CN116916962A

CN116916962A - Antibody drug conjugates

Info

Publication number: CN116916962A
Application number: CN202180075519.4A
Authority: CN
Inventors: 徐鹤; H·M·李; C·阿伦特
Original assignee: Takeda Pharmaceutical Co Ltd
Current assignee: Takeda Pharmaceutical Co Ltd
Priority date: 2020-11-09
Filing date: 2021-11-09
Publication date: 2023-10-20

Abstract

The present disclosure provides antibody drug conjugates comprising STING modulators. Compositions comprising the antibody drug conjugates are also provided. The compounds and compositions are useful for stimulating an immune response in a subject in need thereof.

Description

Antibody drug conjugates

Technical Field

Background

Antibody Drug Conjugates (ADCs) are a rapidly evolving class of targeted therapeutic agents, representing a promising new approach to improve drug selectivity and cytotoxic activity. These therapeutic agents comprise antibodies (or antibody fragments) that can be linked to a payload (payload) drug to form an immunoconjugate. Antibodies direct the binding of the ADC to the targeted cells. The ADC can then be internalized and its payload released, providing therapy to the cell. Because ADC targets cells for it, the side effects of conjugated drugs may be lower than those encountered when the same agent is administered systemically.

The adaptor protein STING (a stimulator of the interferon genes) has been shown to play a role in the innate immune system. Activation of the STING pathway triggers an immune response, producing specific killer T cells that shrink the tumor and provide durable immunity so the tumor does not recur. The activated STING pathway also promotes antiviral responses by producing antiviral and pro-inflammatory cytokines that fight the virus and mobilize the innate and acquired immune systems, ultimately resulting in persistent immunity against pathogenic viruses. The potential therapeutic benefits of enhancing innate and acquired immune responses make STING an attractive drug discovery target. Cyclic dinucleotides can act as STING agonists and are being tested in clinical trials. However, its anionic nature makes its membrane poorly permeable, which may limit its ability to engage STING in cells, often resulting in an undesirable distribution of these compounds within the blood stream.

There remains a need for new STING agonists and improved methods for their delivery to targeted cells.

Disclosure of Invention

In a first aspect, the present disclosure provides a compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

a is an integer from 1 to 20;

ab is an anti-CCR 2 antibody, an anti-CCR 2 antibody fragment, or an anti-CCR 2 antigen binding fragment;

d is a modulator of STING activity comprising a guanine base, a guanine base derivative, an adenine base or an amino group on an adenine base derivative; and is also provided with

L is a linker covalently bonded to Ab; and also covalently bonded to the amino group on D.

In a first embodiment of the first aspect, the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein D-L is represented by formula (Ia):

wherein:

represents the point of attachment to Ab;

b is an integer from 1 to 20;

m is 0, 1, 2, 3 or 4;

n is 0 or 1;

each R ¹ Independently selected from C ₁ -C ₄ Alkyl, O-C ₁ -C ₄ Alkyl and halogen;

R ₂ selected from C ₁ -C ₄ Alkyl and- (CH) ₂ CH ₂ O) _s -CH ₃ The method comprises the steps of carrying out a first treatment on the surface of the Wherein s is an integer of 1 to 10;

R ³ and R is ^3' Each independently selected from hydrogen and C ₁ -C ₃ An alkyl group; and is also provided with

L ¹ Is a cleavable linker fragment.

In a second embodiment of the first aspect, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein D-L is represented by formula (Ia), wherein:

a is an integer from 1 to 8;

b is an integer from 1 to 10; and is also provided with

m is 0.

In a third embodiment of the first aspect, the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein D-L is represented by formula (Ia), wherein:

m is 0;

n is 0; and is also provided with

R ³ And R is ^3' Each hydrogen.

In a third embodiment of the first aspect, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein D-L is represented by formula (Ia), wherein L ¹ Is that

Wherein:

is the point of attachment to the nitrogen atom of formula (Ia);

is the point of attachment to Ab;

t is an integer from 1 to 10;

w is absent or a self-degrading group;

z is absent or is a peptide of 2 to 5 amino acids;

u and U' are independently absent or a spacer; and is also provided with

Q is a heterobifunctional group;

provided that neither W nor Z are present.

In a fourth embodiment of the first aspect, W is a self-degrading group selected from the group consisting of

Wherein:

is the point of attachment to the carbonyl group; and->Is the point of attachment to Z.

In a fifth embodiment of the first aspect, W is

In a sixth embodiment of the first aspect, W is

In a seventh embodiment of the first aspect, Z is a peptide that is capable of being cleaved enzymatically.

In an eighth embodiment of the first aspect, Z is cathepsin cleavable.

In a ninth embodiment of the first aspect, Z is a two amino acid peptide selected from the group consisting of: val-Cit, cit-Val, val-Ala, ala-Val, phe-Lys and Lys-Phe.

In a tenth embodiment of the first aspect, Z is Ala-Val or Val-Ala.

In an eleventh embodiment of the first aspect, U' is absent and U is selected from

Wherein:

is the point of attachment to Z;

is the point of attachment to Q;

p is an integer from 1 to 6;

q is an integer from 1 to 20;

x is O or-CH ₂ -; and is also provided with

Each r is independently 0 or 1.

In a twelfth embodiment of the first aspect, U' is absent and U is

In a thirteenth embodiment of the first aspect, Q is a heterobifunctional group that is linked to U 'or to Ab by chemical or enzyme mediated conjugation when U' is absent.

In a fourteenth embodiment of the first aspect, Q is selected from

Wherein:

is the point of attachment to U or to Z when U is absent; and is also provided with

Is the point of attachment to U 'or to Ab when U' is absent.

In a fifteenth embodiment of the first aspect, Q is:

in a sixteenth embodiment of the first aspect, t is 1.

In a seventeenth embodiment of the first aspect, R ² is-CH ₃ And R is ³ And R is ^3' Each hydrogen.

In an eighteenth embodiment of the first aspect, a is 2 to 6.

In a nineteenth embodiment of the first aspect, b is 1.

In a twentieth embodiment of the first aspect, the amino substituted compound that modulates STING activity is a compound of formula (II):

wherein:

X ¹⁰ is SH or OH;

X ²⁰ is SH or OH;

Y ^a is O, S or CH ₂ ；

Y ^b Is O, S, NH or NR ^a Wherein R is ^a Is C ₁ -C ₄ An alkyl group;

R ¹⁰ is hydrogen, fluorine, OH, NH ₂ 、OR ^b Or NHR ^b ；

R ²⁰ Is hydrogen or fluorine;

R ³⁰ is hydrogen; r is R ⁴⁰ Is hydrogen, fluorine, OH, NH ₂ 、OR ^b Or NHR ^b The method comprises the steps of carrying out a first treatment on the surface of the Or R is ³⁰ And R is ⁴⁰ Together form CH ₂ O；

R ⁵⁰ Is hydrogen or fluorine;

R ^b is C ₁ -C ₆ Alkyl, halo (C) ₁ -C ₆ ) Alkyl or C ₃ -C ₆ Cycloalkyl;

ring A ¹⁰ Is an optionally substituted 5 or 6 membered monocyclic heteroaryl ring containing 1 to 4 heteroatoms selected from N, O or S or an optionally substituted 9 or 10 membered bicyclic heteroaryl ring containing 1 to 5 heteroatoms selected from N, O or S; wherein ring A ¹⁰ Ring bagContaining at least one N atom, and wherein Y ^b With ring A ¹⁰ Is attached to a carbon atom of (2); and is also provided with

Ring B ¹⁰ Is an optionally substituted 9 or 10 membered bicyclic heteroaryl ring containing 2 to 5 heteroatoms selected from N, O or S; wherein ring B ¹⁰ At least two N atoms in the ring;

with the proviso that ring A ¹⁰ Or ring B ¹⁰ Is linked to 'L' in formula (I) via an amino group.

In a twenty-first embodiment of the first aspect, the amino-substituted compound that modulates STING activity is

Wherein the method comprises the steps ofIs the point of attachment to 'L' in formula (I).

In a twenty-second embodiment of the first aspect, the amino-substituted compound that modulates STING activity is a compound of formula (III):

or a pharmaceutically acceptable salt thereof; wherein the method comprises the steps of

X ¹⁰ Is SH or OH;

X ²⁰ is SH or OH;

Y ^c is O, S or CH ₂ ；

Y ^d Is O, S or CH ₂ ；

B ¹⁰⁰ Is formed by (B) ¹ -A) or formula (B) ¹ -a group represented by B):

R ¹³ 、R ¹⁴ 、R ¹⁵ 、R ¹⁶ and R is ¹⁷ Each independently is a hydrogen atom or a substituent;

R ¹⁰⁰⁰ is hydrogen or a bond to a carbonyl group of formula (I);

Y ¹¹ 、Y ¹² 、Y ¹³ 、Y ¹⁴ 、Y ¹⁵ and Y ¹⁶ Each independently is N or CR ^1a Wherein R is ^1a Is hydrogen or a substituent;

Z ¹¹ 、Z ¹² 、Z ¹³ 、Z ¹⁴ 、Z ¹⁵ and Z ¹⁶ Each independently is N or C;

R ¹⁰⁵ is a hydrogen atom or a substituent;

B ²⁰⁰ is formed by (B) ² -A) or formula (B) ² -a group represented by B):

R ²³ 、R ²⁴ 、R ²⁵ 、R ²⁶ and R is ²⁷ Each independently is a hydrogen atom or a substituent;

R ^100' is hydrogen or a bond to a carbonyl group of formula (I);

Y ²¹ 、Y ²² 、Y ²³ 、Y ²⁴ 、Y ²⁵ and Y ²⁶ Each independently is N or CR ^2a Wherein R is ^2a Is hydrogen or a substituent;

Z ²¹ 、Z ²² 、Z ²³ 、Z ²⁴ 、Z ²⁵ and Z ²⁶ Each independently is N or C; and is also provided with

R ²⁰⁵ Is a hydrogen atom or a substituent; wherein R is ¹⁰⁵ And R is ²⁰⁵ Each independently linked to the 2 or 3 position of the 5 membered ring to which it is linked;

the limiting conditions are:

B ¹⁰⁰ or B is a ²⁰⁰ Is linked to 'L' in formula (I) via an amino group.

In a twenty-third embodiment of the first aspect, the amino-substituted compound that modulates STING activity is a compound of formula (IIIa):

R ¹⁰⁰⁰ is hydrogen or a bond to a carbonyl group of formula (I);

R ¹⁰⁵ is a hydrogen atom or a substituent;

r100' is hydrogen or a bond to a carbonyl group of formula (I);

the limiting conditions are:

B ¹⁰⁰ or B is a ²⁰⁰ One of them is:

wherein:

R ¹⁸ is hydrogen or C _1-6 An alkyl group; and is also provided with

R ¹⁹ Is a halogen atom;

and the other is linked to the 'L' group in formula (I) via an-NH-group.

In a twenty-fourth embodiment of the first aspect, the amino-substituted compound that modulates STING activity is a compound of formula (IV):

or a pharmaceutically acceptable salt thereof, wherein:

R ¹ and R is ² Each independently is a hydroxyl group or a halogen atom;

B ¹ the method comprises the following steps:

R ¹⁸ is hydrogen or C _1-6 An alkyl group;

R ¹⁹ Is a halogen atom;

B ² the method comprises the following steps:

and is also provided with

Q ² And Q ⁴ Each independently is an oxygen atom or a sulfur atom.

In a twenty-fifth embodiment of the first aspect, the amino-substituted compound that modulates STING activity is:

or a pharmaceutically acceptable salt thereof, whereinIs the point of attachment to L.

In a twenty-sixth embodiment of the first aspect, the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, having the structure of formula (VI):

wherein a is an integer of 1 to 6.

In a twenty-seventh embodiment of the first aspect, ab is an antibody or fragment thereof that binds to human CCR2 or a portion thereof and is capable of blocking the binding of chemokines to CCR2 and inhibiting the function of CCR 2.

In a twenty-eighth embodiment of the first aspect, the antibody is selected from the group consisting of: monoclonal antibody 1D9 or an antibody that competes with 1D9 for binding to human CCR2 or a portion of CCR 2; MC-21; STI-B020X; uniTI-101 and 4.40A68G.

In a twenty-ninth embodiment of the first aspect, the antibody is monoclonal antibody 1D9 or an antibody that competes with 1D9 for binding to human CCR2 or a portion of CCR 2.

In a thirty-first embodiment of the first aspect, the antibody is a chimeric, humanized, human, mouse, rat, goat or rabbit antibody.

In a thirty-first embodiment of the first aspect, the anti-CCR 2 antibody, anti-CCR 2 antibody fragment or anti-CCR 2 antigen binding fragment comprises: a light chain CDR1 comprising amino acids 24-39 of SEQ ID NO. 1; a light chain CDR2 comprising amino acids 55-61 of SEQ ID NO. 1; a light chain CDR3 comprising amino acids 94-102 of SEQ ID No. 1; a heavy chain CDR1 comprising amino acids 31-35 of SEQ ID NO. 2; a heavy chain CDR2 comprising amino acids 50-68 of SEQ ID NO. 2; and a heavy chain CDR3 comprising amino acids 101-106 of SEQ ID NO. 2.

In a thirty-second embodiment of the first aspect, the anti-CCR 2 antibody, anti-CCR 2 antibody fragment or anti-CCR 2 antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence SEQ ID No. 2.

In a thirty-third embodiment of the first aspect, the antibody, anti-CCR 2 antibody fragment or anti-CCR 2 antigen binding fragment comprises a light chain variable region comprising the amino acid sequence SEQ ID No. 1.

In a thirty-fourth embodiment of the first aspect, the anti-CCR 2 antibody, anti-CCR 2 antibody fragment or anti-CCR 2 antigen binding fragment comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region comprises the amino acid sequence SEQ ID No. 2.

In a thirty-fifth embodiment of the first aspect, the anti-CCR 2 antibody, anti-CCR 2 antibody fragment or anti-CCR 2 antigen binding fragment comprises a heavy chain variable region and a light chain variable region, wherein said light chain variable region comprises the amino acid sequence SEQ ID No. 1.

In a thirty-sixth embodiment of the first aspect, the anti-CCR 2 antibody, anti-CCR 2 antibody fragment or anti-CCR 2 antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence SEQ ID No. 2 and a light chain variable region comprising the amino acid sequence SEQ ID No. 1.

In a thirty-seventh embodiment of the first aspect, the anti-CCR 2 antibody, anti-CCR 2 antibody fragment or anti-CCR 2 antigen binding fragment further comprises a polypeptide selected from the group consisting of human immunoglobulin IgG ₁ 、IgG ₂ 、IgG ₃ 、IgG ₄ 、IgA ₁ And IgA ₂ Heavy chain constant region of heavy chain constant region.

In a thirty-eighth embodiment of the first aspect, the anti-CCR 2 antibody, anti-CCR 2 antibody fragment or anti-CCR 2 antigen binding fragment further comprises a light chain constant region selected from the group consisting of human immunoglobulin iggk and iggλ light chain constant regions.

In a thirty-ninth embodiment of the first aspect, the anti-CCR 2 antibody, anti-CCR 2 antibody fragment or anti-CCR 2 antigen binding fragment binds the same epitope as an antibody comprising the heavy chain variable region of SEQ ID No. 2 and the light chain variable region of SEQ ID No. 1.

