eukaryotic host cells (e.g., mammalian host cells). In some embodiments, the antibodies are produced in prokaryotic host cells (e.g., E. coli).

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to produce an Fc region variant. The Fc region variant can be included in a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, the present application relates to antibody variants that have some, but not all, effector functions, making them ideal candidates for applications where the half-life of the antibody in vivo is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability.

Drug loading of ADC

Drug loading is the average number of drug moieties per antibody. The drug loading can be 1 to 8 drugs (D)/antibody (Ab), i.e., wherein 1, 2, 3, 4, 5, 6, 7, and 8 drug moieties are covalently attached to the antibody. The composition of the ADC includes a collection of antibodies conjugated to a certain range, i.e., 1 to 8 drugs. The average number of drugs per antibody in the ADC preparation from the conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA determination, electrophoresis, and HPLC. The quantitative distribution of the ADC in terms of p can also be determined. The average value of p in a specific ADC preparation can be determined by ELISA (Hamblett et al. (2004) Clin. Cancer Res. 10: 7063-7070; Sanderson et al. (2005) Clin. Cancer Res. 11: 843-852). However, the distribution of p (drug) values cannot be distinguished by the antibody-antigen binding and detection limit of ELISA. In addition, ELISA assays used to detect antibody-drug conjugates cannot determine the position of the drug moiety attached to the antibody, such as the heavy or light chain fragment or the specific amino acid residue. In some cases, separation, purification, and characterization of homogeneous ADC (where p is a certain value from ADC with other drug loadings) can be achieved by means such as reverse phase HPLC or electrophoresis.

For some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, the antibody may have only one or a few cysteine thiol moieties or may have only one or a few sufficiently reactive thiol moieties through which the linker may be attached. Higher drug loadings, such as p>5, may result in aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates.

Typically, during the conjugation reaction, less than the theoretical maximum amount of drug moiety is conjugated to the antibody. Antibodies may contain, for example, many lysine residues that do not react with the linker-drug intermediate (X-L-D) or linker reagent. Only the most reactive lysine groups can react with amine-reactive linker reagents. Additionally, only the most reactive cysteine sulfhydryl groups can react with sulfhydryl-reactive linker reagents or linker-drug intermediates. Typically, antibodies do not contain many (if any) free and reactive cysteine sulfhydryl moieties that can be linked to the drug moiety. Most cysteine sulfhydryl residues in the antibody of the compound exist as disulfide bonds and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP under partial or complete reducing conditions. The loading capacity (drug/antibody ratio "DAR") of the ADC can be controlled in several different ways, including: (i) limiting the molar excess of the linker-drug intermediate or linker reagent relative to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reducing conditions for cysteine sulfhydryl modification.

When more than one nucleophilic or electrophilic group of an antibody reacts with a linker-drug intermediate or a linker reagent and a subsequent dimeric drug moiety reagent, the resulting product is a mixture of antibody-drug conjugates (having a distribution of drug moieties attached to the antibody, such as 1, 2, 3, etc.). Liquid chromatography, such as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC), can separate the compounds in the mixture by drug loading value. Preparations of ADCs with a single drug loading value (p) can be separated, however, these single loading value ADCs may still be heterogeneous mixtures because the drug moiety can be attached to different sites on the antibody through a linker. Therefore, the antibody-drug conjugate compositions of the present application include mixtures of antibody-drug conjugate compounds, wherein the antibody has one or more drug moieties, and wherein the drug moiety can be attached to the antibody at various amino acid residues.

Exemplary Drug Moieties

Examples of drugs containing a tertiary amine or pyridine functional group include, but are not limited to, topotecan, LB-100, IB-01212, Mcl-1 inhibitor, 68Ga-BNOTA-PRGD2, Venetoclax, technetium Tc 99mtilmanocept, E-7016, HM-30181AK, paclitaxel, AS-1896596, EM-015, gadoteridol, BGJ-398, EC-1456, duvitinib, pyrroltinib maleate, GSK-923295, irinotecan, BGP-15, 90Y-edotereptide, mibradil, resminostat, itarnafloxin, NMS-P153, NMS- P937, irinotecan sulfosaccharide, abiraterone, A-366, SB-743921, Gilteritinib, Abemaciclib, IPP-204106, vinflunine, CX-5461, doxycycline, TP-0903, TLK-58747, irinotecan, numonafide, ON-123300, Vintafolide, KX2391, gadopentetate dimeglumine, gedatolisib, tasidol Butyl HCl, Genzyme/Ergomed, TTL-1177, naltrexone, CFI-400945 fumarate, Pictilisib, Felotaxel, OTX-008, banothrix, G-773, Trabicingin, NO-saquinavir, ENMD-2076, gadobutrol, BGB-102, Ga68-labeled, baicalein conjugate, THZ-1, TSR-011, ONC-201, ASP-3 026, galactosylceramide-enhanced vinorelbine, PMX-20005, 111In-RP-782, DSR-6434, vincristine sulfate, J-21475, Apitolisib, MDX-1203, gallium-68-SH-7139, CNX-1351, CT-1578, TG-02, imatinib, luminespib, vindesine-CB-3717 conjugate, AMG511, AZD1152hQPA Accurins, mepaline, CGM-097, TR-100, BGB-324, CEP-37440, OTSSP-167, APR-246, Indotecan, gadoterate meglumine, Lucanthone, Navitoclax, samarium lexidronate (153Sm), DMDAPatA, OCT-1002, irinotecan hydrochloride, BAY-87-2243, SIMM-559, CEP-33779, XL-3 88, SHR-1258, Voruciclib, UNBS-5162, Noscapine, Artemisinin, PHA-665752, Pyrrolobenzodiazepines, Folic acid-vindesine hydrazide conjugate, BRN-103, NSC-134754, AMP-53, PKI-402, Tariquidar, CG-200745, VO-100, PRLX-93936, Cenisertib, SSR-12 5329, CRD-401, SN-24771, Balamod, BIBX-1382, 105509, KW-2152, Lidamycin, CT-17, SN-23490, L-000021649, U-74389G, JNJ-17029259, PAK-200, PFP-6, XR-842, RP-697, NCO-700, B-220, Pazeputin, R-116010, NL-2001, RC-3940-II, BIM-46068, MDL-73811, CHIR-200131, Halitulin, fluazifop, cinchonine, BN-52207, ABT-546, NSC-639366, datidium chloride, WR-63320, LY-329146, dapaphenine, androstane, SR-25989, XR-9051, RSU-1069, N-1379, epelmycin A, PNU-144113, FCE-27726, NSC-357704, PD-171851, DZ-3358, B-9309-068, Goe-7874, Ro-44-5912, MDL-103323, Mofaradine, Ro-46-7864, RU-45144, Win-63320, NC-190, NSC-646958, VA-033, GI-149893, BBR-2378, NSC-639365, Vinfosine, SDZ-62-43 4. BCH-2051, RB-90745, TI-356, ER-37328, SR-26050, CL-329753, LY-326315, AN-1006, CP-117227, R-tiludiprine, RB-90740, SYUIQ-05, SR-16388, SN-28049, SN-30000, topotecan hydrochloride, vinorelbine, WBZ-7, S-44563, GSK-1070916, RPR-203360, demethyldeaminopateamine A, EU-517, AT-9283, spirogermanium hydrochloride, E-7974, Azoacridone, Vinorelbine, GTx-134, Astemizole, Reteptine, Mitonafamide, Lincazolyl, PBT-1, Atimod, Topizan, A-620223, Trodolgoser, Tesetaxel EMD-94283, A-923573, E-7107, NRC-2694, aspiramycin hydrochloride, SMT-14400, PHA-793887, HB-19, S-12363, solidodin, CEP-28122, TKS-040, ABT-306552, ATI-1025, A-928605, PF-3758309, 99mTc-RP-527, MLN-576, K-454, W-198, Debio- 0931, Zosuquida, PF-337210, ABT-737, folatetubulysin conjugate, Elacrida, CP-31398, AV-412, Ilotecan, GSK-1838705A, ABT-839, AEW-541, vincristine, gefitinib, doxorubicin, YHO-13351, pelitinib, cemadotin, A-947864, tozesetin, PD-115934, dolastatin-10, SU-11274, U.S. Suctacine, EHT-1864, dofequida, DX-52-1, RTA-502, canustatinib, metoclopramide, BMS-753493, PD-166285, auristatin PYE, Alchemix, C-1305, ANG-1009, procaine hydrochloride, sirameine, Hoe-33342, Manzamines, OSI-632, STX-1801, BIBF-1000, lutetotecan, ZK-19 1703, tiamulin, VX-322, docetaxel, Ro-28-2653, betacarin, GS-164, JNK-401, tamorizine, pibrosine, S-16020-2, NK-611, lasiroxicin, TOP-008, desclopramide, lortotecan, TAS-103, dextronidupine, BAM-1120, conophylline, ICRF-193, anhydrovinblastine, tubulysin, ectenacil, and L-745631.

Indications and treatments

It is expected that the present application antibody-drug conjugates (ADCs) can be used to treat various diseases or conditions, such as those characterized by overexpression of tumor antigens. Exemplary conditions or hyperproliferative disorders include benign or malignant solid tumors and hematological disorders such as leukemia and lymphoid malignancies. Others include neurons, glial cells, astrocytes, hypothalamus, glands, macrophages, epithelial, mesenchymal, blastocoel, inflammation, angiogenesis, and immunological (including autoimmune) diseases.