In a fortieth embodiment of the first aspect, the anti-CCR 2 antibody comprises the heavy chain region of SEQ ID No. 3.

In a fortieth embodiment of the first aspect, the anti-CCR 2 antibody comprises the light chain region of SEQ ID No. 4.

In a forty-second embodiment of the first aspect, the anti-CCR 2 antibody comprises a heavy chain region of SEQ ID No. 3 and a light chain region of SEQ ID No. 4.

In a second aspect, the present disclosure provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

In a first embodiment of the second aspect, the pharmaceutical composition comprises a compound of formula (I) and an antibody that binds to programmed death 1 (PD-1, CD279, hsem 1 or SLEB 2).

In a second embodiment of the second aspect, the pharmaceutical composition comprises a compound of formula (I) and an antibody that binds to programmed death ligand 1 (PD-L1, CD274 or B7H 1).

In a third aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutically acceptable amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.

In a first embodiment of the third aspect, the method of treating cancer comprises administering to a subject a pharmaceutically acceptable amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and an anti-PD-1 antibody.

In a second embodiment of the third aspect, the method of treating cancer comprises administering to a subject a pharmaceutically acceptable amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and an anti-PD-L1 antibody.

In a third embodiment of the third aspect, the compound of formula (I) or a pharmaceutically acceptable salt thereof and the anti-PD-1 antibody are administered simultaneously.

In a fourth embodiment of the third aspect, the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the anti-PD-1 antibody are administered sequentially.

In a fifth embodiment of the third aspect, the compound of formula (I) or a pharmaceutically acceptable salt thereof and the anti-PD-L1 antibody are administered simultaneously.

In a sixth embodiment of the third aspect, the compound of formula (I) or a pharmaceutically acceptable salt thereof and the anti-PD-L1 antibody are administered sequentially.

In a seventh embodiment of the third aspect, the method further comprises administering radiation to the subject. In an eighth embodiment of the third aspect, the radiation is particle radiation. In a ninth embodiment of the third aspect, the radiation is applied by external beam radiation.

In a fourth aspect, the present disclosure provides a method for stimulating an immune response in a subject in need thereof, the method comprising administering to the subject a pharmaceutically acceptable amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.

Drawings

Figure 1 depicts the preparation of Ab-STING agonist conjugates via random cysteine conjugation.

Figure 2 depicts the preparation of Ab-STING agonist conjugates conjugated via transglutaminase.

Figure 3 depicts the preparation of Ab-STING agonist conjugates conjugated via transglutaminase.

FIG. 4 depicts a mouse PK profile of antibody drug conjugate B-14.

FIG. 5 depicts a mouse PK profile for antibody drug conjugate B-15.

FIG. 6 depicts a mouse PK profile for antibody drug conjugate B-16.

FIG. 7 depicts a mouse PK profile of antibody drug conjugate B-17.

FIG. 8 depicts a mouse PK profile for antibody drug conjugate B-18.

Figure 9 depicts the change in body weight over time of mice dosed with ADC B-17.

Figure 10 depicts the change in body weight over time of mice dosed with ADC B-20.

FIG. 11 depicts a comparison of the anti-tumor activity of antibody drug conjugate B-21 with that of its individual payloads.

Figure 12 depicts changes in CCR2 and CD80 expression in monocytes and MDSCs of non-human primates following administration of antibody drug conjugate B-17.

FIG. 13 depicts changes in serum IL-1RA, IL-6, TNF- α, and IFN- γ in non-human primates following administration of antibody drug conjugate B-17.

Figure 14 depicts a non-human primate PK profile of antibody drug conjugate B-17.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications mentioned herein are incorporated by reference in their entirety.

The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "or" is a logical disjunct (i.e., and/or) and does not indicate an exclusive disjunct unless explicitly indicated by words such as the terms "or," "unless," "alternatively," and the like.

As used herein, the term "about" refers to ± 10%.

Antibody drug conjugates

In some embodiments, the present disclosure provides a compound of formula (I),

or a pharmaceutically acceptable salt thereof, wherein:

a is an integer from 1 to 20;

STING modulator moiety

The present disclosure provides compounds comprising modulators of STING activity. In certain embodiments, a STING modulator is a compound that targets the STING pathway as an antagonist or agonist. In some embodiments, the STING modulator is an agonist. In certain embodiments, STING modulators comprise a guanine base, a guanine base derivative, an adenine base, or an amino group on an adenine base derivative. In some embodiments, the STING modulator is a cyclic dinucleotide or a cyclic dinucleotide-like compound (each is a CDN).

In some embodiments, the STING modulator is a compound of formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

X ¹⁰ is-SH or-OH;

X ²⁰ is-SH or-OH;

Y ^a is-O-, -S-or-CH ₂ -；

Y ^b is-O-, -S-, -NH-or-NR ^a -, wherein R is ^a Is C ₁ -C ₄ An alkyl group;

R ¹⁰ is hydrogen, fluorine, -OH, -NH ₂ 、-OR ^b or-NHR ^b ；

R ²⁰ Is hydrogen or fluorine;

R ³⁰ is hydrogen; r is R ⁴⁰ Is hydrogen, fluorine, -OH, -NH ₂ 、-OR ^b or-NHR ^b The method comprises the steps of carrying out a first treatment on the surface of the Or R is ³⁰ And R is ⁴⁰ Together form-CH ₂ O-；

R ⁵⁰ Is hydrogen or fluorine;

ring A ¹⁰ Is an optionally substituted 5 or 6 membered monocyclic heteroaryl ring containing 1 to 4 heteroatoms selected from N, O or S or an optionally substituted 9 or 10 membered bicyclic heteroaryl ring containing 1 to 5 heteroatoms selected from N, O or S; wherein ring A ¹⁰ Containing at least one N atom in the ring, and wherein Y ^b With ring A ¹⁰ Is attached to a carbon atom of (2); and is also provided with

Ring B ¹⁰ Is an optionally substituted 9 or 10 membered bicyclic heteroaryl ring containing 2-5 heteroatoms selected from N, O or S; wherein ring B ¹⁰ At least two N atoms in the ring;

with the proviso that ring A ¹⁰ Or ring B ¹⁰ Is linked to 'L' in formula (I) via an-NH-group.

Ring a, as described herein ¹⁰ And ring B ¹⁰ May contain one or more substituents and may thus be optionally substituted. Suitable substituents on the unsaturated carbon atoms of the heteroaryl groups include, and are generally selected from, halo, -NO ₂ 、-CN、-R ⁺ 、-C(R ⁺ )＝C(R ⁺ ) ₂ 、-C≡C-R ⁺ 、-OR ⁺ 、-SR ^o 、-S(O)R ^o 、-SO ₂ R ^o 、-SO ₃ R ⁺ 、-SO ₂ N(R ⁺ ) ₂ 、-N(R ⁺ ) ₂ 、-NR ⁺ C(O)R ⁺ 、-NR ⁺ C(S)R ⁺ 、-NR ⁺ C(O)N(R ⁺ ) ₂ 、-NR ⁺ C(S)N(R ⁺ ) ₂ 、-N(R ⁺ )C(＝NR ⁺ )-N(R ⁺ ) ₂ 、-N(R ⁺ )C(＝NR ⁺ )-R ^o 、-NR ⁺ CO ₂ R ⁺ 、-NR ⁺ SO ₂ R ^o 、-NR ⁺ SO ₂ N(R ⁺ ) ₂ 、-O-C(O)R ⁺ 、-O-CO ₂ R ⁺ 、-OC(O)N(R ⁺ ) ₂ 、-C(O)R ⁺ 、-C(S)R ^o 、-CO ₂ R ⁺ 、-C(O)-C(O)R ⁺ 、-C(O)N(R ⁺ ) ₂ 、-C(S)N(R ⁺ ) ₂ 、-C(O)N(R ⁺ )-OR ⁺ 、-C(O)N(R ⁺ )C(＝NR ⁺ )-N(R ⁺ ) ₂ 、-N(R ⁺ )C(＝NR ⁺ )-N(R ⁺ )-C(O)R ⁺ 、-C(＝NR ⁺ )-N(R ⁺ ) ₂ 、-C(＝NR ⁺ )-OR ⁺ 、-N(R ⁺ )-N(R ⁺ ) ₂ 、-C(＝NR ⁺ )-N(R ⁺ )-OR ⁺ 、-C(R ^o )＝N-OR ⁺ 、-P(O)(R ⁺ ) ₂ 、-P(O)(OR ⁺ ) ₂ 、-O-P(O)-OR ⁺ and-P (O) (NR) ⁺ )-N(R ⁺ ) ₂ Wherein R is ⁺ Independently is hydrogen or an optionally substituted aliphatic, aryl, heteroaryl, cycloaliphatic or heterocyclic group, or two independently occurring R ⁺ Together with intervening atoms, form an optionally substituted 5-7 membered aryl, heteroaryl, cycloaliphatic or heterocyclic group. In some embodiments, R ⁺ Independently hydrogen, C _1-6 Aliphatic groups or C _3-6 Cycloaliphatic radicals. Each R ^o Independently is an optionally substituted aliphatic, aryl, heteroaryl, cycloaliphatic or heterocyclic group.

As detailed above, in some embodiments, two independently occurring R' s ⁺ (or any other variable similarly defined in the specification and claims herein), together with intervening atoms, form a single or double ring selected from the group consisting of: 3-13 membered cycloaliphatic groups; 3-12 membered heterocyclyl having 1-5 heteroatoms selected independently from nitrogen, oxygen or sulfur; 6-10 membered aryl; or a 5-10 membered heteroaryl having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, the STING modulator is a compound of formula (IIA):

or a pharmaceutically acceptable salt thereof, wherein R ¹⁰ And R is ⁴⁰ Each independently is hydrogen, fluorine, -OH or-OCH ₂ CF ₃ And ring A ¹⁰ And B ¹⁰ With the proviso that ring A, as defined for compounds of formula (II) ¹⁰ Or ring B ¹⁰ Is linked to 'L' via an-NH-group.

In some embodiments, ring a ¹⁰ Is an optionally substituted 6 membered monocyclic heteroaryl ring containing 1, 2 or 3 nitrogen atoms.

In some embodiments, ring B ¹⁰ The method comprises the following steps:

wherein:

Z ¹⁰ 、Z ²⁰ 、Z ³⁰ and Z ⁴⁰ Each independently is N or CR ²⁰⁰ ；

R ²¹⁰ Is hydrogen or C ₁ -C ₆ Alkyl, halo (C) ₁ -C ₆ ) Alkyl or C ₃ -C ₆ Cycloalkyl;

R ²³⁰ is hydrogen or-NH ₂ The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

R ²⁰⁰ 、R ²²⁰ And R is ²⁴⁰ Each independently is hydrogen, halogen, -OH, -NH ₂ 、-CN、C ₁ -C ₆ Alkyl, halo (C) ₁ -C ₆ ) Alkyl or C ₃ -C ₆ Cycloalkyl groups.

In some embodiments, STING modulators are:

or a pharmaceutically acceptable salt thereof, whereinIs attached to an 'L' group of the parent molecular moietyAnd (5) a dot.

In some embodiments, the STING modulator is a compound of formula (III):

or a pharmaceutically acceptable salt thereof, wherein:

X ¹⁰ is SH or OH;

X ²⁰ is SH or OH;

Y ^c is O, S or CH ₂ ；

Y ^d Is O, S or CH ₂ ；

R ¹⁰⁵ And R is ²⁰⁵ Each independently is hydrogen or a substituent, wherein R ¹⁰⁵ And R is ²⁰⁵ Each independently linked to the 2 or 3 position of the 5 membered ring to which it is linked;

R ¹⁰⁰⁰ is hydrogen or a bond to a carbonyl group of formula (I);

Y ¹¹ 、Y ¹² 、Y ¹³ 、Y ¹⁴ 、Y ¹⁵ and Y ¹⁶ Each independently is N or CR ^1a ；

R ^1a is a hydrogen atom or a substituent;

R ^100' is hydrogen or a bond to a carbonyl group of formula (I);

Y ²¹ 、Y ²² 、Y ²³ 、Y ²⁴ 、Y ²⁵ and Y ²⁶ Each independently is N or CR ^2a ；

R ^2a Is a hydrogen atom or a substituent;

provided that B ¹⁰⁰ Or B is a ²⁰⁰ One of which is linked to the carbonyl group in formula (I) via an-NH-group.

As described herein, the compounds of formula (III) and formula (IIIa) (below) contain substituents at certain positions. Suitable substituents include halogen atoms, cyano groups, nitro groups, optionally substituted hydrocarbyl groups, optionally substituted heterocyclyl groups, acyl groups, optionally substituted amino groups, optionally substituted carbamoyl groups, optionally substituted thiocarbamoyl groups, optionally substituted sulfamoyl groups, optionally substituted hydroxy groups, optionally substituted sulfanyl groups (SH), and optionally substituted silyl groups, wherein the optionally substituted groups have one or more substituents selected from substituent group a:

"substituent group A: "

(1) A halogen atom is used as a halogen atom,

(2) A nitro group, a nitro group and a nitro group,

(3) A cyano group,

(4) An oxo group is present in the polymer,

(5) A hydroxyl group,

(6) Optionally halogenated C _1-6 An alkoxy group, an amino group,

(7)C _6-14 aryloxy (e.g., phenoxy, naphthoxy),

(8)C _7-16 aralkyloxy (e.g., benzyloxy)，

(9) 5-to 14-membered aromatic heterocyclyloxy (e.g., pyridyloxy),

(10) 3 to 14 membered non-aromatic heterocyclyloxy (e.g., morpholinyloxy, piperidinyloxy),

(11)C _1-6 alkyl-carbonyloxy (e.g., acetoxy, propionyloxy),

(12)C _6-14 aryl-carbonyloxy (e.g., benzoyloxy, 1-naphthoyloxy, 2-naphthoyloxy),

(13)C _1-6 alkoxy-carbonyloxy (e.g., methoxycarbonyloxy, ethoxycarbonyloxy, propoxycarbonyloxy, butoxycarbonyloxy),

(14) Mono-or di-C _1-6 Alkyl-carbamoyloxy (e.g., methylcarbamoyloxy, ethylcarbamoyloxy, dimethylcarbamoyloxy, diethylcarbamoyloxy),

(15)C _6-14 aryl-carbamoyloxy (e.g., phenylcarbamoyloxy, naphthylcarbamoyloxy),

(16) 5-to 14-membered aromatic heterocyclylcarbonyloxy (e.g., nicotinoyloxy),

(17) 3 to 14 membered non-aromatic heterocyclylcarbonyloxy (e.g., morpholinylcarbonyloxy, piperidylcarbonyloxy),

(18) Optionally halogenated C _1-6 Alkylsulfonyloxy (e.g., methylsulfonyloxy, trifluoromethylsulfonyloxy),

(19) Optionally by C _1-6 Alkyl substituted C _6-14 Arylsulfonyloxy (e.g., phenylsulfonyloxy, toluenesulfonyloxy),

(20) Optionally halogenated C _1-6 An alkylthio group, which is a group having a hydroxyl group,

(21) A 5-to 14-membered aromatic heterocyclic group,

(22) A 3 to 14 membered non-aromatic heterocyclic group,

(23) A formyl group, a halogen atom,

(24) A carboxyl group,

(25) Optionally halogenated C _1-6 An alkyl-carbonyl group, wherein the alkyl-carbonyl group,

(26)C _6-14 an aryl-carbonyl group,

(27) 5 to 14 membered aromatic heterocyclylcarbonyl,

(28) 3 to 14 membered non-aromatic heterocyclylcarbonyl,

(29)C _1-6 an alkoxy-carbonyl group, wherein the alkoxy-carbonyl group,

(30)C _6-14 aryloxy-carbonyl (e.g., phenoxycarbonyl, 1-naphthyloxycarbonyl, 2-naphthyloxycarbonyl),

(31)C _7-16 aralkyloxy-carbonyl (e.g., benzyloxycarbonyl, phenethyloxycarbonyl),

(32) A carbamoyl group, which is a group having a carboxyl group,

(33) A thiocarbamoyl group, a salt thereof,

(34) Mono-or di-C _1-6 An alkyl-carbamoyl group, which is a group,

(35)C _6-14 aryl-carbamoyl (e.g., phenylcarbamoyl),

(36) 5-to 14-membered aromatic heterocyclylcarbamoyl (e.g., pyridylcarbamoyl, thienylcarbamoyl),

(37) 3 to 14 membered non-aromatic heterocyclylcarbamoyl (e.g., morpholinylcarbamoyl, piperidinylcarbamoyl),

(38) Optionally halogenated C _1-6 An alkylsulfonyl group, an alkyl sulfonyl group,

(39)C _6-14 an aryl sulfonyl group,

(40) 5-to 14-membered aromatic heterocyclylsulfonyl (e.g., pyridylsulfonyl, thienylsulfonyl),

(41) Optionally halogenated C _1-6 An alkylsulfinyl group, an alkyl group,

(42)C _6-14 arylsulfinyl (e.g., phenylsulfinyl, 1-naphthylsulfinyl, 2-naphthylsulfinyl),

(43) 5-to 14-membered aromatic heterocyclic sulfinyl (e.g., pyridylsulfinyl, thienyl sulfinyl),

(44) An amino group, a hydroxyl group,

(45) Mono-or di-C _1-6 Alkylamino (e.g., methylamino, ethylamino, propylamino, isopropylamino, butylamino, dimethylamino, diethylamino, dipropylamino, dibutylamino, N-ethyl-N-methylamino),

(46) Mono-or di-C _6-14 Arylamino groups (e.g., phenylamino groups),

(47) 5-to 14-membered aromatic heterocyclylamino (e.g., pyridylamino),

(48)C _7-16 aralkylamino groups (e.g., benzylamino groups),

(49) A formylamino group, a carbonyl group,

(50)C _1-6 alkyl-carbonylamino (e.g., acetylamino, propionylamino, butyrylamino),

(51)(C _1-6 alkyl) (C) _1-6 Alkyl-carbonyl) amino (e.g., N-acetyl-N-methylamino),

(52)C _6-14 aryl-carbonylamino (e.g., phenylcarbonylamino, naphthylcarbonylamino),

(53)C _1-6 alkoxy-carbonylamino (e.g., methoxycarbonylamino, ethoxycarbonylamino, propoxycarbonylamino, butoxycarbonylamino, tert-butoxycarbonylamino),

(54)C _7-16 Aralkyloxy-carbonylamino groups (e.g., benzyloxycarbonylamino groups),

(55)C _1-6 alkylsulfonylamino (e.g., methylsulfonylamino, ethylsulfonylamino),

(56) Optionally by C _1-6 Alkyl substituted C _6-14 Arylsulfonylamino (e.g., phenylsulfonylamino, tosylamino),

(57) Optionally halogenated C _1-6 An alkyl group, a hydroxyl group,

(58)C _2-6 an alkenyl group,

(59)C _2-6 an alkynyl group, an amino group,

(60)C _3-10 a cycloalkyl group,

(61)C _3-10 cycloalkenyl group, and

(62)C _6-14 aryl groups.

In some embodiments, the STING modulator is a compound of formula (IIIa):

R ¹⁰⁰⁰ is hydrogen or a bond to a carbonyl group of formula (I);

R ¹⁰⁵ is a hydrogen atom or a substituent;

R ^100' is hydrogen or a bond to a carbonyl group of formula (I);

The limiting conditions are:

B ¹⁰⁰ or B is a ²⁰⁰ One of them is:

wherein:

R ¹⁸ is hydrogen or C _1-6 An alkyl group; and is also provided with

R ¹⁹ Is a halogen atom;

and the other is linked to the carbonyl group of formula (I) via an-NH-group.

In some embodiments, the STING modulator is a compound of formula (IV):

or a pharmaceutically acceptable salt thereof, wherein:

R ¹ and R is ² Each independently is a hydroxyl group or a halogen atom;

B ¹ the method comprises the following steps:

R ¹⁸ is hydrogen or C _1-6 An alkyl group;

R ¹⁹ is a halogen atom;

B ² the method comprises the following steps:

and->

Q ² And Q ⁴ Each independently is an oxygen atom or a sulfur atom.

In some embodiments, the cyclic dinucleotide is:

or a pharmaceutically acceptable salt thereof, whereinIs the point of 'L'.

Joint part

The group "L" is a linker. As used herein, the term "linker" refers to any chemical moiety capable of linking an antibody, antibody fragment or antigen binding fragment (Ab) to a drug-containing moiety within a compound of formulas (I) and (IV). The linker may be branched and may be substituted with 1 to 20 drug-containing moieties. In some embodiments, the linker may be substituted with 1 to 10 drug-containing moieties. In some embodiments, the linker may be substituted with 1 to 5 drug-containing moieties. In some embodiments, the linker may be substituted with one or two drug-containing moieties. In some embodiments, the linker may be substituted with one drug-containing moiety.

In some embodiments, the linker "L" is a cleavable linker. In certain embodiments, the linker may be susceptible to acid-induced cleavage, photo-induced cleavage, enzymatic cleavage, and the like, under conditions where the drug and/or antibody may remain active. In some embodiments, the cleavable linker may be cleaved enzymatically. In some embodiments, the cleavable linker may be cleaved by a protease, peptidase, esterase, glycosidase, phosphodiesterase, phosphatase, or lipase. In some embodiments, the cleavable linker may be cleaved by a protease. Examples of proteases include, but are not limited to, cathepsin B, VAGP tetrapeptides and the like.

In certain embodiments, the linker may be any of the linkers disclosed in PCT publications WO 2018/200812, WO 2018/100558, which publications are incorporated by reference in their entirety.

In certain embodiments, "L" has the formula:

wherein:

is the point of attachment to the nitrogen atom; and is also provided with

Is the point of attachment to Ab.

In some embodiments, "L" has the formula:

wherein:

is the point of attachment to the nitrogen atom; />

Is the point of attachment to the antibody.

The group "W" is absent or is a self-degrading group. As used herein, the term "self-degrading" refers to a group undergoing an electronic cascade, resulting in the release of the group to which it is attached. In some embodiments, the self-degrading groups comprise one or more groups that may undergo 1, 4-elimination, 1, 6-elimination, 1, 8-elimination, 1, 6-cyclization elimination, 1, 5-cyclization elimination, 1, 3-cyclization elimination, intramolecular 5-exo-trig cyclization, and/or 6-exo-trig cyclization. In certain embodiments, the self-degrading groups may be any of the self-degrading groups disclosed in PCT publications WO 2018/200812, WO 2018/100558, which publications are incorporated by reference in their entirety.

The group "Z" is absent or a peptide of 2 to 5 amino acids. In certain embodiments, the peptide is a cleavage site for a linker, thereby facilitating drug release following exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al (2003) Nat. Biotechnol. 21:778-784). Examples of peptides having two amino acids include, but are not limited to, alanine-alanine (Ala-Ala), valine-alanine (VA or Val-Ala), valine-citrulline (VC or Val-Cit), alanine-phenylalanine (AF or Ala-Phe), phenylalanine-lysine (FK or Phe-Lys), phenylalanine-homolysine (Phe-Homolys), and N-methyl-valine-citrulline (Me-Val-Cit). Examples of peptides having three amino acids include, but are not limited to, glycine-valine-citrulline (Gly-Val-Cit) and glycine-glycine (Gly-Gly-Gly). The above amino acid combinations may also be present in the reverse order (i.e., cit-Val).

The peptides of the present disclosure may comprise naturally occurring and/or non-naturally occurring amino acid residues. The term "naturally occurring amino acid" refers to Ala, asp, cys, glu, phe, gly, his, he, lys, leu, met, asn, pro, gin, arg, ser, thr, val, trp and Tyr. "unnatural amino acids" (i.e., amino acids that are not naturally occurring) include, as non-limiting examples, homoserine, homoarginine, citrulline, phenylglycine, taurine, iodotyrosine, selenocysteine, norleucine ("Nle"), norvaline ("Nva"), β -alanine, L-or D-naphthylalanine, ornithine ("Orn"), and the like. Peptides may be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor associated proteases, cathepsin B, C or D or plasmin proteases.

Amino acids also include natural and unnatural amino acids in the D form. "D-" refers to an amino acid having a "D" (right-handed) configuration, as opposed to the configuration of a naturally occurring ("L-") amino acid. Natural and unnatural amino acids are commercially available (Sigma Chemical co., advanced Chemtech) or synthesized using methods known in the art.

The groups "U" and "U'" are independently absent or are spacers. As used herein, the term "spacer" refers to a chemical that acts as a linkerPart of science. In the present disclosure, a spacer may link an antibody, antibody fragment, or antigen fragment to a heterobifunctional group and/or a heterobifunctional group to a peptide "Z", or to a group "W" when "Z" is absent. Non-limiting exemplary spacers include-NH-, -S-, -O-, -NHC (=o) CH ₂ CH ₂ -、-S(＝O) ₂ -CH ₂ CH ₂ -、-C(＝O)NHNH-、-C(＝O)O-、-C(＝O)NH-、-CH ₂ -、-CH ₂ CH ₂ -、-CH ₂ CH ₂ CH ₂ -、-CH ₂ ＝CH ₂ -, -C.ident.C-, -CH=N-O-, polyethylene glycol (PEG),

In the compounds of the present disclosure, when "U" is present, it may be a branching group substituted with 1 to 10 "-C (O) -W-Z-" groups. In some embodiments, "U" is substituted with 1 to 5 "-C (O) -W-Z-" groups. In some embodiments, "U" is substituted with 1 or 2 "-C (O) -W-Z-" groups. In some embodiments, "U" is substituted with 1 "-C (O) -W-Z-" group. In certain embodiments, the spacer may be any of the spacers disclosed in PCT publications WO 2018/200812, WO 2018/100558, which publications are incorporated by reference in their entirety.

The group "Q" is a heterobifunctional group. In the present disclosure, the term "heterobifunctional" refers to a chemical moiety that connects a linker as part thereof to an antibody, antibody fragment, or antigen-binding fragment. See, for example, WO 2017/191579. Heterobifunctional groups are characterized by having different reactive groups at both ends of a chemical molecule. The heterobifunctional group may be directly linked to "Ab" or may be linked through a linker "U'". The attachment to the "Ab" may be accomplished by chemical or enzymatic conjugation or a combination of both. Chemical conjugation involves the controlled reaction of accessible amino acid residues on the antibody surface with a reaction handle on "Q" or "U'". Examples of chemical conjugation include, but are not limited to, lysine amide coupling, cysteine coupling, and coupling via unnatural amino acids incorporated by genetic engineering, where the unnatural amino acid residue with the desired reaction handle is mounted to an "Ab". In enzymatic conjugation, the enzyme mediates the coupling of the linker to accessible amino acid residues on an antibody, antibody fragment or antigen-binding fragment. Examples of enzymatic conjugation include, but are not limited to, transpeptidation using sortase (sortase), transpeptidation using microbial transglutaminase, and N-glycan engineering. Chemical conjugation and enzymatic conjugation may also be used sequentially. For example, enzymatic conjugation can also be used to mount a unique reaction handle on the "Ab" for use in subsequent chemical conjugation. In certain embodiments, the heterobifunctional group may be any of those disclosed in PCT publications WO 2018/200812, WO 2018/100558, which publications are incorporated by reference in their entirety.

In some embodiments, "Q" is selected from/>

Wherein the method comprises the steps of

Is the point of attachment to U, or to Z when U is absent; and is also provided with

Is the point of attachment to U ', or to Ab when U' is absent.

In certain embodiments, the present disclosure provides a compound of formula (XX):

or a pharmaceutically acceptable salt thereof, wherein n, m, a, t, D-NH-, R ¹ 、R ² 、R ³ 、R ^3' W, Z and U are as described herein, and wherein Q is a reactive functional group capable of conjugation to an antibody, antibody fragment or antigen binding fragment. Examples of suitable Q groups include, but are not limited to, activated carboxyl groups (such as acid chloride-C (O) -Cl and anhydride), haloacetamides, maleimides, alkynes, cycloalkynes (such as cyclooctyne), oxanorbornadiene (oxanoboadene), norbornene, azide, diaryl tetrazine, monoaryl tetrazine, aldehydes, ketones, hydroxylamine, vinyl sulfone and aziridines. In certain embodiments, the reactive functionality may be any of the reactive functionalities disclosed in PCT publications WO 2018/200812, WO 2018/100558, which publications are incorporated by reference in their entirety.

anti-CCR 2 antibodies, antibody fragments and antigen binding fragments

The group "Ab" is an anti-CCR 2 antibody, an anti-CCR 2 antibody fragment, or an anti-CCR 2 antigen binding fragment. Antibodies are proteins produced by the immune system that are capable of recognizing and binding to a specific antigen. Target antigens typically have many binding sites, also known as epitopes, recognized by CDRs on a variety of antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen may have more than one corresponding antibody. The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies, single domain antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. The antibody may be a murine antibody, a human antibody, a humanized antibody, a chimeric antibody or an antibody derived from another species. (Janeway, c., convers, p., walport, m., shomchik (2001) immune Biology, 5 th edition, garland Publishing, new York).

Useful anti-CCR 2 antibodies, antibody fragments and antigen-binding fragments include antibodies (immunoglobulins) or functional fragments thereof (e.g., antigen-binding fragments) that bind to mammalian CC-chemokine receptor 2 (also known as CCR2, CKR-2, CD192, MCP-1RA or MCP-1 RB) or a portion of said receptor. In one embodiment, the antibody or fragment thereof is specific for human or rhesus CCR2 or a portion thereof. In another embodiment, the antibody or fragment blocks the binding of a ligand (e.g., MCP-1, MCP-2, MCP-3, MCP-4) to a receptor and inhibits a function associated with the binding of the ligand to the receptor (e.g., leukocyte migration). For example, as described herein, antibodies and fragments thereof useful in the present disclosure bind to human or rhesus CCR2 or a portion thereof, and may block the binding of chemokines (e.g., MCP-1, MCP-2, MCP-3, MCP-4) to a receptor and inhibit functions associated with the binding of chemokines to a receptor. In one embodiment, the antibody is monoclonal antibody (mAb) LS132.1D9 (1D 9) or an antibody that competes with 1D9 for binding to human CCR2 or a portion of human CCR2. Functional fragments of the foregoing antibodies are also contemplated.