In certain embodiments, the ADCs of the present application comprising anti-NaPi2b antibodies (such as those described above) are used in a method of treating a solid tumor, such as an ovarian solid tumor.

In another embodiment, the ADCs of the present application comprising anti-CD33 antibodies (e.g., those described herein) are used in a method of treating hematological malignancies, such as non-Hodgkin lymphoma (NHL), diffuse large hematopoietic lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, acute myeloid leukemia (AML), and myeloid leukemia (MCL), including B-cell related cancers and proliferative diseases. See US8226945; Li et al. (2013) Mol. Cancer. Ther. 12(7):1255-1265; Polson et al. (2010) Leukemia 24:1566-1573; Polson et al. (2011) Expert Opin. Investig. Drugs 20(1):75-85, the contents of which are incorporated herein by reference.

In another embodiment, the ADC of the present application comprising an anti-MUC16 antibody (such as those described herein) is used in a method for treating ovarian cancer, breast cancer, and pancreatic cancer. Cancer can be associated with the expression or activity of the MUC16/CA125/O772P polypeptide. See WO2007/001851; US7989595; US8449883; US7723485; Chen et al. (2007) Cancer Res. 67(10):4924-4932; Junutula et al. (2008) Nature Biotech. 26(8):925-932, the contents of which are incorporated herein by reference.

In certain embodiments, the present application ADC comprising an anti-HER2 antibody (such as those described above) is used in a method for treating cancer, such as breast cancer or gastric cancer, more particularly HER2+ breast cancer or gastric cancer, wherein the method comprises administering the ADC to a patient in need of such treatment. In one such embodiment, the ADC comprises the anti-HER2 antibody trastuzumab or pertuzumab.

Typically, disease to be treated or illness are hyperproliferative diseases such as cancer. The example of the cancer to be treated in the present application includes but is not limited to carcinoma, lymphoma, blastoma, sarcoma and leukemia or lymphoid malignancies. The more specific example of such cancer includes squamous cell carcinoma (such as epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, stomach cancer or gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrium or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer and head and neck cancer.

Autoimmune diseases that antibody-drug conjugates can be used to treat include rheumatological diseases (e.g., rheumatoid arthritis, Sjögren's syndrome, scleroderma, lupus such as systemic lupus erythematosus (SLE) and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, antiphospholipid antibody syndrome, and psoriatic arthritis), osteoarthritis, autoimmune gastrointestinal and liver diseases (e.g., inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease), vasculitis (e.g., ANCA-associated vasculitis (including Churg-Strauss vasculitis), Wegener's granulomatosis, and polyarteritis), autoimmune neurological disorders (e.g., multiple sclerosis, opsoclonus-myoclonus syndrome, myasthenia gravis, , neuromyelitis optica, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathy), kidney diseases (e.g., glomerulonephritis, Goodpastor's syndrome, and Buerger's disease), autoimmune skin diseases (e.g., psoriasis, urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneous lupus erythematosus), blood diseases (e.g., thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases (e.g., inner ear disease and hearing loss), Behçet's disease, Raynaud's syndrome, organ transplantation, and autoimmune endocrine disorders (e.g., diabetes-related autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), Addison's disease, and autoimmune thyroid diseases (e.g., Graves' disease and thyroiditis)). More preferred such diseases include, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis.

For the prevention or treatment of disease, the appropriate dose of the ADC will depend on the type of disease to be treated, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The molecule is appropriately administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of the molecule is an initial candidate dose for administration to the patient, whether, for example, by one or more separate administrations or by continuous infusion. Depending on the above factors, a typical daily dose may be in the range of about 1 μg/kg to 100 mg/kg or more. An exemplary dose of the ADC to be administered to a patient is in the range of about 0.1 to about 10 mg/kg of patient body weight.

Experimental part

Example 1 (MC-Sq-Cit-PAB-Dolastatin 10)

[4-[[(2S)-2-[[1-[5-(2,5-dioxopyrrol-1-yl)pentylcarbamoyl]cyclobutanecarbonyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl-[(1S)-1-[[(1S)-1-[[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[[(1S)-2-phenyl-1-thiazol-2-yl-ethyl]amino]propyl]pyrrolidin-1-yl]-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-dimethyl-ammonium

In a vial, N1′-[(1S)-1-[[4-(chloromethyl)phenyl]carbamoyl]-4-ureido-butyl]-N1-[5-(2,5-dioxopyrrol-1-yl)pentyl]cyclobutane-1,1-dicarboxamide (100 mass%, 1 eq, 0.04713 mmol, 1.000, 27.76 mg), dolastatin 10 (100 mass%, 37 mg, 0.04713 mmol, 1.000, 37 mg), and tetrabutylammonium iodide (65.65 mass%, 0.3 eq, 0.01414 mmol, 0.3000, 7.956 mg) were combined and dissolved in N,N-dimethylformamide (100 mass%, 50 μL, 0.646 mmol, 13.7, 47.3 mg, 0.05 mL). The mixture was stirred at room temperature for 3 hours and no reaction was observed. To the reaction mixture was added N,N-diisopropylethylamine (100% by mass, 1.5 equivalents, 0.07069 mmol, 1.500,9.136 mg, 0.0123 mL). The reaction mixture was stirred at room temperature overnight. LC/MS of the reaction mixture showed 16% product. 1 equivalent of base was added, which improved product formation and gave a clearer LC/MS peak.

The reaction mixture was diluted with DMF and then directly purified on HPLC under formic acid conditions. A pure fraction of [4-[[(2S)-2-[[1-[5-(2,5-dioxopyrrol-1-yl)pentylcarbamoyl]cyclobutanecarbonyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl-[(1S)-1-[[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[[(1S)-2-phenyl-1 [-thiazol-2-yl-ethyl]amino]propyl]pyrrolidin-1-yl]-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-dimethyl-ammonium (100% by mass, C, 5 mg, 0.0037350 mmol, 0.07925, 5 mg) m/z = 1338.1 (7.9% yield of a pure fraction) and an impure fraction were obtained. ¹ H NMR(400MHz,DMSO-d6)δ10.47(d,J＝2.5Hz,1H),8.92(q,J＝9.0Hz,1H),8.31(s,4H), 8.05-7.91(m,0H),7.91-7.75(m,4H),7.67(s,0H),7.63(d,J＝3.2Hz,0H),7.62-7.5 4(m,0H),7.54(s,0H),7.58-7.45(m,2H),7.33-7.11(m,5H),7.08-6.95(m,2H),6.1 3(t,J＝5.6Hz,1H),5.75(s,1H),5.57-5.37(m,3H),4.76-4.55(m,3H),4.41(dt,J＝1 5.5,6.0Hz,1H),3.98(q,J＝6.3Hz,2H),3.61-3.28(m,4H),3.28-3.07(m,9H),3.07- 2.87(m,10H),2.84-2.68(m,1H),2.49-2.31(m,6H),2.31-1.90(m,0H),1.88-1.54( m,7H),1.41(ddt,J＝28.5,22.6,10.5Hz,10H),1.18(tdd,J＝19.3,7.8,4.5Hz,3H),1 .12-1.02(m,5H),1.02-0.91(m,5H),0.91-0.82(m,6H),0.79(td,J=7.4,3.0Hz,4H).

Example 2 (MC-Sq-Cit-PAB-Tubulysin M)

(2R)-2-((2S,3S)-1-(((1R,3R)-1-acetoxy-1-(4-((2R,4S)-4-carboxy-1-phenylpentan-2-ylcarbamoyl)thiazol-2-yl)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-ylcarbamoyl)-1-(4-((S)-2-(1-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentylcarbamoyl)cyclobutanecarboxamido)-5-ureidopentanamido)benzyl)-1-methylpiperidinium

In a vial, N1'-[(1S)-1-[[4-(chloromethyl)phenyl]carbamoyl]-4-ureido-butyl]-N1-[5-(2,5-dioxopyrrol-1-yl)pentyl]cyclobutane-1,1-dicarboxamide (100 mass%, 17 mg, 0.02886 mmol, 1.000, 17 mg), (2S,4R)-4-[[2-[(1R,3R)-1-acetoxy-4-methyl-3-[methyl-[(2S,3S)-3-methyl-2-[[(2R)-1-methylpiperidin [2-Carboxyl]amino]pentanoyl]amino]pentyl]thiazole-4-carbonyl]amino]-2-methyl-5-phenyl-pentanoic acid (100% by mass, 1 equivalent, 0.02886 mmol, 1.000, 21.01 mg) and tetrabutylammonium iodide (65.65% by mass, 0.3 equivalent, 0.008657 mmol, 0.3000, 4.872 mg) were dissolved in N,N-dimethylformamide (100% by mass, 75 μL, 0.970 mmol, 33.6, 70.9 mg, 0.075 mL). The resulting reaction mixture was stirred at room temperature for 1 hour, and no desired product was observed. N,N-Diisopropylethylamine (100% by mass, 1.1 equivalent, 0.03174 mmol, 1.100, 4.103 mg, 0.00554 mL) was added to the reaction mixture, and a small amount of product was formed. The reaction mixture was then stirred at room temperature overnight before an additional 1 equivalent of base was added and the mixture was stirred until the end of the day. The desired product was then observed, diluted and purified on HPLC to avoid any loss of product.