In some embodiments, a humanized immunoglobulin or antigen binding fragment thereof having binding specificity for CCR2 is employed, the immunoglobulin comprising an antigen binding region of non-human origin (e.g., rodent) and at least a portion of an immunoglobulin of human origin (e.g., human framework region, gamma-type human constant region). In one embodiment, the humanized immunoglobulin or fragment thereof competes with 1D9 for binding to CCR2. In one embodiment, the antigen binding region of the humanized immunoglobulin is derived from monoclonal antibody 1D9 (e.g., an immunoglobulin comprising variable regions of light and heavy chains, as shown below).

For example, the humanized immunoglobulin or antigen binding fragment thereof may comprise: an antigen binding region comprising at least one Complementarity Determining Region (CDR) of non-human origin; and a Framework Region (FR) derived from the human framework region. In one aspect, a humanized immunoglobulin having binding specificity for CCR2 comprises: a light chain comprising at least one CDR derived from an antibody that binds CCR2 of non-human origin and an FR derived from a light chain of human origin (e.g., from HF-21/28); and heavy chains comprising CDRs from an antibody that binds CCR2 that is of non-human origin and FR from a heavy chain of human origin (e.g., from 4b4' cl). In another aspect, the light chain comprises three CDRs derived from the light chain of the 1D9 antibody, and the heavy chain comprises three CDRs derived from the heavy chain of the 1D9 antibody.

In one embodiment, the humanized immunoglobulin having binding specificity for CCR2 comprises CDR1, CDR2, and CDR3 of the light chain of a 1D9 antibody and human light chain FR, and comprises CDR1, CDR2, and CDR3 of the heavy chain of a 1D9 antibody and human heavy chain FR. In one embodiment, the humanized immunoglobulin comprises a humanized heavy chain and a light chain as described herein, e.g., a humanized light chain comprising a variable region of a light chain as set forth below, a humanized heavy chain comprising a variable region of a heavy chain as set forth below. Humanized immunoglobulins comprising one or more humanized light and/or heavy chains are also contemplated.

The amino acid sequences of the kappa light chain variable region (VL) of the humanized 1D9 antibody are shown below. CDRs are highlighted in bold:

the amino acid sequences of the heavy chain variable regions (VH) of the humanized 1D9 antibodies are shown below. CDRs are highlighted in bold:

in certain embodiments, an anti-CCR 2 antibody, anti-CCR 2 antibody fragment, or anti-CCR 2 antigen binding fragment comprises: a light chain CDR1 comprising amino acids 24-39 of SEQ ID NO. 1; a light chain CDR2 comprising amino acids 55-61 of SEQ ID NO. 1; a light chain CDR3 comprising amino acids 94-102 of SEQ ID No. 1; a heavy chain CDR1 comprising amino acids 31-35 of SEQ ID NO. 2; a heavy chain CDR2 comprising amino acids 50-68 of SEQ ID NO. 2; and a heavy chain CDR3 comprising amino acids 101-106 of SEQ ID NO. 2.

In some embodiments, the anti-CCR 2 antibody, anti-CCR 2 antibody fragment or anti-CCR 2 antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence SEQ ID No. 2.

In some embodiments, the anti-CCR 2 antibody, anti-CCR 2 antibody fragment or anti-CCR 2 antigen binding fragment comprises a light chain variable region comprising the amino acid sequence SEQ ID No. 1.

In some embodiments, an anti-CCR 2 antibody, anti-CCR 2 antibody fragment, or anti-CCR 2 antigen binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence SEQ ID No. 2.

In some embodiments, an anti-CCR 2 antibody, anti-CCR 2 antibody fragment, or anti-CCR 2 antigen binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises the amino acid sequence SEQ ID No. 1.

In some embodiments, an anti-CCR 2 antibody, anti-CCR 2 antibody fragment, or anti-CCR 2 antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 2 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 1.

In certain embodiments, the anti-CCR 2 antibody, anti-CCR 2 antibody fragment, or anti-CCR 2 antigen binding fragment further comprises a heavy chain constant region. In some embodiments, the heavy chain constant region is selected from human immunoglobulin IgG ₁ 、IgG ₂ 、IgG ₃ 、IgG ₄ 、IgA ₁ And IgA ₂ Heavy chain constant region.

In some embodiments, the anti-CCR 2 antibody, anti-CCR 2 antibody fragment, or anti-CCR 2 antigen binding fragment further comprises a light chain constant region. In some embodiments, the light chain constant region is selected from the group consisting of human immunoglobulin iggκ and iggλ light chain constant regions.

In certain embodiments, an anti-CCR 2 antibody fragment, or an anti-CCR 2 antigen binding fragment binds the same epitope as an antibody comprising the heavy chain variable region of SEQ ID No. 2 and the light chain variable region of SEQ ID No. 1.

"percent identity" refers to the degree of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). The percent identity can be determined by aligning two sequences, introducing a gap to maximize identity between the sequences. Alignment can be generated using procedures known in the art. For purposes herein, the alignment of nucleotide sequences may be performed with the blastn program set by default parameters, and the alignment of amino acid sequences may be performed with the blastp program set by default parameters (see national center for biotechnology information (National Center for Biotechnology Information; NCBI) of the world wide web ncbi.nlm.nih.gov).

By "binds to the same epitope as a reference CCR2 antibody" is meant an antibody that binds to the same CCR2 amino acid residue as a reference CCR2 antibody. The ability of CCR2 antibodies to bind the same epitope as the reference CCR2 antibody was determined by a hydrogen/deuterium exchange assay (see Coales et al Rapid Commun. Mass spectra 2009; 23:639-647).

In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human CCR2, comprises the six CDRs of the antibodies set forth in SEQ ID No. 1 and SEQ ID No. 2, and comprises a VH having a sequence at least 80% identical to the VH sequence of SEQ ID No. 2 and a VL having a sequence at least 80% identical to the VL sequence of SEQ ID No. 1. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human CCR2, comprises the six CDRs of the antibodies set forth in SEQ ID No. 1 and SEQ ID No. 2 (i.e., the three VH CDRs of the antibodies set forth in SEQ ID No. 2 and the three VL CDRs of SEQ ID No. 1), and comprises a VH having a sequence at least 85% identical to the VH sequence of SEQ ID No. 2 and a VL having a sequence at least 85% identical to the VL sequence of SEQ ID No. 1.

In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human CCR2, comprises the six CDRs of the antibodies set forth in SEQ ID No. 1 and SEQ ID No. 2 (i.e., the three VH CDRs of SEQ ID No. 2 and the three VL CDRs of SEQ ID No. 1), and comprises a VH having a sequence at least 90% identical to the VH sequence of SEQ ID No. 2 and a VL having a sequence at least 90% identical to the VL sequence of SEQ ID No. 1. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human CCR2, comprises the six CDRs of the antibodies set forth in SEQ ID No. 1 and SEQ ID No. 2 (i.e., the three VH CDRs of the antibody and the three VL CDRs of SEQ ID No. 1), and comprises a VH having a sequence at least 95% identical to the VH sequence of SEQ ID No. 2 and a VL having a sequence at least 95% identical to the VL sequence of SEQ ID No. 1.

In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human CCR2, comprises the six CDRs of the antibodies set forth in SEQ ID No. 1 and SEQ ID No. 2 (i.e., the three VH CDRs of SEQ ID No. 2 and the three VL CDRs of SEQ ID No. 1), and comprises a VH having a sequence at least 96% identical to the VH sequence of SEQ ID No. 2 and a VL having a sequence at least 96% identical to the VL sequence of SEQ ID No. 1. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human CCR1, comprises the six CDRs of the antibodies set forth in SEQ ID No. 1 and SEQ ID No. 2 (i.e., the three VH CDRs of SEQ ID No. 2 and the three VL CDRs of SEQ ID No. 1), and comprises a VH having a sequence at least 97% identical to the VH sequence of SEQ ID No. 2 and a VL having a sequence at least 97% identical to the VL sequence of SEQ ID No. 2. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human CCR2, comprises the six CDRs of the antibodies set forth in SEQ ID No. 1 and SEQ ID No. 2 (i.e., the three VH CDRs of SEQ ID No. 2 and the three VL CDRs of SEQ ID No. 1), and comprises a VH having a sequence at least 98% identical to the VH sequence of SEQ ID No. 2 and a VL having a sequence at least 98% identical to the VL sequence of SEQ ID No. 1. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human CCR2, comprises the six CDRs of the antibodies set forth in SEQ ID No. 1 and SEQ ID No. 2 (i.e., the three VH CDRs of SEQ ID No. 2, the three VL CDRs of SEQ ID No. 1), and comprises a VH having a sequence at least 99% identical to the VH sequence of SEQ ID No. 2 and a VL having a sequence at least 99% identical to the VL sequence of SEQ ID No. 1.

In certain embodiments, an antibody or antigen binding fragment thereof described herein binds to human CCR2, comprises the six CDRs of the antibodies set forth in SEQ ID No. 1 and SEQ ID No. 2 (i.e., the three VH CDRs of SEQ ID No. 2 and the three VL CDRs of SEQ ID No. 1), comprises a VH having a sequence at least 80% identical to the VH sequence of SEQ ID No. 2 and a VL having a sequence at least 80% identical to the VL sequence of SEQ ID No. 1, and binds to human, cynomolgus monkey, rat and/or mouse CCR 2. In certain embodiments, an antibody or antigen binding fragment thereof described herein binds to CCR2, comprises the six CDRs of the antibodies set forth in SEQ ID No. 1 and SEQ ID No. 2 (i.e., the three VH CDRs of SEQ ID No. 2 and the three VL CDRs of SEQ ID No. 1), comprises a VH having a sequence at least 85% identical to the VH sequence of SEQ ID No. 2 and a VL having a sequence at least 85% identical to the VL sequence of SEQ ID No. 1, and binds to human, cynomolgus monkey, rat and/or mouse CCR 2.

In certain embodiments, an antibody or antigen binding fragment thereof described herein binds to human CCR2, comprises six CDRs of the antibodies set forth in SEQ ID No. 1 and SEQ ID No. 2 (i.e., three VH CDRs of the antibody and three VL CDRs of SEQ ID No. 1), comprises a VH having a sequence at least 90% identical to the VH sequence of SEQ ID No. 2 and a VL having a sequence at least 90% identical to the VL sequence of SEQ ID No. 1, and binds to human, cynomolgus monkey, rat and/or mouse CCR 2. In certain embodiments, an antibody or antigen binding fragment thereof described herein binds to human CCR2, comprises the six CDRs of the antibodies set forth in SEQ ID No. 1 and SEQ ID No. 2 (i.e., the three VH CDRs of SEQ ID No. 2 and the three VL CDRs of SEQ ID No. 1), comprises a VH having a sequence at least 95% identical to the VH sequence of SEQ ID No. 2 and a VL having a sequence at least 95% identical to the VL sequence of SEQ ID No. 1, and binds to human, cynomolgus monkey, rat and/or mouse CCR 2.

In certain embodiments, an antibody or antigen binding fragment thereof described herein binds to human CCR2, comprises six CDRs of the antibodies set forth in SEQ ID No. 1 and SEQ ID No. 2 (i.e., three VH CDRs of SEQ ID No. 2 and three VL CDRs of SEQ ID No. 1), comprises a VH having a sequence at least 96% identical to the VH sequence of SEQ ID No. 2 and a VL having a sequence at least 96% identical to the VL sequence of SEQ ID No. 1, and binds to human, cynomolgus monkey, rat and/or mouse CCR 2. In certain embodiments, an antibody or antigen binding fragment thereof described herein binds to human CCR2, comprises the six CDRs of the antibodies set forth in SEQ ID No. 1 and SEQ ID No. 2 (i.e., the three VH CDRs of SEQ ID No. 2 and the three VL CDRs of SEQ ID No. 1), comprises a VH having a sequence at least 97% identical to the VH sequence of SEQ ID No. 2 and a VL having a sequence at least 97% identical to the VL sequence of SEQ ID No. 1, and binds to human, cynomolgus monkey, rat and/or mouse CCR 2. In certain embodiments, an antibody or antigen binding fragment thereof described herein binds to human CCR2, comprises the six CDRs of the antibodies set forth in SEQ ID No. 1 and SEQ ID No. 2 (i.e., the three VH CDRs of SEQ ID No. 2 and the three VL CDRs of SEQ ID No. 1), comprises a VH having a sequence at least 98% identical to the VH sequence of SEQ ID No. 2 and a VL having a sequence at least 98% identical to the VL sequence of SEQ ID No. 1, and binds to human, cynomolgus monkey, rat and/or mouse CCR 2. In certain embodiments, an antibody or antigen binding fragment thereof described herein binds to human CCR2, comprises the six CDRs of the antibodies set forth in SEQ ID No. 1 and SEQ ID No. 2 (i.e., the three VH CDRs of SEQ ID No. 2 and the three VL CDRs of SEQ ID No. 1), comprises a VH having a sequence at least 99% identical to the VH sequence of SEQ ID No. 2 and a VL having a sequence at least 99% identical to the VL sequence of SEQ ID No. 1, and binds to human, cynomolgus monkey, rat and/or mouse CCR 2.

In certain embodiments, the compounds of formula (I) are combined with an antibody, antibody fragment or antigen-binding fragment that binds PD-1 and/or an antibody, antibody fragment and/or antigen-binding fragment that binds PD-L1. PD-1 is an immune checkpoint protein expressed on activated T cells, B cells and monocytes that, upon binding to its ligand PD-L1, modulates the immune system, for example by promoting apoptosis of antigen-specific T cells and reducing apoptosis of regulatory T cells. PD-L1 can be expressed by tumors to aid in detection and elimination of tumor evasion immune system. Antagonistic inhibition of PD-1/PD-L1 interactions advantageously increases T cell activation and enhances recognition and elimination of tumor cells by the immune system. In certain embodiments, the anti-PD-1 antibody is selected from the group consisting of: palbociclizumab (Pembrolizumab), nivolumab (Nivolumab), cimetidine Li Shan antibody (Cemiplimab), picomab (pimvalimab), sabadizumab (Spartalizumab), cerilizumab (Camrelizumab), singdil Li Shan antibody (sintillimab), tirelizumab (tisrelizumab), terepuzumab Li Shan antibody (Toripalimab), costalimab (Dostarlimab), ependymab (Ezabenlimab), INCMGA0012, AMP-224, AMP-514, SYM-021, LZM-009, CS-1003, SYN-125, GNR-051, MW-11, TY-101, BAT-1306, F520, sasanlimab), pa An Puli antibody (pentimab), terpriumab (cozetimab) CX-188, sitopril mab (Zimberelimab) and Tepolrimab (Tebotelimab) or antibodies that can compete with palbociclizumab, nawuzumab, simip Li Shan, pimipramimab, stbadizumab, carilizumab, xindi Li Shan, tirilizumab, terlipressin Li Shan, dustrilizumab, ebenlimab, INCMGA0012, AMP-224, AMP-514, SYM-021, LZM-009, CS-1003, SYN-125, GNR-051, MW-11, TY-101, BAT-1306, F520, sashan Li Shan, pa An Puli mab, prlimumab, CX-188, sacalimumab or Tepollimumab for binding to a portion of human PD-1 or PD-1.

In some embodiments, the anti-PD-1 antibody is a pamphlet Li Zhushan antibody.