(2S,4R)-4-[[2-[(1R,3R)-1-acetoxy-3-[[(2S,3S)-2-[[(2R)-1-[[4-[[(2S)-2-[[1-[5-(2,5-dioxopyrrol-1-yl)pentylcarbamoyl]cyclobutanecarbonyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-1-methyl-piperidin-1-ium-2-carbonyl]amino]-3-methyl-pentanoyl]-methyl-amino]-4-methyl-pentyl]thiazole-4-carbonyl]amino]-2-methyl-5-phenyl-pentanoic acid (100% by mass, 11.7 mg, 0.0091292 mmol, 0.3164, 11.7 mg) was isolated. 31.64% yield. HRMS: m/z=1281.6780. ^1H NMR(500MHz,DMSO-d6)δ10.80(s,2H),9.50(s,2H),8.48(s,6H),8.36(s,3H),8.12( s,2H),8.05(s,1H),7.75(s,3H),7.33-7.26(m,4H),7.26-7.18(m,5H),7.13(d,J=7. 6Hz,5H),6.99(d,J＝5.7Hz,2H),6.42(s,2H),5.73(s,2H),5.56(s,3H),4.64(s,4H) ,4.57(t,J＝7.8Hz,2H),4.38(dq,J＝15.0,5.4Hz,2H),4.13(dd,J＝13.0,7.6Hz,2H),3 .05(q,J＝6.9,6.2Hz,12H),3.00-2.87(m,10H),2.84-2.70(m,3H),2.50(s,2H),2.4 0(hept,J＝13.1,10.5Hz,9H),2.25(d,J＝11.2Hz,1H),2.18-1.94(m,0H),1.91-1.64( m,0H),1.58(s,0H),1.42(ddt,J＝23.8,16.7,8.4Hz,15H),1.32-1.23(m,12H),1.17( p,J＝8.3,7.4Hz,6H),1.11-0.99(m,6H),0.94-0.85(m,12H),0.80(q,J＝7.6Hz,10H).

Example 3 (Nitro-MM-PDS-PAB-Tubulysin M)

(2S,4R)-4-[[2-[(1R,3R)-1-acetoxy-4-methyl-3-[methyl-[(2S,3S)-3-methyl-2-[[(2R)-1-methyl-1-[[4-[[(2R)-2-[(6-nitro-3-pyridinyl)disulfanyl]propoxy]carbonylamino]phenyl]methyl]piperidin-1-ium-2-carbonyl]amino]pentanoyl]amino]pentyl]thiazole-4-carbonyl]amino]-2-methyl-5-phenyl-pentanoic acid

Step 1: Monomethyl-nitro-PDS-PAB-Cl

Monomethyl (R)-2-((6-nitropyridin-3-yl)disulfanyl)propyl 4-(chloromethyl)phenylcarbamate

Monomethyl (R)-2-((6-nitropyridin-3-yl)disulfanyl)propyl 4-(hydroxymethyl)phenylcarbamate (-nitro-PDS-PAB-OH) (100% by mass, 50 mg, 0.1265 mmol, 1.000, 50 mg) was dissolved in N,N-dimethylformamide (100% by mass, 0.2 mL, 3 mmol, 20,200 mg, 0.2 mL) in a vial. The resulting reaction mixture was cooled in an ice bath and a solution of thionyl chloride (100% by mass, 1.2 equivalents, 0.1517 mmol, 1.200, 18.05 mg, 0.01106 mL) in dichloromethane (100% by mass, 50 μL, 0.7800 mmol, 6.16 g, 66.25 mg, 0.05 mL) was added dropwise. The reaction mixture was removed from the ice bath and stirred at room temperature for 30 minutes. LCMS showed the desired product. One additional equivalent of thionyl chloride (solution in DCM) was added dropwise to the reaction mixture and stirred at room temperature overnight. A new LCMS peak was formed, which was close to the product peak and had a mass of 438. The reaction mixture was diluted with ethyl acetate, quenched with water, and the organic layer was washed twice with water, dried over MgSO ₄ and concentrated on silica gel and purified by flash chromatography (eluted with 30% EtOAc/heptane). A white solid was isolated. LC/MS showed a substance with an alcohol precursor as an impurity. Monomethyl (R)-2-((6-nitropyridin-3-yl)disulfanyl)propyl 4-(chloromethyl)phenylcarbamate (-nitro-PDS-PAB-Cl) (100% by mass, B, 38 mg, 0.09181 mmol, 0.7260, 38 mg) (72.6% yield). m/z=414.00.

In a vial, [(2R)-2-[(6-nitro-3-pyridyl)disulfanyl]propyl] N-[4-(chloromethyl)phenyl]carbamate (100 mass%) and (2S,4R)-4-[[2-[(1R,3R)-1-acetoxy-4-methyl-3-[methyl-[(2S,3S)-3-methyl-2-[[(2R)-1-methylpiperidine-2-carbonyl]amino]pentanoyl]amino]pentyl]thiazole-4-carbonyl]amino]-2-methyl-5-phenyl-pentanoic acid (100 mass%, 9.6 mg, 0.013 mmol, 1.0, 9.6 mg) were dissolved in N,N-dimethylformamide (100 mass%). Tetrabutylammonium iodide (65.65% by mass, 0.2 equivalents, 0.0026 mmol, 0.20, 1.5 mg) was added to the reaction mixture, followed by N,N-diisopropylethylamine (100% by mass, 1 equivalent, 0.013 mmol, 1.0, 1.7 mg, 0.0023 mL). The reaction mixture was warmed to 38°C for 20 minutes and then stirred at room temperature for 2 hours. LC/MS showed the desired product. The reaction was stopped at about halfway to avoid decomposition, diluted with DMF and purified directly on HPLC under acidic conditions. (2S,4R)-4-[[2-[(1R,3R)-1-acetoxy-4-methyl-3-[methyl-[(2S,3S)-3-methyl-2-[[(2R)-1-methyl-1-[[4-[[(2R)-2-[(6-nitro-3-pyridyl)disulfanyl]propoxy]carbonylamino]phenyl]methyl]piperidin-1-ium-2-carbonyl]amino]pentanoyl]amino]pentyl]thiazole-4-carbonyl]amino]-2-methyl-5-phenyl-pentanoic acid was isolated. (100 mass%, 4.6 mg, 0.0041576 mmol, 0.32, 4.6 mg) (32% yield), m/z = 1106.4.

Example 4 (MC-Sq-Cit-PAB Duocarmycin DM)

Preparation of 2-[[2-[(1S)-1-(chloromethyl)-5-hydroxy-1,2-dihydrobenz[e]indole-3-carbonyl]-1H-indol-5-yl]oxy]ethyl-[[4-[[(2S)-2-[[1-[5-(2,5-dioxopyrrol-1-yl)pentylcarbamoyl]cyclobutanecarbonyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-dimethyl-ammonium

Step 1: (S)-N-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)-N-(1-(4-(hydroxymethyl)phenylamino)-1-oxo-5-ureidopentan-2-yl)cyclobutane-1,1-dicarboxamide

plan

program

Compound 1 (150 g, 1.53 mol) was added to a stirred solution of compound 2 (201 g, 1.53 mol) in HOAc (1000 _mL ). The mixture was stirred at room temperature for 2 h and then heated to reflux for 8 h. The organic solvent was removed under reduced pressure, and the residue was extracted with EtOAc (500 _mL x 3) and washed with H₂O. The combined organic layers were dried over _Na₂SO₄ and concentrated to afford the product. The resulting product was washed with petroleum ether to afford compound 3 as a white solid (250 g, 77.4%).

DPPA (130 g, 473 mmol) and TEA (47.9 g, 473 mmol) were added to a solution of compound 3 (100 g, 473 mmol) in t-BuOH (200 mL). The mixture was heated at reflux under _N₂ for 8 h. The mixture was concentrated and the residue was purified by silica gel column chromatography (PE:EtOAc = 3:1) to yield compound 4 (13 g, 10%).

To a solution of compound 4 (28 g, 992 mmol) in anhydrous EtOAc (30 mL) was added HCl/EtOAc (50 mL) dropwise. The mixture was stirred at room temperature for 5 h, filtered, and the solid dried to afford compound 5 (16 g, 73.7%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.02 (s, 2H), 6.99 (s, 2H), 3.37-3.34 (m, 2H), 2.71-2.64 (m, 2H), 1.56-1.43 (m, 4H), 1.23-1.20 (m, 2H).

To a mixture of compound 6 (17.50 g, 0.10 mol) in a mixture of dioxane and H ₂ O (50 mL/75 mL) was added K ₂ CO ₃ (34.55 g, 0.25 mol). Fmoc-Cl (30.96 g, 0.12 mol) was slowly added at 0°C. The reaction mixture was allowed to warm to room temperature over 2 h. The organic solvent was removed under reduced pressure, and the aqueous slurry was adjusted to pH 3 with 6 M HCl solution and extracted with EtOAc (100 mL x 3). The organic layer was dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to yield the desired product 7 (38.0 g, 95.6%). (Compound 7 was later commercially available.)