In certain embodiments, the anti-PD-L1 antibody is selected from the group consisting of: abutilizumab (Avelumab), durvalimab You Shan (Durvaumab), ke Xili mab (Cosibelimab), MSB-2311, ZKAB-001, FAZ-053, MDX-1105, CBT-502, IMC-001, RC-98, KL-A167, GR-1405, lodalimab (lodaplimab), shu Geli mab (Sugemalimab), en Wo Lishan antibody (Envanmalimab), opucolimab (Opucolimab) and Galivalimab (Garivulimab) or may compete with Abizumab, avaulimab, duvalili You Shan antibody, ke Xili mab, MSB-2311, ZKAB-001, FAZ-053, MDX-1105, CBT-502, IMC-001, RC-98, KL-A, GR-1405, shu Geli, llolimab, or a PD-Wo Lishan of Ulimab or a portion of Ulimab.

In some embodiments, the anti-PD-L1 antibody is alemtuzumab.

Other anti-PD-1 antibodies that may be used in combination with the compounds of formula (I) are NAT105 (abcam ab 5287); CAL20 (abcam ab 237728); EPR20665 (abcam ab 214421); NAT 105-chimeric (abcam ab 216352); EPR4877 (2) (abcam ab 137132); EP23119-111 (abcam ab 243644); SP269 (abcam ab 227681); PDCD1/1410R (abcam ab 218475); EH12.22H7 (abcam ab 223562); PDCD1/922 (abcam ab 216037); j43 (abcam ab 95789); j43.1 (abcam ab 218768); SPM597 (abcam ab 218474); j116 (abcam ab 171267); RMP1-14 (abcam ab 171265); EPR18017-203 (abcam ab 242810); EPR18017-253 (abcam ab 242562); EPR22234-127 (abcam ab 259656); EPR22234-42 (abcam ab 259655); MAB10861 (R & D Systems); MAB10864 (R & DSsystems); MAB1086 (R & D Systems); MAB10863 (R & D Systems); MAB8578 (R & D Systems); MAB77381 (R & D Systems); MAB7738 (R & D Systems); MAB10866 (R & D Systems); MAB10865 (R & DSsystems); MAB10867 (R & D Systems); SJ01-91 (HUABIO); 1F2 (HUABIO); 3a11 PD-1 blocking Ab (HUABIO); j43 (MyBioSource); RMP1-30 (MyBioSource); 8A1 (bio inc.); BSR1 (Abeomics); PDCD1/922 (Abeomics); PD1.3,1.3 (Miltenyi Biotec); abx174170 (Abbexa); PDCD1 (Fitzgerald Industries intl.); j116 (United States Biological); BSR1 (Nordic BioSite); PDCD1 (BosterBio); 10B3 (ProSci inc.); 4C7 (ProSci inc.); mhT28 blocking (Sino Biological inc.); HF06 neutralization (Sino Biological inc.); or TK12-02 (Creative Diagnostics) or an antibody that competes with any of the foregoing antibodies for binding to PD-1 or a portion of PD-1.

Other anti-PD-L1 antibodies that can be used in combination with the compounds of formula (I) are 28-8 (abcam ab 205921); EPR19759 (abcam ab 213524); CAL10 (abcam ab 237726); 73-10 (abcam ab 228415); EPR20529 (abcam ab 213480); SP142 (abcam ab 228462); BLR020E (abcam ab 243877); RM1012 (abcam ab 282458); EPR23546-160 (abcam ab 252436); ABM4E54 (abcam ab 210931); PDL1/2744 (abcam ab 269674); MIH5 (abcam ab 269253); 29E,2A3 (abcam ab 259283); MIH6 (abcam ab 80276); BMS-5-28 (abcam ab 278010); EPR23939-25 (abcam ab 278009); MAB1561 (R & DSsystems); MAB90871 (R & D Systems); MAB1562 (R & D Systems); MAB90783 (R & D Systems); MAB10348 (R & D Systems); MAB1561R (R & D Systems); MAB9078 (R & D Systems); MAB10355 (R & DSsystems); MIH1 (Invitrogen); MIH5 (Invitrogen); RM320 (Invitrogen); JJ08-95 (Invitrogen); 485 (Invitrogen); MA5-37856 (Invitrogen); 10D4 (Invitrogen); 15 (Invitrogen); 1-111A (Invitrogen); 2B11D11 (Proteintech); OTI2C7 (OriGene); UMAB228 (OriGene); OR-5H8 (OriGene); OTI9E12 (OriGene); UMAB229 (OriGene); OTI11G4 (OriGene); OTI2C11 (OriGene); OTI14H4 (OriGene); OTI7D4 (OriGene); OTI9E1 (OriGene); OTI11G4 (OriGene); OTI2F5 (OriGene); OTI9A5 (OriGene); OTI3F5 (OriGene); OTI4G4 (OriGene); OTI9E5 (OriGene); OTI13G7 (OriGene); OTI9E10 (OriGene); OTI20G10 (OriGene); OR-5E3 (OriGene); OTI4D4 (OriGene); OTI13D11 (OriGene); OTI8C8 (OriGene); OTI16H9 (OriGene); OTI12G7 (OriGene); OTI1B12 (OriGene); OTI2E3 (OriGene); OTI2B12 (OriGene); OR-5E4 (OriGene); BLR020E (Bethyl Laboratories); 3F2 (Abnova); 3D2 (Abnova); 2E6 (Abnova); 2E11 (Abnova); 1H3 (Abnova); 2C4 (Abnova); ac10 (Abnova); 3C10 (Abnova); or 4C11 (Abnova) or an antibody that competes with any of the foregoing antibodies for binding to PD-L1 or a portion of PD-L1.

The term "antibody" as used herein also refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds to an antigen of a target of interest or portion thereof, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune disease. The immunoglobulins disclosed herein can be of any type (e.g., igG, igE, igM, igD and IgA), class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2) or subclass of immunoglobulin molecule. The immunoglobulin may be derived from any species. However, in one aspect, the immunoglobulin is of human, murine or rabbit origin.

The term "single domain antibody", also known as nanobody, is an antibody fragment consisting of a single monomer variable antibody domain, having a molecular weight of about 12kDa to about 15kDa. Monomeric antibodies may be based on heavy chain variable domains or light chains. Examples of single domain antibodies include, but are not limited to, V _H H fragment and V _NAR Fragments. See, e.g., harmsen M.M. et al Applied Microbiology and Biotechnology 77 (1): 13-22.

An "antibody fragment" comprises a portion of an intact antibody, typically the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, fab ', F (ab') ₂ And Fv fragments; a diabody; a linear antibody; fragments generated from Fab expression libraries, anti-idiotype (anti-Id) antibodies, CDRs (complementarity determining regions) and epitope-binding fragments, single chain antibody molecules that immunospecifically bind to any one of a cancer cell antigen, a viral antigen, or a microbial antigen; and multispecific antibodies formed from antibody fragments.

An "intact antibody" is an antibody comprising an antigen binding variable region (antigen-binding variable region) and a light chain constant domain (CL) and heavy chain constant domains CH1, CH2 and CH 3. The constant domain may be a natural sequence constant domain (e.g., a human natural sequence constant domain) or an amino acid sequence variant thereof.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies have high specificity against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to its specificity, monoclonal antibodies have the advantage that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method described first by Kohler et al, nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "monoclonal antibodies" can also be used, for example, by Clackson et al (1991) Nature,352:624-628; the techniques described in Marks et al (1991) J.mol.biol.222:581-597 were isolated from phage antibody libraries.

Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al (1984) Proc.Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen binding sequences derived from a non-human primate (e.g., old world monkey, ape, etc.) and human constant region sequences.

Various methods have been employed to generate monoclonal antibodies (MAbs). Hybridoma technology refers to a cloned cell line that produces a single type of antibody using cells of various species, including mice (rats), hamsters, rats, and humans. Another method for preparing MAbs uses genetic engineering, including recombinant DNA techniques. Monoclonal antibodies made by these techniques include, inter alia, chimeric antibodies and humanized antibodies. Chimeric antibodies combine DNA coding regions from more than one type of species. For example, a chimeric antibody may have a variable region from a mouse and a constant region from a human. Humanized antibodies, although containing non-human parts, are mainly derived from humans. As with chimeric antibodies, humanized antibodies may contain fully human constant regions. However, unlike chimeric antibodies, the variable regions may be partially of human origin. The non-human synthetic portion of a humanized antibody is typically derived from the CDRs in a murine antibody. In any case, these regions are critical for the antibody to recognize and bind to a particular antigen. Although useful for diagnosis and short-term therapy, murine antibodies cannot be administered to humans for long periods of time without increasing the risk of deleterious immunogenic reactions. This reaction is known as human anti-mouse antibody (HAMA) and occurs when the human immune system recognizes and attacks a mouse antibody as a foreign. HAMA reactions can cause toxic shock or even death.

Chimeric and humanized antibodies reduce the likelihood of HAMA reactions by minimizing the non-human portion of the administered antibody. Furthermore, chimeric and humanized antibodies may have the additional benefit of activating secondary human immune responses such as antibody-dependent cellular cytotoxicity.

An intact antibody may have one or more "effector functions," which refers to biological activity attributable to the Fc region of the antibody (either the native sequence Fc region or the amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors; BCR), and the like.

Complete antibodies can be classified into different "classes" according to the amino acid sequence of the constant domain of their heavy chain. There are five main classes of intact antibodies: igA, igD, igE, igG and IgM, and several of these classes can be further divided into "subclasses" (isotypes), e.g., igG1, igG2, igG3, igG4, igA, and IgA2. The heavy chain constant domains corresponding to different antibody classes are referred to as α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional configurations of different immunoglobulin classes are well known.

Useful non-immunoreactive protein, polypeptide, or peptide antibodies include, but are not limited to, transferrin, epidermal growth factor ("EGF"), bombesin, gastrin releasing peptide, platelet derived growth factor, IL-2, IL-6, transforming growth factor ("TGF") (such as TGF-alpha and TGF-beta), vaccinia growth factor ("VGF"), insulin and insulin-like growth factors I and II, lectins, and apoproteins from low density lipoproteins.

Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the serum of immunized animals. Various procedures well known in the art may be used to generate polyclonal antibodies against an antigen of interest. For example, for the production of polyclonal antibodies, various host animals may be immunized by injection with an antigen of interest or derivative thereof, including, but not limited to, rabbits, mice, rats, and guinea pigs. Depending on the host species, various adjuvants may be used to increase the immune response and include, but are not limited to, freund's (complete and incomplete) adjuvants, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (BCG) and Corynebacterium parvum (corynebacterium parvum). Such adjuvants are also well known in the art.

Useful monoclonal antibodies are homogeneous populations of antibodies directed against a particular antigenic determinant (e.g., a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical, a nucleic acid, or a fragment thereof). Monoclonal antibodies (mabs) directed against the antigen of interest can be prepared by using any technique known in the art for producing antibody molecules from continuous cell line cultures. These techniques include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975,Nature 256,495-497), the human B cell hybridoma technique (Kozbor et al, 1983,Immunology Today 4:72) and the EBV-hybridoma technique (Cole et al, 1985,Monoclonal Antibodies and Cancer Therapy,Alan R.Liss,Inc, pages 77-96). Such antibodies may be of any immunoglobulin class, including IgG, igM, igE, igA and IgD, and any subclass thereof. The mAb-producing hybridomas used in the present disclosure can be cultured in vitro or in vivo.

Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies can be made by any of a number of techniques known in the art (e.g., teng et al, 1983, proc. Natl. Acad. Sci. U.S. A.80,7308-7312; kozbor et al, 1983,Immunology Today 4,72-79; and Olsson et al, 1982, meth. Enzymol.92, 3-16).

The antibody may also be a bispecific antibody. Methods for preparing bispecific antibodies are known in the art. Traditional generation of full length bispecific antibodies is based on co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al, 1983,Nature 305:537-539). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, typically using an affinity chromatography step, is quite cumbersome and the product yields are low. A similar procedure is disclosed in WO 93/08829 and Traunecker et al, EMBO J.10:3655-3659 (1991).

According to various methods, an antibody variable domain (antibody-antigen combining site) having a desired binding specificity is fused to an immunoglobulin constant domain sequence. The fusion may have an immunoglobulin heavy chain constant domain comprising a hinge region, C _H2 And C _H3 At least a portion of the zone. First heavy chain constant region (C _H1 ) May contain the necessary sites for light chain binding, which are present in at least one fusion. Will have the encoded immunoglobulin heavy chain fused Nucleic acids of the sequences of the immunoglobulin light chain and, if desired, of the substance are inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields, this provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments. However, when expressing at least two polypeptide chains in equal ratios yielding high yields or when the ratios are not of particular importance, it is possible to insert the coding sequences of two or all three polypeptide chains in one expression vector.

Bispecific antibodies can have a hybrid immunoglobulin heavy chain (with a first binding specificity) in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from the non-desired immunoglobulin chain combination, since the presence of the immunoglobulin light chain in only half of the bispecific molecule provides an easy way of separation (WO 94/04690; suresh et al Methods in Enzymology,1986,121:210; rodrigues et al 1993,J.of Immunology 151:6954-6961; carter et al 1992, bio/Technology10:163-167; carter et al 1995,J.of Hematotherapy 4:463-470; merchant et al 1998,Nature Biotechnology 16:677-681). Using such techniques, bispecific antibodies can be prepared for conjugation to ADCs in the treatment or prevention of diseases as defined herein.

The hybrid or bifunctional antibodies may be derived biologically (i.e. by cell fusion techniques) or chemically (in particular with cross-linking agents or disulfide-bond forming reagents) and may comprise whole antibodies or fragments thereof (EP 105360; WO 83/03679; EP 217577).

The antibody may be a functionally active fragment, derivative or analogue of an antibody that immunospecifically binds to a cancer cell antigen, a viral antigen or a microbial antigen or other antibody that binds to a tumor cell or matrix. In this regard, "functionally active" means that a fragment, derivative or analog is capable of eliciting an anti-idiotype antibody that recognizes the same antigen as the antibody from which the fragment, derivative or analog was derived. In particular, in exemplary embodiments, the antigenicity of an idiotype of an immunoglobulin molecule may be enhanced by deleting the framework and CDR sequences at the C-terminus of the CDR sequences that specifically recognize the antigen. To determine which CDR sequences bind an antigen, a synthetic peptide containing the CDR sequences can be used in a binding assay with the antigen by any binding assay known in the art (e.g., BIA nuclear assay) (see, e.g., kabat et al, 1991,Sequences of Proteins ofImmunological Interest, fifth edition, national Institute of Health, bethesda, md.; kabat E et al, 1980,J.of Immunology 125 (3): 961-969).

Other useful antibodies include antibody fragments, such as but not limited to F (ab') 2 fragments, which contain a variable region, a light chain constant region, and a heavy chain CH1 domain, which can be produced by pepsin digestion of an antibody molecule; and Fab fragments which can be produced by reduction of the disulfide bonds of the F (ab') 2 fragment. Other useful antibodies are antibody heavy and light chain dimers, or any minimal fragment thereof (such as Fvs or Single Chain Antibodies (SCAs)) (e.g., as described in U.S. Pat. No. 4,946,778;Bird,1988,Science 242:423-42; huston et al, 1988,Proc.Natl.Acad.Sci.USA 85:5879-5883; and Ward et al, (1989) Nature 334:544-54), or any other molecule having the same specificity as the antibody.