To a solution of compound 7 (4 g, 10 mmol) in a mixture of DCM and MeOH (100 mL/50 mL) was added 4-amino-phenyl-methanol (8) (1.6 g, 13 mmol, 1.3 equiv) and EEDQ (3.2 g, 13 mmol, 1.3 equiv). After the mixture was stirred at room temperature under N ₂ for 16 h, it was concentrated to give a brown solid. MTBE (200 mL) was added and stirred at 15 ° C for 2 h. The solid was collected by filtration and washed with MTBE (50 mL × 2) to give the crude product 9 as an orange solid (4.2 g, 84%).

LCMS (ESI): m/z 503.0 [M+1].

To a stirred solution of compound 9 (4.2 g, 8.3 mmol) in anhydrous DMF (20 ml) was added piperidine (1.65 mL, 17 mmol, 2 equivalents) dropwise at room temperature. The mixture was stirred at room temperature for 30 min, and a solid precipitate formed. Anhydrous DCM (50 mL) was added, and the mixture immediately became transparent. The mixture was stirred at room temperature for an additional 30 min, and LCMS showed that compound 9 was consumed. It was concentrated to dryness under reduced pressure (ensuring no piperidine remained), and the residue was partitioned between EtOAc and H ₂ O (50 mL/20 mL). The aqueous phase was washed with EtOAc (50 mL x 2) and concentrated to give 10 as an oily residue (2.2 g, 94%) (containing a small amount of DMF).

To a solution of compound 11 (8 g, 29.7 mmol) in DME (50 mL) was added a solution of compound 10 (6.0 g, 21.4 mmol) and NaHCO ₃ (7.48 g, 89.0 mmol) in water (30 mL). The mixture was stirred at room temperature for 16 h, then concentrated to dryness under reduced pressure and the residue was purified by column chromatography (DCM:MeOH=10:1) to give crude compound 12 as a white solid (6.4 g, 68.7%).

LCMS (ESI): m/z 435.0 [M+1].

To a stirred solution of compound 12 (6.4 g, 14.7 mmol) in a mixture of THF and MeOH (20 mL/10 mL) was added a solution of LiOH·H ₂ O (1.2 g, 28.6 mmol) in H ₂ O (20 mL) at room temperature. The reaction mixture was stirred at room temperature for 16 h, the solvent was removed under reduced pressure, and the resulting residue was purified by preparative HPLC to give compound 13 (3.5 g, yield: 58.5%).

LCMS (ESI): m/z 406.9 [M+1]. ¹ H NMR (400 MHz, Methanol-d ₄ ) δ 8.86 (d, J=8.4 Hz, 2H), 8.51 (d, J=8.4 Hz, 2H), 5.88-5.85 (m, 1H), 5.78 (s, 2H), 4.54-4.49 (m, 3H), 4.38-4.32 (m, 1H), 3.86-3.75 (m, 1H), 3.84-3.80 (m, 2H), 3.28-3.21 (m, 1H), 3.30-3.24 (m, 1H), 3.00-2.80 (m, 1H), 2.37-2.28 (m, 2H).

DIPEA (1.59 g, 12.3 mmol) and BOP-Cl (692 mg, 2.71 mmol) were added to a solution of compound 13 (1.0 g, 2.46 mmol) in DMF (10 mL) at 0°C, followed by compound 5 (592 mg, 2.71 mmol). The mixture was stirred at 0°C for 0.5 h. The reaction mixture was quenched with citric acid solution (10 mL) and extracted with DCM/MeOH (10:1). The organic layer was dried and concentrated, and the residue was purified by silica gel column chromatography (DCM:MeOH = 10:1) to give compound 14 (1.0 g, 71%).

¹ H NMR (400MHz, DMSO-d ₆ ): δ10.00(s,1H),7.82-7.77(m,2H),7.53(d,J＝8.4Hz,2H),7.19(d,J＝8.4Hz,2H),6.96 (s,2H),5.95(t,J＝6.4Hz,1H),5.39(s,2H),5.08(t,J＝5.6Hz,1H),4.40-4.35(m,3H),4. 09(d,J＝4.8Hz,1H),3.01(d,J＝3.2Hz,2H),3.05-2.72(m,4H),2.68-2.58(m,3H),2.40-2 .36(m,4H),1.72-1.70(m,3H),1.44-1.42(m,1H),1.40-1.23(m,6H),1.21-1.16(m,4H).

Step 2: (S)-N-(1-(4-(chloromethyl)phenylamino)-1-oxo-5-ureidopentan-2-yl)-1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoylamino)cyclobutanecarboxamide

To a solution of compound 14 (2.0 g, 3.5 mmol) in NMP (50 mL) was added _SOCl₂ (1.25 g, 10.5 mmol) dropwise at 0°C. The reaction mixture was stirred at 20°C for 30 min, then diluted with water (50 mL) and extracted with EtOAc (50 mL x 3). The organic layer was dried, concentrated, and purified by flash column chromatography (DCM:MeOH = 20:1) to afford product 15 (1.0 g, 48.4%) as a gray solid. LCMS: (5-95, AB, 1.5 min), 0.696 min, m/z = 589.0 [M+1] ⁺ .

Step 3: 2-[[2-[(1S)-1-(Chloromethyl)-5-hydroxy-1,2-dihydrobenz[e]indole-3-carbonyl]-1H-indol-5-yl]oxy]ethyl-[[4-[[(2S)-2-[[1-[5-(2,5-dioxopyrrol-1-yl)pentylcarbamoyl]cyclobutanecarbonyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-dimethyl-ammonium

N1'-[(1S)-1-[[4-(Chloromethyl)phenyl]carbamoyl]-4-ureido-butyl]-N1-[5-(2,5-dioxopyrrol-1-yl)pentyl]cyclobutane-1,1-dicarboxamide 15 (100 mass%, 1 eq, 0.04310 mmol, 1.000, 25.39 mg) and [(1S)-1-(chloromethyl)-5-hydroxy-1,2-dihydrobenz[e]indol-3-yl]-[5-(2-dimethylaminoethyloxy)-1H-indol-2-yl]methanone (100 mass%, 20 mg, 0.04310 mmol, 1.000, 20 mg) were combined in N,N-dimethylformamide (100 mass%) and the resulting mixture was stirred for several days. Next, tetrabutylammonium iodide (65.65% by mass, 0.3 equivalents, 0.01293 mmol, 0.3000, 7.276 mg) was added and the mixture was stirred overnight. No exchange of iodide and chloride was observed. N,N-diisopropylethylamine (100% by mass, 1.2 equivalents, 0.05172 mmol, 1.200, 6.685 mg, 0.00902 mL) was then added and the mixture was stirred for 4 hours. The reaction mixture contained a main peak on LCMS, which corresponded to the desired product. The reaction mixture was then diluted with DMF and directly purified and isolated on HPLC under acidic conditions. Concentrating the fractions at room temperature degraded some of the material. Therefore, the material was purified again. 2-[[2-[(1S)-1-(chloromethyl)-5-hydroxy-1,2-dihydrobenz[e]indole-3-carbonyl]-1H-indol-5-yl]oxy]ethyl-[[4-[[(2S)-2-[[1-[5-(2,5-dioxopyrrol-1-yl)pentylcarbamoyl]cyclobutanecarbonyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-dimethyl-ammonium (100 mass%, 11.8 mg, 0.0116 mmol, 0.269, 11.8 mg). 26.9% yield. m/z = 1017.35

Example 5: MC-Sq-Cit-PAB-Vinblastine

(5S,7S,9S)-9-((3aR,3a1R,4R,5S,5aR,10bR)-4-acetoxy-3a-ethyl-5-hydroxy-8-methoxy-5-(methoxycarbonyl)-6-methyl-3a,3a1,4,5,5a,6,11,12-octahydro-1H-indolizino[8,1-cd]carbazol-9-yl)-3-(4-((S)-2- (1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutanecarboxamido)-5-ureidopentanamido)benzyl)-5-ethyl-5-hydroxy-9-(methoxycarbonyl)-1,2,3,4,5,6,7,8,9,10-decahydro-3,7-methano[1]azacycloundeca[5,4-b]indol-3-ium