In addition, recombinant antibodies (such as chimeric and humanized monoclonal antibodies) comprising human and non-human portions, which can be made using standard recombinant DNA techniques, are useful antibodies. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as molecules having variable regions derived from murine monoclonal antibodies and human immunoglobulin constant regions. (see, e.g., cabin et al, U.S. Pat. No. 4,816,567; and Boss et al, U.S. Pat. No. 4,816,397). Humanized antibodies are antibody molecules derived from non-human species that have one or more Complementarity Determining Regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule. (see, e.g., queen, U.S. Pat. No. 5,585,089) such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, e.g., using the methods described in: WO 87/02671; EP 184,187; EP 171496; EP 173494; WO 86/01533; U.S. Pat. nos. 4,816,567; EP 12023; berter et al, 1988,Science 240:1041-1043; liu et al, 1987,Proc.Natl.Acad.Sci.USA 84:3439-3443; liu et al, 1987, J.Immunol.139:3521-3526; sun et al, 1987,Proc.Natl.Acad.Sci.USA 84:214-218; nishimura et al, 1987, cancer.Res.47:999-1005; wood et al, 1985,Nature 314:446-449; and Shaw et al, 1988,J.Natl.Cancer Inst.80:1553-1559; morrison,1985,Science 229:1202-1207; oi et al, 1986,BioTechniques 4:214; U.S. Pat. nos. 5,225,539; jones et al, 1986,Nature 321:552-525; verhoeye et al (1988) Science 239:1534; and Beidler et al, 1988, J.Immunol.141:4053-4060.

Fully human antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but that can express human heavy and light chain genes. Transgenic mice are immunized in a normal manner with a selected antigen (e.g., all or a portion of a polypeptide of the disclosure). Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma techniques. Transgenic mice have human immunoglobulin transgenes that rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using this technique, it is possible to produce both IgG, igA, igM and IgE antibodies that are therapeutically useful. For an overview of this technology for the production of human antibodies, see Lonberg and Huszar (1995, int. Rev. Immunol. 13:65-93). A detailed discussion of such techniques for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies. See, for example, U.S. patent No. 5,625,126;5,633,425;5,569,825;5,661,016;5,545,806. Other human antibodies are commercially available from, for example, abgenix, inc. (Freemont, calif.) and Genpharm (San Jose, calif.).

Fully human antibodies that recognize selected epitopes can be generated using a technique known as "guided selection". In this approach, the selection of fully human antibodies recognizing the same epitope is guided by the use of selected non-human monoclonal antibodies (e.g., mouse antibodies). (Jespers et al, (1994) Biotechnology 12:899-903). Human antibodies can also be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J.mol. Biol.,227:381 (1991); marks et al, J.mol. Biol.,222:581 (1991)).

An antibody may be a fusion protein of an antibody or a functionally active fragment thereof, e.g., in which the antibody is fused via a covalent bond (e.g., a peptide bond) at the N-terminus or C-terminus to an amino acid sequence of another protein (or a portion thereof, such as a portion of at least 10, 20, or 50 amino acids of a protein) that is not an antibody. The antibody or fragment thereof may be covalently linked to another protein at the N-terminus of the constant region.

Antibodies include analogs and derivatives that are modified by any type of molecule, i.e., covalent attachment, provided that such covalent attachment retains the antibody's antigen-binding immunospecificity. For example, but not by way of limitation, derivatives and analogs of antibodies include derivatives and analogs of antibodies that are further modified, for example, by: glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by any known protecting/blocking group, proteolytic cleavage, linkage to a cellular antibody unit or other protein, and the like. Any of a number of chemical modifications may be made by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, and the like. In addition, the analog or derivative may contain one or more unnatural amino acids.

Antibodies in antibody drug conjugates include antibodies having modifications (e.g., substitutions, deletions, or additions) in amino acid residues that interact with the Fc receptor. In particular, antibodies include antibodies having modifications in amino acid residues identified as being involved in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g., WO 97/34631). Antibodies immunospecific for cancer cell antigens may be obtained commercially, for example, from Genentech (San Francisco, calif.) or produced by any method known to those skilled in the art (e.g., chemical synthesis or recombinant expression techniques). Nucleotide sequences encoding antibodies immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or similar databases, literature publications or by conventional cloning and sequencing.

The antibody of the ADC may be a monoclonal antibody (e.g., a murine monoclonal antibody), a chimeric antibody, or a humanized antibody. The antibody may be an antibody fragment, such as a Fab fragment.

Known anti-CCR 2 antibodies for the treatment or prevention of cancer may be conjugated to ADCs. Antibodies immunospecific for cancer cell antigens are commercially available or produced by any method known to those skilled in the art (e.g., recombinant expression techniques). Nucleotide sequences encoding antibodies immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or similar databases, literature publications or by conventional cloning and sequencing. Examples of antibodies that may be used to treat cancer include, but are not limited to, STI-B020X (anti-CCR2 monoclonal antibody, sorrento Therapeutics), MC-21 (anti-CCR2 humanized antibody, university of Regensburt/MRC; described in European patent No. 2004692, which is incorporated herein by reference), 4.40A68G (Pfizer/Amgen; described in U.S. patent No. 8710191, which is incorporated herein by reference), uniTI-101 (CSF-1R X CCR2 bispecific antibody, elstar Therapeutics), and WO97/31949, which is incorporated herein by reference).

The term "amino acid sequence variant" refers to a polypeptide whose amino acid sequence differs to some extent from that of the native sequence polypeptide. Typically, the amino acid sequence variant should have at least about 70% sequence identity to at least one receptor binding domain of a natural antibody or to at least one ligand binding domain of a natural receptor, and typically it will have at least about 80%, more typically at least about 90% sequence homology to such receptor or ligand binding domain. Amino acid sequence variants have substitutions, deletions and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Amino acids are designated by conventional names, single-letter and three-letter codes.

"sequence identity" is defined as the percentage of identical residues in an amino acid sequence variant after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for alignment are well known in the art. One such computer program is "Align 2", written by Genentech, inc, which was submitted to the U.S. copyright office No. 20559 of Washington, d.c.20559, along with user documentation at 12, 10, 1991.

The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. An exemplary FcR is a native sequence human FcR. Furthermore, fcR may be a receptor that binds an IgG antibody (gamma receptor) and includes receptors of the fcγri, fcγrii and fcγriii subclasses, including allelic variants and alternatively spliced forms of these receptors. Fcyrii receptors include fcyriia ("activating receptor") and fcyriib ("inhibiting receptor") which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activation receptor FcgammaRIIA contains an immunoreceptor tyrosine activation motif in its cytoplasmic domain (immunoreceptor tyrosine-based activation motif; ITAM). The inhibitory receptor fcyriib contains an Immunoreceptor Tyrosine Inhibitory Motif (ITIM) in its cytoplasmic domain. (for reviews see M.Daeron, annu.Rev.Immunol.,15:203-234 (1997)). FcR is reviewed in Ravetch and Kinet, annu. Rev. Immunol.,9:457-92 (1991); capel et al, immunomethods,4:25-34 (1994); and de Haas et al, J.Lab.Clin.Med.,126:330-41 (1995). Other fcrs (including fcrs to be identified in the future) are encompassed herein by the term "FcR. The term also includes the neonatal receptor FcRn, which is responsible for transfer of maternal IgG to the fetus (Guyer et al, J.Immunol.,117:587 (1976) and Kim et al, J.Immunol.,24:249 (1994)).

"complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to cleave a target in the presence of complement. The complement activation pathway is initiated by the binding of a first component of the complement system (C1 q) to a molecule (e.g., an antibody) that is complexed with a cognate antigen. To assess complement activation, CDC assays may be performed, for example, as described in Gazzano-Santoro et al, J.Immunol. Methods,202:163 (1996).

"Natural antibodies" are typically heterotetrameric glycoproteins of about 150,000 daltons, which are composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, whereas in heavy chains of different immunoglobulin isotypes the number of disulfide bonds varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain.

The term "variable" refers to the fact that the sequences of certain portions of the variable domains vary widely between antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed in the antibody variable domains. It focuses on three of the light and heavy chain variable domains in what are termed hypervariable regions. The more highly conserved parts of the variable domains are called Framework Regions (FR). The variable domains of the natural heavy and light chains each comprise four FR, principally in a β -sheet configuration, connected by three hypervariable regions that form a loop connecting the β -sheet structure, and in some cases form part of the β -sheet structure. The hypervariable regions in each chain are held together tightly by the FR and together with the hypervariable regions in the other chain contribute to the formation of the antigen binding site of the antibody (see Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5 th edition Public Health Service, national Institutes of Health, bethesda, md.). The constant domains are not directly involved in binding of antibodies to antigens, but exhibit various effector functions, such as antibody involvement in Antibody Dependent Cellular Cytotoxicity (ADCC).

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions typically comprise amino acid residues of the "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain, and residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; kabat et al, supra) and/or residues of the "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain, and residues 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; chothia and Lesk (1987) J.Mol.biol., 196:901-917). "framework region" or "FR" residues are variable domain residues other than the hypervariable region residues as defined herein.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments (each of which has a single antigen-binding site) and a residue "Fc" fragment (the name of which reflects its ability to crystallize readily). Papain treatment gives F (ab') 2 fragments which have two antigen binding sites and are still capable of cross-linking antigens.

"Fv" is the smallest antibody fragment that contains the complete antigen recognition and antigen binding site. This region consists of a dimer of one heavy and one light chain variable domain in close, non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. In summary, six hypervariable regions confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, but with less affinity than the complete binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments in that several residues are added at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is herein the designation of Fab' wherein the cysteine residue of the constant domain bears at least one free thiol group. F (ab ') 2 antibody fragments were originally generated as paired Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The "light chain" of antibodies from any vertebrate species can be classified into one of two distinct types (called kappa and lambda) based on the amino acid sequence of their constant domains.

"Single chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Fv polypeptides may also comprise polypeptide linkers between the VH and VL domains, which enable the scFv to form the desired structure for antigen binding. For reviews of scFv, see Pluckaphun, the Pharmacology of Monoclonal Antibodies, volume 113, prosenburg and Moore, springer-Verlag, new York, pages 269-315 (1994). anti-ErbB 2 antibody scFv fragments are described in WO 93/16185, U.S. Pat. No. 5,571,894 and 5,587,458.

The term "diabody" refers to a small antibody fragment having two antigen-binding sites, said fragment comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between two domains on the same strand, the domains are forced to pair with the complementary domain of the other strand and create two antigen binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93/11161; hollinger et al (1993) Proc.Natl. Acad.Sci.USA 90:6444-6448.

A "humanized" version of a non-human (e.g., rodent) antibody is a chimeric antibody that contains minimal sequences derived from a non-human immunoglobulin. Humanization is a method of transferring murine antigen binding information to non-immunogenic human antibody receptors and has resulted in a number of therapeutically useful drugs. The humanization process generally begins by transferring all six murine Complementarity Determining Regions (CDRs) into the human antigen framework (Jones et al, (1986) Nature 321:522-525). These CDR-grafted antibodies generally do not retain their original antigen binding affinity, and indeed, affinity is often severely impaired. In addition to the CDRs, select non-human antibody framework residues must be incorporated to maintain the appropriate CDR configuration (Chothia et al (1989) Nature 342:877). It has been demonstrated that transferring critical mouse framework residues to human recipients to support the structural configuration of grafted CDRs restores antigen binding and affinity (Riechmann et al, (1992) J.mol.biol.224,487-499; foote and Winter, (1992) J.mol.biol.224:487-499; prest a et al, (1993) J.Immunol.151,2623-2632; werther et al, (1996) J.Immunol.methods 157:4986-4995; and prest et al (2001) Thromb.Haemost.85:379-389). In most cases, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit or a non-human primate having the desired specificity, affinity, and capability. In some cases, framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibody may comprise residues present in the recipient antibody or the donor antibody. These modifications were made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR is that of a human immunoglobulin sequence. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, for further details, U.S. patent No. 6,407,213; jones et al (1986) Nature 321:522-525; riechmann et al (1988) Nature 332:323-329; and Presta, (1992) curr.op.struct.biol.,2:593-596.

A "parent antibody" is an antibody in which one or more amino acid residues of the amino acid sequence are replaced with one or more cysteine residues. The parent antibody may comprise a native or wild-type sequence. The parent antibody may have pre-existing amino acid sequence modifications (such as additions, deletions, and/or substitutions) relative to other naturally occurring, wild-type, or modified forms of the antibody. The parent antibody is directed against the target antigen of interest. Antibodies directed against non-polypeptide antigens such as tumor-associated glycolipid antigens (see U.S. Pat. No. 5,091,178) are also contemplated.

An "isolated" antibody is an antibody that has been identified and isolated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In certain embodiments, the antibodies will be purified to (1) greater than 95% by weight of the antibody (as determined by the Lowry method) or greater than 99% by weight, (2) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by use of a gas phase protein sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using coomassie blue or silver stains. Isolated antibodies include antibodies that are in situ within recombinant cells, as at least one component of the natural environment of the antibody will not be present. Typically, however, the isolated antibody will be prepared by at least one purification step.

An antibody that "binds" a molecular target or antigen of interest is an antibody that is capable of binding the antigen with sufficient affinity such that the antibody can be used to target cells expressing the antigen.

The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "treatment" may also mean extending survival as compared to expected survival in the case of untreated. The subjects in need of treatment include those already with the condition or disorder and those prone to have the condition or disorder or those in whom the condition or disorder is to be prevented.

"phage display" is a technique whereby a variant polypeptide is displayed as a fusion protein with a coating protein on the surface of a phage (e.g., filamentous phage) particle. One utility of phage display is the fact that libraries of large randomized protein variants can be rapidly and efficiently sorted for sequences that bind to target molecules with high affinity. Display of peptide and protein libraries on phage has been used to screen millions of polypeptides for specific binding properties. Multivalent phage display methods have been used to display small random peptides and small proteins, typically by fusion with PIII or PVIII of filamentous phage. Wells and Low man, curr.Opin.Structure.biol., 3:355-362 (1992), and references cited therein. In monovalent phage display, a library of proteins or polypeptides is fused to a phage coat protein or portion thereof and expressed at low levels in the presence of wild type protein. The avidity effect is reduced relative to multivalent phage, so that sorting is based on intrinsic ligand avidity, and phagemid (phagemid) vectors are used, which simplify DNA manipulation. Lowman and Wells, methods: A companion to Methods in Enzymology,3:205-0216 (1991). Phage display includes techniques for producing antibody-like molecules (janeway, c., convers, p., walport, m., shomchik (2001) Immunobiology, 5 th edition, garland Publishing, new York, pages 627-628).

A "phagemid" is a plasmid vector having a bacterial origin of replication (e.g., co1E 1) and a copy of the intergenic region of the phage. Phagemids can be used on any known phage, including filamentous phage and lambda phage. Plasmids also typically contain selectable markers for antibiotic resistance. The DNA segments cloned into these vectors may be propagated as plasmids. When the cells carrying these vectors have all the genes necessary for the production of phage particles, the replication pattern of the plasmid is changed to rolling circle replication to produce a copy of plasmid DNA and package the phage particles. Phagemids can form infectious or non-infectious phage particles. This term includes phagemids comprising a phage-coated protein gene or fragment thereof linked as a gene fusion to a heterologous polypeptide gene such that the heterologous polypeptide gene is displayed on the surface of a phage particle. The compounds described herein may be in the form of pharmaceutically or pharmaceutically acceptable salts. In some embodiments, such salts are derived from inorganic or organic acids or bases. For reviews of suitable salts see, for example, berge et al, J.Pharm. Sci.,1977,66,1-19 and Remington: the Science and Practice of Pharmacy, 20 th edition, A.Gennaro (ed.), lippincott Williams & Wilkins (2000).