To a vial was added N1'-[(1S)-1-[[4-(chloromethyl)phenyl]carbamoyl]-4-ureido-butyl]-N1-[5-(2,5-dioxopyrrol-1-yl)pentyl]cyclobutane-1,1-dicarboxamide (100 mass%, 1 eq, 0.04400 mmol, 1.000, 25.92 mg) and vinblastine (89.21 mass%, B, 40 mg, 0.04400 mmol, 1.000, 40 mg) in N,N-dimethylformamide (100 mass%, 100 μL, 1.29 mmol, 29.4, 94.5 mg, 0.1 mL). To this mixture were added tetrabutylammonium iodide (65.65 mass%, 0.3 equivalents, 0.01320 mmol, 0.3000, 7.428 mg) and N,N-diisopropylethylamine (100 mass%, 1 equivalent, 0.04400 mmol, 1.000, 5.687 mg, 0.00767 mL) and the mixture was stirred for several days. 1 equivalent of base was added to further drive the reaction. The reaction mixture was diluted with DMF and directly purified on HPLC under acidic conditions. One pure fraction was isolated to give the final product (100 mass%, 8.1 mg, 0.0059 mmol, 0.13, 8.1 mg) m/z = 1364.85 (13% yield). Several impure fractions were also isolated. ¹ H NMR (500MHz, DMSO-d6) δ10.58(s,1H),9.84(s,1H),8.78(s,1H),8.43(s,2H),8.11(d,J＝7.9Hz,1H),7.91( t,J＝5.6Hz,1H),7.74(d,J＝8.1Hz,2H),7.66(d,J＝8.1Hz,1H),7.47(d,J＝8.1Hz,2H),7.35(d,J＝8.0Hz,1H), 7.14-6.99(m,2H),6.98(d,J＝2.4Hz,1H),6.57(s,1H),6.41(s,1H),6.31(t,J＝6.6Hz,1H),5.80(dd,J＝10. 5,4.9Hz,1H),5.54(s,2H),5.28(d,J＝10.1Hz,1H),5.13(s,2H),4.60(d,J＝12.4Hz,2H),4.40(dt,J＝17.3,6 .1Hz,2H),4.18-4.07(m,2H),3.78(s,3H),3.62(t,J＝14.1Hz,6H),3.36(t,J＝7.0Hz,2H),3.20(dq,J＝29.0 ,8.5,6.3Hz,2H),3.07(q,J＝6.6Hz,2H),2.98(q,J＝6.4Hz,2H),2.93-2.86(m,1H),2.83-2.71(m,2H),2.67( s,3H),2.48-2.33(m,5H),2.30(d,J＝14.8Hz,1H),2.12-2.04(m,1H),1.97(s,3H),1.70(ddtd,J＝44.3,19.2 ,9.5,8.5,4.4Hz,4H),1.56-1.31(m,14H),1.26-1.14(m,3H),0.81(t,J＝7.4Hz,4H),0.60(t,J＝7.3Hz,3H).

Examples 6 and 7

Example 6: MC-Sq-Cit-PAB-morpholine-linked-gefitinib

(S)-4-(3-(4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)propyl)-4-(4-(2-(1-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentylcarbamoyl)cyclobutanecarboxamido)-5-ureidopentanamido)benzyl)morpholin-4-ium

Example 7: MC-Sq-Cit-PAB-Pyrimidine-linked-Gefitinib

(S)-4-(3-chloro-4-fluorophenylamino)-1-(4-(2-(1-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentylcarbamoyl)cyclobutanecarboxamido)-5-ureidopentanamido)benzyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-1-ium

Solution 1

Scheme 2. Synthesis of intermediates

A solution of compound 1 (113.88 mg, 0.20 mmol) and compound 2 (100 mg, 0.22 mmol) in DMF (3.0 mL) was stirred at 23 ° C for 18.0 h. The reaction mixture was concentrated and purified by silica gel chromatography (10-20% MeOH/DCM) to give compound 3A (9 mg, 4.3% yield) as a yellow solid and compound 3 (8 mg, 3.5% yield) as a white solid. ¹ H of 3a NMR: (400MHz, DMSO-d6) δ10.10(s,1H),8.31(s,1H),8.17(s,1H),7.87-7.66(d,J＝8.8Hz ,2H),7.69-7.58(m,6H),7.39-7.02(m,8H),6.86(s,1H),6.0(s,1H),5.43(s,2H),5.33(s ,2H),4.25-4.12(m,6H),3.74(s,4H),3.53-3.51(m,5H),2.99-2.92(m,2H),2.40-2.32( m,5H),1.89-1.85(m,2H),1.60-1.36(m,4H); LCMS(5-95AB/1.5min): RT＝0.673min,[M+H] ^+931.3 . 3 of ¹ H NMR: (DMSO, 400MHz) δ: 10.59 (s, 1H), 10.05 (s, 1H), 8.50 (s, 2H), 8.46 (s, 1H), 8.18-8.16 (d ,J＝8.0Hz,1H),8.00(s,1H),7.88-7.71(m,7H),7.514-7.24(m,8H),6.29(s,1H),5.54(s,2H ),4.69(s,2H),4.30-4.14(m,6H),3.97-3.95(m,8H),3.57-3.51(m,6H),2.98-2.97(d,J＝5. 6Hz, 2H), 1.68-1.60 (m, 2H), 1.47-1.40 (m, 2H); LCMS (5-95AB/1.5min): RT＝0.662min, [M+H] ^+931.3 .

To a solution of compound 3 (50.0 mg, 0.050 mmol) in DMF (1.5 mL) was added piperidine (9.13 mg, 0.110 mmol) and the mixture was stirred at 23 ° C for 3.0 h. LCMS showed 50.6% product. The mixture was concentrated and the residue was washed twice with MTBE (6.0 mL) to give compound 4 (31 mg, 0.044 mmol, 81.4%) as a white solid and used directly. LCMS (5-95AB/1.5 min): RT = 0.553 min, [M+H] ⁺ 709.2.

To a solution of compound 4 (31.0 mg, 0.040 mmol) in DMF (3.0 mL) was added compound 5 (41.4 mg, 0.090 mmol) and DIEA (11.3 mg, 0.090 mmol). After the mixture was stirred at 23°C for 18.0 h, LCMS showed 52% of the product. The residue was purified by preparative HPLC (FA, acetonitrile 20%-50%) to give (S)-4-(3-(4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)propyl)-4-(4-(2-(1-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentylcarbamoyl)cyclobutanecarboxamido)-5-ureidopentanamido)benzyl)morpholin-4-ium, Example 6 (10.4 mg, 23.6% yield), as a white solid. LCMS (5-95AB/1.5 min): RT = 0.736 min, [M+H] ⁺ 1001.1.

To a solution of compound 3A (110.0 mg, 0.120 mmol) in DMF (2.5 mL) was added piperidine (20.1 mg, 0.240 mmol). The mixture was stirred at 23 ° C for 3.0 h. LCMS showed 50.7% product. The mixture was concentrated to remove DMF and washed twice with MTBE (10.0 mL) to give crude compound 4A (85 mg, 100% yield) as a yellow solid which was used directly in the next step.

To a solution of compound 4A (40 mg, 0.060 mmol) in DMF (3 mL) were added compound 5 (53.4 mg, 0.110 mmol) and DIEA (14.6 mg, 0.113 mmol). The mixture was stirred at 23°C for 18.0 h. The resulting residue was purified by preparative HPLC (FA, acetonitrile 20%-50%) to afford (S)-4-(3-chloro-4-fluorophenylamino)-1-(4-(2-(1-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentylcarbamoyl)cyclobutanecarboxamido)-5-ureidopentanamido)benzyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-1-ium, Example 7 (10.3 mg, 17.9% yield), as a yellow solid. LCMS (5-95AB/1.5min): RT=0.740min, [M] ⁺ 1001.6.

To a flask containing compound 6 (1000 mg, 1.99 mmol) was added HBr/HOAc solution (4.0 mL) dropwise at 0 ° C. It was stirred at 25 ° C for 3.0 h. LCMS (5-95AB/1.5min) showed 90.2% product. The mixture was concentrated, EtOAc (300 mL) was added and concentrated to remove a small amount of acid, and the residue was then poured into ice-water (50.0 mL) and the solid was collected by filtration to give compound 2 (726 mg, 41.3% yield) as a gray solid. LCMS (5-95AB/1.5min): RT = 0.772 min, [M+H] ⁺ 567.0.

To a solution of compound 7 (200 mg, 0.65 mmol) in DCM (12 mL) were added compound 8 (179 mg, 0.97 mmol) and DIC (123 mg, 0.97 mmol), and the mixture was stirred at 23 ° C for 1.5 h. LCMS showed 70.9% product. The mixture was concentrated and washed twice with THF (2.0 mL) to give compound 5 (120 mg, 0.263 mmol, 40.5% yield) as a colorless oil that was used directly in the next step.

Methods for preparing ADCs

Preparation of cysteine-engineered antibodies for conjugation by reduction and reoxidation

Under certain conditions, cysteine engineered antibodies can be made reactive for conjugation with the present linker-drug intermediates by treatment with a reducing agent such as DTT (Cleland's reagent, dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine hydrochloride; Getz et al. (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, MA). Full-length cysteine engineered monoclonal antibodies (ThioMabs) expressed in CHO cells (Gomez et al. (2010) Biotechnology and Bioeng. 105(4):748-760; Gomez et al. (2010) Biotechnol. Prog. 26:1438-1445) are reduced (e.g., overnight at room temperature with approximately a 50-fold excess of DTT) to reduce disulfide bonds that can form between newly introduced cysteine residues and cysteines present in the culture medium.

Light chain amino acids are numbered according to Kabat (Kabat et al., Sequences of proteins of immunological interest, (1991) 5th ed., U.S. Department of Health and Human Services, National Institutes of Health, Bethesda, MD). Heavy chain amino acids are numbered according to the EU numbering system (Edelman et al. (1969) Proc. Natl. Acad. of Sci. 63(1):78-85), except where the Kabat system is noted. Single-letter amino acid abbreviations are used.

Full-length cysteine-engineered monoclonal antibodies (ThioMab) expressed in CHO cells have cysteine adducts (cystine) or glutathionylation on the engineered cysteines due to cell culture conditions. To release the reactive sulfhydryl groups of the engineered cysteines, ThioMab was dissolved in 500 mM sodium borate and 500 mM sodium chloride at approximately pH 8.0 and reduced with approximately 50-100-fold excess of 1 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride (Getz et al. (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, MA) at 37° C. for approximately 1-2 hours. Alternatively, DTT can be used as a reducing agent. Interchain disulfide bond formation was monitored by non-reducing SDS-PAGE or by denaturing reversed-phase HPLC PLRP column chromatography. The reduced ThioMab was diluted and loaded onto a HiTrap SP FF column in 10 mM sodium acetate (pH 5) and eluted with PBS containing 0.3 M sodium chloride or 50 mM Tris-Cl (pH 7.5) containing 150 mM sodium chloride.