In the present disclosure, the group "Ab" (i.e., antibody fragment, and/or antigen fragment) may be conjugated to more than one drug-containing moiety. In some embodiments, "Ab" may be conjugated to 1 to 20 drug-containing moieties. In some embodiments, "Ab" may be conjugated to 1 to 10 drug-containing moieties. In some embodiments, "Ab" may be conjugated to 1 to 5 drug-containing moieties. In some embodiments, "Ab" may be conjugated to 1 or 2 drug-containing moieties. In some embodiments, "Ab" may be conjugated to one drug-containing moiety.

In some aspects of the disclosure, the ADC is combined with an antibody that binds to PD-1 and/or an antibody that binds to PDL-1.

The compounds described herein may be in the form of pharmaceutically or pharmaceutically acceptable salts. In some embodiments, such salts are derived from inorganic or organic acids or bases. For a review of suitable salts see, for example, bere et al, J.Pharm. Sci.,1977,66,1-19 and Remington: the Science and Practice of Pharmacy, 20 th edition, A.Gennaro (ed.), lippincott Williams & Wilkins (2000).

Examples of suitable acid addition salts include acetates, adipates, alginates, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorite, camphorsulfonate, cyclopentapropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, caproate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectate (pecinate), persulfate, 3-phenylpropionate, bittering, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.

Examples of suitable base addition salts include ammonium salts; alkali metal salts such as sodium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine; and salts with amino acids such as arginine, lysine, and the like.

For example, berge lists the following FDA approved commercial salts: anionic acetate, benzenesulfonate (besylate; benzoates, bicarbonates, bitartrates, bromides, calcium edetate (edetate), camphorsulfonates (camsylates; camphorsulfonate), carbonates, chlorides, citrates, dihydrochloride, edetate (edetate), ethanedisulfonate (1, 2-ethanedisulfonate), lauryl sulfate (estolate; laurel sulfate), ethanesulfonate, fumarate, glucoheptonate, gluconate, glutamates, glycolyl p-aminophenylarsonate, hexylresorcinol, hydrabamine, N '-di- (dehydroabietyl) ethylenediamine, hydrobromide, hydrochloride, hydroxynaphthalene formate, iodide, hydroxyethane sulfonate (2-hydroxyethane sulfonate), lactate, lactose, malate, maleate, mandelate, methanesulfonate, methasulfonate, methyl bromide, methyl nitrate, methyl sulfate, mucinate, naphthalene sulfonate (2-naphthalene sulfonate), nitrate, dihydroxynaphthalene sulfonate, pantothenate, polyaldehyde, hemi-succinate, sulfonate, sulfosuccinate, saussure, and the like, and the cations of (N, N' -dehydroabietyl) sulfonate, sulfoxylate, and the like, n' -dibenzylethylenediamine), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine; and the metal cations aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.

Berge additionally lists the following non-FDA approved commercial (outside the united states) salts: anionic adipates, alginates, aminosalicylates, anhydromethylene citrates, arecolines, aspartate, bisulfate, butylbromide, camphorites, digluconates, dihydrobromites, disuccinates, glycerophosphate, hemisulfates, hydrofluoric acid, hydroiodites, methylenebis (salicylate), napadisylates (1, 5-naphthalenedisulfonate), oxalates, pectates, persulfates, benzene Ding Xianba specific salts of earth acids (phenylethyllbarbitrates), picrates, propionates, thiocyanates, tosylate and undecanoates; organic cations phenethylamine (N-benzyl phenethylamine), clemizole (1-p-chlorobenzyl-2-pyrrolidin-1' -ylmethyl benzimidazole), diethylamine, piperazine and tromethamine (tris (hydroxymethyl) aminomethane); barium and bismuth metal cations.

The compounds described herein may also include suitable carriers, excipients, and auxiliaries, which may vary depending on the manner of administration.

In some embodiments, the pharmaceutical composition may be formulated as a suitable parenteral dosage form. The formulations may be prepared by various methods known in the art. The pharmaceutical composition may be administered directly into the blood stream, muscle or directly into an organ. Suitable means for parenteral administration include intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous administration. Suitable devices for parenteral administration include needle syringes, needleless syringes and infusion techniques.

Parenteral compositions are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffers. However, the composition may also be formulated as a sterile nonaqueous solution or in dry form to be used with a suitable vehicle, such as sterile pyrogen-free water.

Preparation of the parenteral compositions under sterile conditions (e.g., by lyophilization) can be readily accomplished using standard techniques well known to those skilled in the art.

Formulations for parenteral administration may be formulated for immediate release and/or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release and programmed release. Thus, the compositions may be formulated as solid, semi-solid, or thixotropic liquids for administration as an implantable depot providing modified release of the active agent.

Parenteral formulations may be admixed with other pharmaceutically acceptable excipients used in parenteral dosage forms, such as, but not limited to, preservatives.

In another embodiment, the pharmaceutical composition may be formulated into a suitable oral dosage form, such as a tablet, capsule, powder, granule, suspension, solution, emulsion, and the like. Other suitable carriers may be present, such as disintegrants, diluents, chelating agents, binders, glidants, lubricants, fillers, extenders, leavening agents, anti-adherents, and the like.

The oral dosage formulation may also contain other suitable pharmaceutical excipients such as sweeteners, vehicles/wetting agents, colorants, flavors, preservatives, viscosity increasing/thickening agents, and the like.

The dosage of the pharmaceutical composition of the present disclosure can be tailored to the individual patient.

The term "radiation" refers to photon radiation or particle radiation. In some embodiments, the radiation may be photon radiation (x-rays and gamma rays). In such embodiments, the photons may be generated as a high energy photon beam from a radioactive source such as cobalt or a linear accelerator. In some embodiments, the radiation may be particle radiation (such as electrons, protons, neutrons, carbon ions, alpha particles, and beta particles). Particle emission may be generated by a linear accelerator. In some embodiments, the radiation may be an electron beam. In some embodiments, the radiation may be a proton beam. In some embodiments, the radiation may be a neutron beam.

In some embodiments, the radiation may be delivered by external beam radiation. In some embodiments, the external beam radiation may be three-dimensional conformal radiation therapy (3D-CRT). In some embodiments, the external beam radiation may be Intensity Modulated Radiation Therapy (IMRT). In some embodiments, the external beam radiation may be Image Guided Radiation Therapy (IGRT). In some embodiments, the external beam radiation may be modulated intensity proton therapy (IMPT). In some embodiments, the external beam irradiation may be Stereotactic Radiosurgery (SRS). In some embodiments, the external beam therapy may be fractionated stereotactic radiotherapy. In some embodiments, the external beam radiation may be body stereotactic radiotherapy (SBRT). An example of a machine providing SBRT is Gamma And->In some embodiments, radiation may be administered using three-dimensional conformal or body stereotactic radiation therapy delivery.

In some embodiments, the radiation may be delivered by in vivo radiation therapy (internal radiation therapy) (brachytherapy). In such embodiments, the in vivo radiation therapy may be, for example, interval radiation using small particles, seeds, wires, or tubes placed near the cancer or tumor site. In such embodiments, the in vivo radiation therapy may be, for example, intracavity radiation using a radioactive material container that may be placed in a body cavity.

Methods of use of the compounds and compositions

Certain compounds described herein are STING agonists and are therefore useful for stimulating an immune response in a subject thereof. The composition can be used for treating cancer.

The compounds of the present disclosure exhibit STING modulating/agonistic activity. Certain compounds of the present disclosure are excellent in terms of efficacy expression, pharmacokinetics (e.g., absorption, distribution, metabolism, excretion), solubility (e.g., water solubility), interaction with other agents (e.g., drug metabolizing enzyme inhibition), safety (e.g., acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, carcinogenicity, central toxicity), and/or stability (e.g., chemical stability, stability to enzymes), and are useful as agents.

The compounds of the present disclosure are useful for increasing STING activity in a mammal (e.g., mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, human).

The compounds of the present disclosure are useful as agents, such as for the prevention or treatment of diseases that may be affected by STING (sometimes abbreviated herein as "STING-related diseases"), such as cancers, e.g., colorectal cancer (e.g., colorectal cancer, rectal cancer, anal cancer, familial colorectal cancer, hereditary non-polyposis colorectal cancer, gastrointestinal stromal tumor), lung cancer (e.g., non-small cell lung cancer, malignant mesothelioma), mesothelioma, pancreatic cancer (e.g., pancreatic ductal carcinoma, pancreatic endocrine tumor), pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer (e.g., papillary adenocarcinoma, mucous adenocarcinoma, adenosquamous carcinoma), duodenal cancer, small intestine cancer, breast cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast carcinoma), ovarian carcinoma (e.g., ovarian epithelial carcinoma, extragonadal germ cell tumor, ovarian low malignant potential tumor (ovarian low-malignant potential tumor)), testicular tumor, prostate carcinoma (e.g., hormone-dependent prostate carcinoma, non-hormone-dependent prostate carcinoma, castration-resistant prostate carcinoma), liver cancer (e.g., hepatocellular carcinoma, primary liver cancer, extrahepatic cholangiocarcinoma), thyroid carcinoma (e.g., medullary thyroid carcinoma), renal carcinoma (e.g., renal cell carcinoma (e.g., clear cell renal cell carcinoma), transitional cell carcinoma of the renal pelvis and ureter), uterine carcinoma (e.g., cervical carcinoma, uterine sarcoma), pregnancy choriocarcinoma, brain tumor (e.g., medulloblastoma, glioma), uterine cancer, pineal astrocytoma, hairy astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, pituitary adenoma), retinoblastoma, skin carcinoma (e.g., basal cell tumor, malignant melanoma), sarcoma (e.g., rhabdomyosarcoma, leiomyosarcoma, soft tissue sarcoma, spindle cell sarcoma), malignant bone tumor, bladder cancer, blood cancer (e.g., multiple myeloma, leukemia (e.g., acute myelogenous leukemia), malignant lymphoma, hodgkin's disease, chronic myeloproliferative disease), primary unknown cancer; a cancer growth inhibitor; an inhibitor of cancer metastasis; apoptosis promoters; agents for treating pre-cancerous lesions (e.g., myelodysplastic syndrome); etc.

In certain embodiments, the compounds of the present disclosure are useful as agents for colorectal cancer, breast cancer, skin cancer, malignant lymphoma, or lung cancer.

In certain embodiments, the compounds of the present disclosure may be used concurrently with antibody therapy. In some embodiments, the antibody therapy comprises an anti-PD-1 antibody. In some embodiments, the antibody therapy comprises an anti-PD-L1 antibody.

In certain embodiments, the compounds of the present disclosure may be used concurrently with antibody therapy and radiation therapy. In some embodiments, the radiation therapy may be photon radiation therapy. In some embodiments, the radiation therapy may be particle radiation therapy.

Furthermore, the compounds of the present disclosure may be used concurrently with non-drug therapies. In particular, the compounds of the present disclosure or the compositions of the present disclosure may be combined with non-drug therapies such as (1) surgery, (2) hypertension chemotherapy using angiotensin II or the like, (3) gene therapy, (4) hyperthermia, (5) cryotherapy, (6) laser cautery, and (7) radiation therapy.

For example, by using the compound of the present disclosure before or after the above-mentioned surgery or the like, or before or after a combination treatment of two or three thereof, effects such as prevention of occurrence of drug resistance, prolongation of disease-free survival, inhibition of metastasis or recurrence of cancer, prolongation of life, and the like can be obtained.

In some embodiments, the present disclosure relates to a method of treating cancer in a patient by administering to a patient in need of such treatment a compound of formula (I), or a pharmaceutically acceptable salt thereof, in combination with radiation.

In some embodiments, the present disclosure relates to a method of treating cancer in a patient by administering to a patient in need of such treatment a combination of a compound of formula (I), or a pharmaceutically acceptable salt thereof, one or more checkpoint inhibitors, and radiation. In some embodiments, the one or more checkpoint inhibitors comprise an antibody. In some embodiments, the one or more checkpoint inhibitors comprise an anti-PD-1 antibody, in some embodiments, the one or more checkpoint inhibitors comprise an anti-PD-L1 antibody.

In some embodiments, the present disclosure relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in combination with a checkpoint inhibitor and radiation for treating cancer in a patient.

In some embodiments, the present disclosure relates to a composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, for treating cancer in a patient, wherein the patient is also treated with one or more checkpoint inhibitors and radiation. In some embodiments, the present disclosure relates to a composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, for treating cancer in a patient, wherein the compound of formula (I), or a pharmaceutically acceptable salt thereof, is combined with one or more checkpoint inhibitors and radiation. In some embodiments, the compound of formula (I) may be administered simultaneously or sequentially with the checkpoint inhibitor, radiation, and/or a combination thereof. In some embodiments, the present disclosure relates to methods of treating cancer comprising administering to a patient in need of such treatment a therapeutically effective amount of a combination of a compound of formula (I), one or more checkpoint inhibitors, and radiation.

In some embodiments, the radiation may be administered at least 5 hours prior to administration of the checkpoint inhibitor and/or the compound of formula (I). In some embodiments, the radiation may be administered at least 10 hours prior to administration of the checkpoint inhibitor and/or the compound of formula (I). In some embodiments, the radiation may be administered at least 20 hours prior to administration of the checkpoint inhibitor and/or the compound of formula (I). In some embodiments, the radiation may be administered at least 40 hours prior to administration of the checkpoint inhibitor and/or the compound of formula (I). In some embodiments, the radiation may be administered at least 80 hours prior to administration of the checkpoint inhibitor and/or the compound of formula (I).

In some embodiments, the radiation may be administered daily on days 1-5 of the week and repeated for 2 to 8 weeks. In some embodiments, radiation may be administered daily on days 1-5 of the week and repeated for 6 to 8 weeks. In some embodiments, radiation may be administered daily for 2 weeks on days 1-5 of each week. In some embodiments, radiation may be administered daily for 3 weeks on days 1-5 of each week. In some embodiments, radiation may be administered daily for 4 weeks on days 1-5 of each week. In some embodiments, radiation may be administered daily for 5 weeks on days 1-5 of each week. In some embodiments, radiation may be administered daily for 1-5 days per week and repeated for 6 weeks. In some embodiments, radiation may be administered daily for 7 weeks on days 1-5 of each week. In some embodiments, radiation may be administered daily for 8 weeks on days 1-5 of each week.

In some embodiments, radiation may be administered on any two of days 1-5 per week and repeated for 5 to 8 weeks. In some embodiments, radiation may be administered on any two of days 1-5 per week and repeated for 6 to 8 weeks. In some embodiments, radiation may be administered on any two of days 1-5 per week and repeated for 5 weeks. In some embodiments, radiation may be administered on any two of days 1-5 per week and repeated for 6 weeks. In some embodiments, radiation may be administered on any two of days 1-5 per week and repeated for 7 weeks. In some embodiments, radiation may be administered on any two of days 1-5 per week and repeated for 8 weeks.