Disulfide bonds are reestablished between cysteine residues present in the parent Mab by reoxidation. The eluted reduced ThioMab is treated with 15× or 2 mM dehydroascorbic acid (dhAA) at pH 7 for 3 hours or in 50 mM Tris-Cl (pH 7.5) or with a 2 mM copper sulfate (CuSO ₄ ) aqueous solution at room temperature overnight. Other oxidants and oxidation conditions known in the art can be used. Ambient air oxidation can also be effective. This mild partial reoxidation step effectively forms intrachain disulfides with high accuracy. The buffer is exchanged by elution on Sephadex G25 resin and eluted with PBS with 1 mM DTPA. The sulfhydryl/Ab value is checked by measuring the reduced antibody concentration by absorbance of the solution at 280 nm and by measuring the sulfhydryl concentration by reacting with DTNB (Aldrich, Milwaukee, WI) and measuring the absorbance at 412 nm.

Liquid chromatography/mass spectrometry analysis was performed on a TSQ Quantum Triple quadrupole ^™ mass spectrometer (ThermoElectron, San Jose California) with an extended mass range. Samples were chromatographed on a PRLP-1000A microporous column (50 mm × 2.1 mm, Polymer Laboratories, Shropshire, UK) heated to 75°C. A linear gradient of 30-40% B (solvent A: 0.05% TFA in water, solvent B: 0.04% TFA in acetonitrile) was used and the eluent was directly ionized using an electrospray source. Data were collected by a data system and deconvoluted using (Novatia, LLC, New Jersey). Prior to LC/MS analysis, the antibody or drug conjugate (50 micrograms) was treated with PNGase F (2 units/ml; Prozyme, San Leandro, CA) at 37°C for 2 hours to remove N-linked carbohydrates.

Hydrophobic interaction chromatography (HIC) samples were injected onto a Butyl HIC NPR column (2.5 μm particle size, 4.6 mm × 3.5 cm) (Tosoh Bioscience) and eluted at 0.8 ml/min with a linear gradient from 0-70% B (A: 1.5 M ammonium sulfate in 50 mM potassium phosphate (pH 7), B: 50 mM potassium phosphate (pH 7), 20% isopropanol). An Agilent 1100 series HPLC system equipped with a multi-wavelength detector and Chemstation software was used to resolve and quantify antibody species with varying drug/antibody ratios. The cysteine-engineered antibodies of this application can be prepared according to the general method described above.

Conjugation of Linker-Drug Intermediate to Antibody (Procedure 1)

When expressed in CHO cells, the engineered antibody cysteine is blocked as a mixed disulfide with glutathione and/or cysteine. These cysteines must be "deblocked" before conjugation. The antibody (5-12 mg/mL) to be deblocked in 20 mM succinate, 150 mM NaCl, 2 mM EDTA is added to 75-100 mM Tris (pH 7.5-8) (using 1 M Tris). Cosolvent (DMSO, DMF or DMA) is added to the antibody solution, followed by addition of a linker-drug (in DMSO or DMF) to a final concentration of 10-13% organic solvent and 2.5-10 × linker-drug relative to antibody concentration. The reaction is carried out at room temperature for 1-12 hours (until maximum conjugation is reached). The conjugation reaction mixture is purified by cation exchange chromatography and/or gel filtration using a disposable column (Smaxi or Zeba, respectively). If the crude conjugate is significantly aggregated (e.g., >10%) according to analytical SEC, additional purification is performed by preparative gel filtration (S200 column). The conjugate is then exchanged into formulation buffer (20 mM His-acetate, pH 5.5, 240 mM sucrose) using gel filtration or dialysis. Tween-20 is then added to the purified conjugate to a final concentration of 0.02%. The final conjugate concentration range is 2.4-7.5 mg/mL (the % yield of the antibody from the deblocking is 34-81%). The conjugate is analyzed by LCMS to obtain a measured value of the drug-antibody ratio (DAR), which ranges from 1.3-2.1 (average value of 1.8). The presence of high molecular weight aggregates of the conjugate is also analyzed using analytical SEC (Zenix or Shodex column); the purified final conjugate shows 0-10% aggregation. The conjugate is also evaluated for endotoxin contamination, which does not exceed 1.3 EU/mg in all cases. Free unconjugated drug did not exceed 1% of the final conjugate.

Conjugation of Linker-Drug Intermediate to Antibody (Method 2, Alternative Method)

After the reduction and reoxidation procedures of the above-described embodiments, the antibody is dissolved in PBS (phosphate buffered saline) buffer and cooled on ice. The excessive linker-drug intermediate with sulfhydryl reactive functional groups such as maleimido or bromoacetamide of about 1.5 molar concentration to 20 equivalents is dissolved in DMSO, diluted in acetonitrile and water and added to the reoxidized antibody of the cooling reduction in PBS. After about 1 hour, excessive maleimide is added to quench the reaction and seal any unreacted antibody sulfhydryl groups. The conjugate mixture can be loaded and eluted to remove excessive drug-linker intermediate and other impurities by HiTrap SP FF post. The reaction mixture is concentrated by centrifugal ultrafiltration and purified and desalted by eluting with G25 resin in PBS by cysteine engineering antibody drug conjugates, filtered and frozen by 0.2 μm filter under aseptic conditions.

The ADC of the present application can be prepared according to the procedures described in the above sections.

Determination

Selected linkers were then tested and found to be active in in vitro and in vivo assays. The cleavage data are shown in the table below.

Cathepsin B cleavage assay

As with peptide linkers, non-peptide linkers for ADCs are expected to be cleavable in the lysosome for proper drug release. Lysosomes, as the digestive organelles of cells, are rich in proteases that exhibit optimal hydrolytic activity at acidic pH. Cathepsin B is a representative lysosomal protease and has been shown to contribute to the activation of ADC peptide linkers. As an initial screen, purified cathepsin B was used to develop an assay for identifying cleavable linker-drug constructs suitable for antibody conjugation. Norfloxacin was used to represent the drug component of the linker-drug. The percentage of cleavage relative to a control peptide (e.g., Val-Cit) and the kinetic parameters ( _Km and _Vmax ) of the cleavage reaction were measured at a given time point. A detailed description of the assay is shown below. This assay allows identification of a variety of structurally distinct linkers with proteolytic activity and subsequent use in the preparation of ADCs.

The cathepsin B cleavage activity of substrate using experimental linker-drug is measured by using LC/MS to monitor the release of norfloxacin.By the linker-drug (3 times of serial dilutions) of different concentrations containing 20nM cathepsin B (EMD Millipore catalog number (Cat. No.) 219364, human liver), 10mM MES pH 6.0,1mM DTT, 0.03% CHAPS and 25nM norfloxacin-d5 internal standard (Santa Cruz Biotechnology, catalog number (Cat. No.) sc-301482) in 20 μ L reaction mixtures incubated.By reaction mixture 37 DEG C of incubations 1 hour, then add 60 μ L 2% formic acid to react with quenching.By the reaction mixture that stops by injecting 2 μ L on WatersAcquity UPLC BEH Phenyl post (2.1mm × 50mm, Waters catalog number (Cat. No.) 186002884) come analytical sample. The sample was purified on a Water Acquity UPLC using a 2-minute linear gradient (0% to 80%) of acetonitrile and 0.1% formic acid. Norfloxacin and the norfloxacin-d5 internal standard (norfloxacin 320→233 m/z, norfloxacin-d5 325→233 m/z) were detected using an AB Sciex QTrap 5500 triple quadrupole mass spectrometer operating in positive MRM mode. Quantified norfloxacin (normalized to the internal standard) was plotted against linker-drug concentration, and the resulting plot was curve-fitted using GraphPad Prism software using a Michaelis-Menten fit to obtain the kinetic constants _Km and _Vmax .

In vitro cell proliferation assay

The potency of the ADC was measured by a cell proliferation assay (CELLTITERGLO ^™ Luminescent Cell Viability Assay, Promega Corp. Technical Bulletin TB288; Mendoza et al. (2002) Cancer Res. 62:5485-5488) using the following protocol:

1. Deposit a 100 μl aliquot of cell culture containing approximately 10 ⁴ cells (SKBR-3, BT474, MCF7, or MDA-MB-468) in culture medium into each well of a 96-well plate with opaque walls.

2. Prepare control wells containing culture medium and no cells.

3. Add ADC to the experimental wells and incubate for 3-5 days.

4. Equilibrate the plate to room temperature for approximately 30 minutes.

5. Add a volume of CELLTITER GLO ^™ reagent equal to the volume of cell culture medium present in each well.

6. Mix the contents on an orbital shaker for 2 minutes to induce cell lysis.

7. Incubate the plate at room temperature for 10 minutes to stabilize the luminescent signal.

8. Luminescence was recorded and reported in the graphs as RLU = relative luminescence units.

Data are plotted as mean luminescence + standard deviation error bars for each replicate. The protocol is a modification of CELLTITERGLO ^™ luminescent cell culture medium: SK-BR-3 are grown in 50/50/10% FBS/glutamine/250 μg/mL G-418, and OVCAR-3 are grown in RPMI/20% FBS/glutamine.

In vivo assay

1. The efficacy of an anti-Napi2B antibody-drug conjugate (ADC) was investigated in a mouse xenograft model of Igrov-1 (human ovarian cancer).

Female CB-17SCID beige mice (Charles River Laboratories; San Diego, CA) were each inoculated with 5 million Igrov-1 cells in the chest mammary fat pad area. When xenograft tumors reached an average tumor volume of 100-300 mm ³ (referred to as day 0), the animals were randomly divided into groups of 7-10 mice per group and received a single intravenous injection of ADC. The tumors and body weights of the mice were measured 1-2 times per week throughout the study. When the weight loss was >20% of its initial body weight, the mice were immediately euthanized. All animals were euthanized before the tumor reached 3000 mm ³ or showed signs of impending ulceration.

2. The efficacy of anti-Napi2B antibody-drug conjugates (ADCs) was investigated in the OVCAR3-X2.1 (human ovarian cancer) mouse xenograft model. The OVCAR3 cell line was obtained from ATCC (American Type Culture Collection; Manassas, VA) and the subline OVCAR3-X2.1 was generated at Genentech for optimal growth in mice.

Female CB-17SCID beige mice (Charles River Laboratories; San Diego, CA) were each inoculated with 10,000,000 OVCAR3-X2.1 cells in the chest mammary fat pad area. When the xenograft tumor reached an average tumor volume of 100-300mm ³ (referred to as Day 0), the animals were randomly divided into groups of 7-10 mice per group and received a single intravenous injection of ADC. The tumor and body weight of the mice were measured 1-2 times per week during the entire study. When weight loss was >20% of its initial body weight, the mice were euthanized immediately. All animals were euthanized before the tumor reached 3000mm ³ or showed signs of ulceration.

3. The efficacy of anti-CD22 antibody-drug conjugates (ADCs) was investigated in mouse xenograft models of BJAB-luc (human Burkitt's lymphoma) or WSU-DLCL2 (human diffuse large B-cell lymphoma). The BJAB cell line was obtained from DSMZ (German Collection of Microorganisms and Cell Cultures; Braunschweig, Germany) and a subline, BJAB-luc, was generated at Genentech to stably express the luciferase gene. The WSU-DLCL2 cell line was also obtained from DSMZ.

Female CB-17 SCID mice (Charles River Laboratories; San Diego, CA) were each inoculated subcutaneously with 20 million BJAB-luc or WSU-DLCL2 cells in the flank area. When xenograft tumors reached an average tumor volume of 100-300 mm ³ (referred to as day 0), the animals were randomly divided into groups of 7-10 mice per group and received a single intravenous injection of ADC. The tumors and body weights of the mice were measured 1-2 times per week throughout the study. When weight loss was >20% of its initial body weight, the mice were immediately euthanized. All animals were euthanized before the tumor reached 3000 mm ³ or showed signs of impending ulceration.

4. The efficacy of anti-CD22 antibody-drug conjugate (ADC) was investigated in a mouse xenograft model of WSU-DLCL2 (human diffuse large B-cell lymphoma). The WSU-DLCL2 cell line was obtained from DSMZ (German Collection of Microorganisms and Cell Cultures; Braunschweig, Germany).

Female CB-17 SCID mice (Charles River Laboratories; San Diego, CA) were each inoculated subcutaneously with 20 million WSU-DLCL2 cells in the flank area. When xenograft tumors reached an average tumor volume of 100-300 mm ³ (referred to as Day 0), the animals were randomly divided into groups of 7-10 mice per group and received a single intravenous injection of ADC. The tumors and body weights of the mice were measured 1-2 times per week throughout the study. When weight loss was >20% of its initial body weight, the mice were immediately euthanized. All animals were euthanized before the tumors reached 3000 mm ³ or showed signs of impending ulceration.

Biological data

ADC linker-drug structure

sequence

NaPi2b humanized antibody:

In one embodiment, the Napi2b antibody of the ADC of the present application comprises three light chain hypervariable regions and three heavy chain hypervariable regions (SEQ ID NOs: 1-6), the sequences of which are shown below.

In one embodiment, the Napi2b antibody of the ADC of the present application comprises a variable light chain sequence of SEQ ID NO: 7 and a variable heavy chain sequence of SEQ ID NO: 8.

In one embodiment, the Napi2b antibody of the ADC of the present application comprises a light chain sequence of SEQ ID NO: 9 and a heavy chain sequence of SEQ ID NO: 10.

Anti-CD33 humanized antibody:

In one embodiment, the anti-CD33 antibody of the ADC of the present application comprises three light chain hypervariable regions and three heavy chain hypervariable regions, the sequences of which (SEQ ID NOs: 11-16) are shown below.

In one embodiment, the anti-CD33 antibody of the ADC of the present application comprises the variable light chain sequence of SEQ ID NO: 17 and the variable heavy chain sequence of SEQ ID NO: 18.

In one embodiment, the anti-CD33 antibody of the ADC of the present application comprises the light chain sequence of SEQ ID NO: 19 and the heavy chain sequence of SEQ ID NO: 20.

In one embodiment, the anti-CD33 antibody of the ADC of the present application comprises three light chain hypervariable regions and three heavy chain hypervariable regions, the sequences of which (SEQ ID NOs: 19-24) are shown below.

In one embodiment, the anti-CD33 antibody of the ADC of the present application comprises the variable light chain sequence of SEQ ID NO: 25 and the variable heavy chain sequence of SEQ ID NO: 26.

In one embodiment, the anti-CD33 antibody of the ADC of the present application comprises the variable light chain sequence of SEQ ID NO: 27 and the variable heavy chain sequence of SEQ ID NO: 28.

In one embodiment, the anti-CD33 antibody of the ADC of the present application comprises the variable light chain sequence of SEQ ID NO: 29 and the variable heavy chain sequence of SEQ ID NO: 30.

In one embodiment, the anti-CD33 antibody of the ADC of the present application comprises the variable light chain sequence of SEQ ID NO: 31 and the variable heavy chain sequence of SEQ ID NO: 32.

Anti-CD22 humanized antibody:

In one embodiment, the anti-CD22 antibody of the ADC of the present application comprises three light chain hypervariable regions and three heavy chain hypervariable regions (SEQ ID NOs: 41-46), the sequences of which are shown below.

In one embodiment, the anti-CD22 antibody of the ADC of the present application comprises the variable light chain sequence of SEQ ID NO: 47 and the variable heavy chain sequence of SEQ ID NO: 48.

In one embodiment, the anti-CD22 antibody of the ADC of the present application comprises the light chain sequence of SEQ ID NO: 49 and the heavy chain sequence of SEQ ID NO: 50.

ADC in vivo data

Anti-CD22 10F4v3LC K149C MC-Sq-Cit-PAB-Tubulysin M (ADC2-2) shows target-specific killing in BJAB and WSU-DLCL2

Anti-CD22 10F4v3LC K149C MC-Sq-Cit-PAB-Dolastatin 10 (ADC1-2) shows target-specific killing in WSU-DLCL2

Anti-Napi2b 10H1.11.4B LC K149C MC-Sq-Cit-PAB-Tubulysin M (ADC2-1) shows target-specific killing in IGROV-1 and OVCAR-3x2.1 in the EdUProlif assay

Anti-Napi2b 10H1.11.4B LC K149C MC-Sq-Cit-PAB-Dolastatin 10 (ADC1-1) shows target-specific killing in IGROV-1 and OVCAR-3x2.1 in the EdUProlif assay

ADC in vivo data

The following ADCs were tested in the above in vivo assays and found to be active. The activities of the ADCs are shown in Figure 1 and the description below.

Figure 1 shows a comparison of the efficacy of various CD22 ADCs in SCID mice bearing WSU-DLCL2 human diffuse large B-cell lymphoma. Both CD22 ADC1-2 and ADC2-2 showed dose-dependent inhibition of tumor growth compared to the vehicle group, with ADC1-2 showing stronger anti-tumor activity than ADC2-2. The non-binding control NaPi2b ADC had minimal effect on tumor growth.

Sequence Listing

<110> Genentech

<120> Quaternary ammonium compounds and antibody-drug conjugates thereof

<130> P32456-WO

<140>

<141>

<150> 62/087,127

<151> 2014-12-03

<160> 50

<170> PatentIn version 3.5

<210> 1

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 1

Arg Ser Ser Glu Thr Leu Val His Ser Ser Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 2

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 2

Arg Val Ser Asn Arg Phe Ser

1 5

<210> 3

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 3

Phe Gln Gly Ser Phe Asn Pro Leu Thr

1 5

<210> 4

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 4

Gly Phe Ser Phe Ser Asp Phe Ala Met Ser

1 5 10

<210> 5

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 5

Ala Thr Ile Gly Arg Val Ala Phe His Thr Tyr Tyr Pro Asp Ser Met

1 5 10 15

Lys Gly

<210> 6

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 6

Ala Arg His Arg Gly Phe Asp Val Gly His Phe Asp Phe

1 5 10

<210> 7

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 7

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Glu Thr Leu Val His Ser

20 25 30

Ser Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser Phe Asn Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg

<210> 8

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 8

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asp Phe

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Gly Arg Val Ala Phe His Thr Tyr Tyr Pro Asp Ser Met

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Arg Gly Phe Asp Val Gly His Phe Asp Phe Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 9

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 9

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Glu Thr Leu Val His Ser

20 25 30

Ser Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser Phe Asn Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 10

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 10

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asp Phe

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Gly Arg Val Ala Phe His Thr Tyr Tyr Pro Asp Ser Met

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Arg Gly Phe Asp Val Gly His Phe Asp Phe Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Cys Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 11

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 11

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp

1 5 10 15

<210> 12

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 12

Leu Gly Val Asn Ser Val Ser

1 5

<210> 13

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 13

Met Gln Ala Leu Gln Thr Pro Trp Thr

1 5

<210> 14

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide e"

<400> 14

Asn His Ala Ile Ser

1 5

<210> 15

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 15

Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 16

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 16

Glu Trp Ala Asp Val Phe Asp Ile

1 5

<210> 17

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 17

Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Val Asn Ser Val Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 18

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 18

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Ile Phe Ser Asn His

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Phe

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Trp Ala Asp Val Phe Asp Ile Trp Gly Gln Gly Thr Met

100 105 110

Val Thr Val Ser Ser

115

<210> 19

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 19

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 20

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 20

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 21

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 21

Leu Gln His Asn Ser Tyr Pro Trp Thr

1 5

<210> 22

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 22

Gly Asn Tyr Met Ser

1 5

<210> 23

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 23

Leu Ile Tyr Ser Gly Asp Ser Thr Tyr Tyr Tyr Ala Asp Ser Val Lys Gly

1 5 10 15

<210> 24

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 24

Asp Gly Tyr Tyr Val Ser Asp Met Val Val

1 5 10

<210> 25

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 26

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 26

Glu Val Gln Leu Val Glu Ser Gly Gly Ala Leu Ile Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Ile Ser Gly Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Leu Ile Tyr Ser Gly Asp Ser Thr Tyr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Asn Ile Ser Arg Asp Ile Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys Val

85 90 95

Arg Asp Gly Tyr Tyr Val Ser Asp Met Val Val Trp Gly Lys Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 27

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 27

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 28

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 28

Glu Val Gln Leu Val Glu Ser Gly Gly Ala Leu Ile Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Ile Ser Gly Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Leu Ile Tyr Ser Gly Asp Ser Thr Tyr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Ile Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys Val

85 90 95

Arg Asp Gly Tyr Tyr Val Ser Asp Met Val Val Trp Gly Lys Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 29

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 29

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 30

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 30

Glu Val Gln Leu Val Glu Ser Gly Gly Ala Leu Ile Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Ile Ser Gly Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Leu Ile Tyr Ser Gly Asp Ser Thr Tyr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Ser Ile Ser Arg Asp Ile Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys Val

85 90 95

Arg Asp Gly Tyr Tyr Val Ser Asp Met Val Val Trp Gly Lys Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 31

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 31

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 32

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 32

Glu Val Gln Leu Val Glu Ser Gly Gly Ala Leu Ile Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Ile Ser Gly Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Leu Ile Tyr Ser Gly Asp Ser Thr Tyr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Ala Ile Ser Arg Asp Ile Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys Val

85 90 95

Arg Asp Gly Tyr Tyr Val Ser Asp Met Val Val Trp Gly Lys Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 33

<400> 33

000

<210> 34

<400> 34

000

<210> 35

<400> 35

000

<210> 36

<400> 36

000

<210> 37

<400> 37

000

<210> 38

<400> 38

000

<210> 39

<400> 39

000

<210> 40

<400> 40

000

<210> 41

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 41

Arg Ser Ser Gln Ser Ile Val His Ser Val Gly Asn Thr Phe Leu Glu

1 5 10 15

<210> 42

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 42

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 43

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 43

Phe Gln Gly Ser Gln Phe Pro Tyr Thr

1 5

<210> 44

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 44

Gly Tyr Glu Phe Ser Arg Ser Trp Met Asn

1 5 10

<210> 45

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 45

Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Ser Gly Lys Phe Lys

1 5 10 15

Gly

<210> 46

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 46

Asp Gly Ser Ser Trp Asp Trp Tyr Phe Asp Val

1 5 10

<210> 47

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 47

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Val Gly Asn Thr Phe Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser Gln Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg

<210> 48

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 48

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Glu Phe Ser Arg Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Ser Gly Lys Phe

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Ser Ser Trp Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 49

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 49

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Val Gly Asn Thr Phe Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser Gln Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Cys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 50

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223> /Comment="Description of artificial sequence: synthetic peptide"

<400> 50

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Glu Phe Ser Arg Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Ser Gly Lys Phe

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Ser Ser Trp Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

Claims (20)

1. An antibody-drug conjugate prepared by conjugating an antibody to a linker-drug intermediate compound selected from the group consisting of: 2. The antibody-drug conjugate of claim 1, wherein the antibody is a cysteine engineered antibody. 3. The antibody-drug conjugate of claim 1, wherein the antibody binds to one or more polypeptides selected from the group consisting of: BMPR1B; E16; STEAP1; 0772P; MPF; NaPi2b; Sema 5b; PSCA hlg; ETBR; MSG783; STEAP2; TrpM4; CRIPTO; CD21; CD79b; FcRH2; HER2; NCA; MDP; IL20Rα; brevican; EphB2R; ASLG659; PSCA; GEDA; BAFF-R; CD22; CD79a; CXCR5; HLA-DOB; P2X5; CD72; LY64; FcRH1; IRTA2; TENB2; PMEL17; TMEFF1; GDNF-Ra1; Ly6E; TMEM46; Ly6G6D; LGR5; RET; LY6K; GPR19; GPR54; ASPHD1; tyrosinase; TMEM118; GPR172A; CD33; and CLL1. 4. The antibody-drug conjugate of claim 3, wherein the antibody binds to NaPi2b. 5. The antibody-drug conjugate of claim 4, wherein the NaPi2b antibody comprises HVR-L1 consisting of the amino acid sequence of SEQ ID NO: 1; HVR-L2 consisting of the amino acid sequence of SEQ ID NO: 2; HVR-L3 consisting of the amino acid sequence of SEQ ID NO: 3; HVR-H1 consisting of the amino acid sequence of SEQ ID NO: 4; HVR-H2 consisting of the amino acid sequence of SEQ ID NO: 5; and HVR-H3 consisting of the amino acid sequence of SEQ ID NO: 6. 6. The antibody-drug conjugate of claim 5, wherein the NaPi2b antibody comprises a VL domain consisting of the amino acid sequence of SEQ ID NO: 7; and a VH domain consisting of the amino acid sequence of SEQ ID NO: 8. 7 . The antibody-drug conjugate of claim 5 , wherein the light chain consists of the amino acid sequence of SEQ ID NO: 9 and the heavy chain consists of the amino acid sequence of SEQ ID NO: 10. 8. The antibody-drug conjugate of claim 3, wherein the antibody binds to CD22. 9. The antibody-drug conjugate of claim 8, wherein the CD22 antibody comprises HVR-L1 consisting of the amino acid sequence of SEQ ID NO: 41; HVR-L2 consisting of the amino acid sequence of SEQ ID NO: 42; HVR-L3 consisting of the amino acid sequence of SEQ ID NO: 43; HVR-H1 consisting of the amino acid sequence of SEQ ID NO: 44; HVR-H2 consisting of the amino acid sequence of SEQ ID NO: 45; and HVR-H3 consisting of the amino acid sequence of SEQ ID NO: 46. 10. The antibody-drug conjugate of claim 8, wherein the CD22 antibody comprises a VL domain consisting of the amino acid sequence of SEQ ID NO: 47; and a VH domain consisting of the amino acid sequence of SEQ ID NO: 48. 11. The antibody-drug conjugate of claim 8, wherein the light chain consists of the amino acid sequence of SEQ ID NO: 49 and the heavy chain consists of the amino acid sequence of SEQ ID NO: 50. 12 . A pharmaceutical composition comprising the antibody-drug conjugate of claim 1 and a pharmaceutically acceptable carrier thereof. 13. Use of the pharmaceutical composition of claim 12 in the preparation of a medicament for treating cancer, wherein the treatment comprises administering a therapeutically effective amount of the pharmaceutical composition of claim 12 to a patient. 14. Use of the pharmaceutical composition of claim 12 in the preparation of a medicament for treating lymphoma, wherein the treatment comprises administering a therapeutically effective amount of the pharmaceutical composition of claim 12 to a patient. 15. Use of the pharmaceutical composition of claim 12 in the preparation of a medicament for treating blastoma, wherein the treatment comprises administering a therapeutically effective amount of the pharmaceutical composition of claim 12 to a patient. 16. Use of the pharmaceutical composition of claim 12 in the preparation of a medicament for treating sarcoma, wherein the treatment comprises administering a therapeutically effective amount of the pharmaceutical composition of claim 12 to a patient. 17. Use of the pharmaceutical composition of claim 12 in the preparation of a medicament for treating leukemia, wherein the treatment comprises administering a therapeutically effective amount of the pharmaceutical composition of claim 12 to a patient. 18. Use of the pharmaceutical composition of claim 12 in the preparation of a medicament for treating lymphatic malignancies, wherein the treatment comprises administering a therapeutically effective amount of the pharmaceutical composition of claim 12 to a patient. 19. A linker-drug intermediate compound selected from: 20. A method for preparing the antibody-drug conjugate of claim 1, comprising conjugating the antibody to the linker-drug intermediate of claim 19.