In some embodiments, the checkpoint inhibitor may be administered once every twelve weeks, once every four weeks, once every three weeks, once every two weeks, once a week, twice a week, three times a week or daily. In some embodiments, the checkpoint inhibitor may be administered once every two weeks. In some embodiments, the checkpoint inhibitor may be administered once every three weeks. In some embodiments, the checkpoint inhibitor may be administered once every four weeks. In some embodiments, the checkpoint inhibitor may be administered once every twelve weeks.

In certain embodiments, the radiation is administered at least 40 hours prior to administration of the checkpoint inhibitor and/or the compound of formula (I). In certain embodiments, the radiation is administered at least 30 hours prior to administration of the checkpoint inhibitor and/or the compound of formula (I). In certain embodiments, the radiation is administered at least 20 hours prior to administration of the checkpoint inhibitor and/or the compound of formula (I). In certain embodiments, the radiation is administered at least 10 hours prior to administration of the checkpoint inhibitor and/or the compound of formula (I). In certain embodiments, the radiation is administered at least 5 hours prior to administration of the checkpoint inhibitor and/or the compound of formula (I). In certain embodiments, the radiation is administered at least 1 hour prior to administration of the checkpoint inhibitor and/or the compound of formula (I).

In some embodiments, the compound of formula (I) and/or checkpoint inhibitor may be administered to the patient 1 day to 3 months after the patient receives radiation therapy. In some embodiments, the compound of formula (I) and/or checkpoint inhibitor may be administered to the patient 1 day to 2 months after the patient receives radiation therapy. In some embodiments, the compound of formula (I) and/or checkpoint inhibitor may be administered to the patient 1 day to 1 month after the patient receives radiation therapy. In some embodiments, the compound of formula (I) and/or checkpoint inhibitor may be administered to the patient from 1 day to 15 days after the patient receives radiation therapy. In some embodiments, the compound of formula (I) and/or checkpoint inhibitor may be administered to the patient from 1 day to 7 days after the patient receives radiation therapy.

In some embodiments, the radiation may be administered in divided doses of about 1Gy to about 100 Gy. In some embodiments, the radiation may be administered in divided doses of about 1Gy to about 50 Gy. In some embodiments, the radiation may be administered in divided doses of about 1Gy to about 20 Gy. In some embodiments, the radiation may be administered in divided doses of about 5Gy to about 20 Gy. In some embodiments, the radiation may be administered in divided doses of about 6Gy to about 18 Gy. In some embodiments, the radiation may be administered in divided doses of about 8Gy to about 16 Gy. In some embodiments, the radiation may be administered in divided doses of about 5Gy to about 10 Gy. In some embodiments, the radiation may be administered in divided doses of about 10Gy to about 15 Gy. In some embodiments, the radiation may be administered in divided doses of about 15Gy to about 20 Gy. In some embodiments, the radiation may be administered in divided doses of about 8Gy or about 16 Gy.

In some embodiments, the radiation may be administered in divided doses of about 1 Gy. In some embodiments, the radiation may be administered in divided doses of about 2 Gy. In some embodiments, the radiation may be administered in divided doses of about 3 Gy. In some embodiments, the radiation may be administered in divided doses of about 4 Gy. In some embodiments, the radiation may be administered in divided doses of about 5 Gy. In some embodiments, the radiation may be administered in divided doses of about 6 Gy. In some embodiments, the radiation may be administered in divided doses of about 7 Gy. In some embodiments, the radiation may be administered in divided doses of about 8 Gy. In some embodiments, the radiation may be administered in divided doses of about 9 Gy. In some embodiments, the radiation may be administered in divided doses of about 10 Gy. In some embodiments, the radiation may be administered in divided doses of about 11 Gy. In some embodiments, the radiation may be administered in divided doses of about 12 Gy. In some embodiments, the radiation may be administered in divided doses of about 13 Gy. In some embodiments, the radiation may be administered in divided doses of about 14 Gy. In some embodiments, the radiation may be administered in divided doses of about 15 Gy. In some embodiments, the radiation may be administered in divided doses of about 16 Gy. In some embodiments, the radiation may be administered in divided doses of about 17 Gy. In some embodiments, the radiation may be administered in divided doses of about 18 Gy. In some embodiments, the radiation may be administered in divided doses of about 19 Gy. In some embodiments, the radiation may be administered in divided doses of about 20 Gy.

In some embodiments, the radiation may be administered in several portions. In some embodiments, the radiation may be administered in 1 to 10 times. In some embodiments, the radiation may be administered in 1 to 5 times. In some embodiments, the radiation may be administered 1, or 2, or 3, or 4, or 5 times. In some embodiments, the radiation may be administered in 1 or 3 times.

In some embodiments, the radiation may be administered 1-3 times in divided doses of about 1-5 Gy. In some embodiments, the radiation may be administered 1-3 times in divided doses of about 5-10 Gy. In some embodiments, the radiation may be administered 1-3 times in divided doses of about 10-15 Gy. In some embodiments, the radiation may be administered 1-3 times in divided doses of about 15-20 Gy. In some embodiments, the radiation may be administered 1-3 times in divided doses of about 5-10Gy or 1-3 times in divided doses of about 15-20 Gy. In some embodiments, the radiation may be administered 1 time in divided doses of about 8 Gy. In some embodiments, the radiation may be administered 3 times in divided doses of about 8 Gy. In some embodiments, the radiation may be administered 1 time in divided doses of about 16 Gy. In some embodiments, the radiation may be administered 1 time at a fractionated dose of about 8Gy, or 3 times at a fractionated dose of about 8Gy, or 1 time at a fractionated dose of about 16 Gy.

In addition, it is possible to combine treatment with a compound of the present disclosure or a combination of the present disclosure with supportive therapy: (i) Administration of antibiotics (e.g., beta-lactams such as pantoprene (pansporin) and the like; macrolides such as clarithromycin and the like) against complications of various infectious diseases; (ii) Administering a high caloric infusion, an amino acid preparation or a general vitamin preparation for the amelioration of malnutrition; (iii) administering morphine for pain relief; (iv) Administering an agent directed to ameliorating side effects (such as nausea, vomiting, anorexia, diarrhea, leukopenia, thrombocytopenia, reduced hemoglobin concentration, hair loss, liver disease, kidney disease, DIC, fever, etc.); and (v) administering an agent against multidrug resistance or the like that inhibits cancer.

Examples

Definition of the definition

Ab antibody

ACN acetonitrile

ADA anti-drug antibodies

ADC antibody drug conjugates

BLQ is below quantization limit

C degree centigrade

CCR 2C-C motif chemokine receptor 2

CR complete reaction

CD cluster of differentiation

DAR drug antibody ratio

DMA N, N-dimethylacetamide

DMSO dimethyl sulfoxide

DTT dithiothreitol

Epsilon extinction coefficient

E0.1% 0.1% solution extinction coefficient

EC ₅₀ Half maximum effective concentration

EDTA ethylenediamine tetraacetic acid

h hours

HIC hydrophobic interaction chromatography

hIgG human immunoglobulin G

HPLC high pressure liquid chromatography

IACUC laboratory animal Care and use Committee

IFN interferon

IgG immunoglobulin G

IgM immunoglobulin M

IL interleukins

IP interferon gamma-induced proteins

LC liquid chromatography

LCMS liquid chromatography-mass spectrometry

Mu M micromolar

MCP monocyte chemotactic protein

MDSC myeloid derived suppressor cells

mL of

MS mass spectrum

Maximum tolerated dose of MTD

NA is not applicable

OAc acetate salt

PBS phosphate buffered saline

PEG polyethylene glycol

QTOF quadrupole time of flight

rt room temperature

SEC size exclusion chromatography

Stimulus of STING interferon gene

TCEP (tris (2-carboxyethyl) phosphine)

TNF tumor necrosis factor

TPPTS 3,3',3 "-phosphanetrialkyltris (benzenesulfonic acid) trisodium salt

Tris (hydroxymethyl) aminomethane

UFLC ultra-fast liquid chromatography

UV ultraviolet light

Analysis method

Analytical SEC conditions:

SEC spectra were recorded at 280nm on either a Hewlett-Packard HP1100 or Agilent1100 series LC system with a diode array detector using a SEC column (typically Tosoh Biosep TSK Gel, G3000SWxl; P/N8541;250A;5um;7.8mm x 300mm). The mobile phase was 100mM sodium phosphate, 300mM sodium chloride (pH 6.8), 10% acetonitrile (v/v) or 1xPBS. A typical run was at a flow rate of 1mL/min for 20min.

Analytical HIC conditions:

HIC spectra were recorded at 280nm on a Hewlett-Packard HP1100 or Agilent1100 series LC system with diode array detector using an HIC column (typically Tosoh Butyl-NPR,4.6x 35mm,2.5um,P/N: 14947). Mobile phase a was 25mM sodium phosphate, 1.5M ammonium sulfate (pH 7), and mobile phase B was 75%25mM sodium phosphate (pH 7), 25% isopropyl alcohol. For a typical 20min run, a 12min linear gradient of 95%/5% a/B to 100% B was used between the initial and final intervals of isocratic flow.

LC-QTOF condition:

an inversion column heated to 80 c (typically Agilent, PLRP-S,5 μm, 2.1 mm. Times.50 mm) and LCMS spectra were recorded. To best characterize the compounds, various gradients and run times were chosen. The mobile phase is based on ACN/water gradient and contains 0.1% formic acid. One example of a solvent gradient used is 95% mobile phase a (mobile phase a=99% water+1% acn+0.1% formic acid) to 100% mobile phase B (mobile phase b=95% acn+5% water+0.1% formic acid), with the conditions shown in table 1.

TABLE 1

The samples were intact or reduced (20 uL 1-5 mg/mL ADC solution treated with 4uL 0.5M DTT at 37℃for 30 min). The raw data was deconvolved over the appropriate mass range using Agilent BioConfirm software to obtain protein molecular weights, and the DAR was calculated using an Agilent DAR calculator.

LC/MS conditions:

LC/MS/MS analysis was performed using a Shimadzu UFLC-20 AD XR binary pump and SIL-30AC MP autosampler system and AB SCIEX Triple Quad 4500ESI mass spectrometry.

Typically, after passing through a Waters Xselect C18 CSH 3.5u 2.1mm ID x 30mm column, a 5uL sample aliquot is injected into the LC/MS/MS. Mobile phase a contained 0.1% formic acid in water and mobile phase B contained 0.1% formic acid in 5% water and 95% acetonitrile. The total run time was 3min at 1.5mL/min with a linear gradient of 100% a to 100% B over a 1.5min flow rate. Initially, the instrument was run at 100% aqueous mobile phase solvent for 0.5min and then increased to 100% organic solvent for the next 1.5 min.

Preparation type SEC:

preparative SEC purification was recorded on a Gilson preparative HPLC system with a UV detector using SEC columns (typically GE Superdex 200Increase 10/300 GL). The mobile phase was 1xPBS (pH 7.4). A typical run was at a flow rate of 1mL/min for 30min. Fraction collection was triggered based on UV threshold (at 214 and 280 nm).

ADC concentration:

after subtracting the UV absorbance of the corresponding linker-payload construct, the ADC concentration was calculated from the UV absorbance at 280nm measured by the NanoDrop (2000c;Fisher Scientific) coefficient.

Table 2 lists linker-payload constructs for ADC preparations. The compound contains compound No. 14 (described in WO2018/100558 A2) or compound I-5c (described in WO 2019/092660) as payload. The synthesis of linker-payloads is described in PCT application PCT/IB 2020/054400.

TABLE 2

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Anti-human CCR2 monoclonal antibodies are produced as described in US 7,473,421 B2, which comprise humanized variable domains of 1D9 mouse monoclonal antibody heavy and light chains and constant domains of human IgG1 heavy and human kappa light chains (humanized 1D9 is also referred to as TAK-202, hereinafter may also be referred to as hig 1 isotype). The hIgG4 isotype of humanized 1D9 was prepared in a manner similar to that described in Anticancer Research, month 3-4, volume 26, phase 2A 1057-1063.

Humanized 1D9 sequences

Heavy chain:

light chain:

humanized 1D9 hIgG4 isotype sequence

Heavy chain:

light chain:

example 1

Procedure for the preparation of Ab-STING agonist conjugates via random cysteine conjugation

To a solution of anti-CCR 2 antibody (humanized 1D9, 10 mg/mL) in 50mM histidine, 125mM arginine and pH 6.1 buffer was added TCEP (in H ₂ 1mM solution in O, 2-3 equivalents). ReactionThe mixture was purged with argon and incubated at rt or 37 ℃ for 1-3h with gentle shaking. The desired linker-payload construct (5 mM solution in DMA, 6-9 eq.) was then slowly added to the mixture. The reaction was purged with argon and incubated at rt or ℃ for an additional 1-2h with gentle shaking. The reaction mixture was purified according to the preparative SEC method described herein to give ADC. The ADC concentration, percent aggregation, and DAR were determined by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

A schematic of this procedure is shown in fig. 1.

Other antibody conjugates were prepared using similar procedures as described above.

Example 2

Preparation of additional Ab-STING agonist conjugates via random cysteine conjugation

Using the linker-payload constructs and antibodies shown as starting materials, the antibody drug conjugates listed in table 3 were prepared as described in example 1.

TABLE 3 Table 3

Example 3

Procedure for the preparation of Ab-STING agonist conjugates via transglutaminase conjugation

Deglycosylation: a solution of anti-CCR 2 antibody (humanized 1D9, generated as described in U.S. Pat. No. 7,473,421 B2, 60 mg/mL) in 50mM histidine, 125mM arginine, pH 6.1 buffer was diluted with an equal volume of pH 7.2 PBS. N-glycosidase F (N-glycydase F) (New England Biolabs, P0704S,500,000 units/mL, 300 units per 1mg of antibody) was added to the solution, and the reaction mixture was heated to 37℃and gently mixed overnight. The resulting deglycosylated humanized 1D9 was buffer exchanged with PBS pH 7.2.

Transglutaminase conjugation: to the deglycosylated humanized 1D9 PBS solution (10-20 mg/mL) prepared above was added a 0.1M DMSO solution of amine-PEG-azide (40 eq.) followed by transglutaminase (ACTIVA) ^TM Ajinomoto, 5-10 mg per 1mg antibody). The reaction mixture was heated to 37 ℃ and gently mixed overnight. The product was purified according to the preparative SEC method described herein to give humanized 1D 9-NH-PEG-azide.

Strain-promoted azide-alkyne cycloaddition: to the solution of the humanized 1D 9-NH-PEG-azide conjugate prepared above (2-15 mg/mL in PBS) was added a solution of strained alkyne containing linker-payload construct in 4-10 mM DMSO (3-5 equivalents, where DMSO < 10% of total solvent volume). The resulting solution was gently stirred at rt overnight. The product was purified according to the preparative SEC method described herein to give ADC. The ADC concentration, percent aggregation, and DAR were determined by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

A schematic diagram of this procedure is shown in fig. 2 (rg=n ₃ ). Other antibody conjugates were prepared using similar procedures as described above.

Example 4

Preparation of Ab-STING agonist conjugates conjugated via transglutaminase

Using the starter linker-payload constructs shown as starting materials in the table, the antibody drug conjugates listed in table 4 were prepared as described in example 3.

TABLE 4 Table 4

Example 5

Transglutaminase conjugation (after modification following the procedure described in Tumey, L.N. et al mol. Pharmaceuticals 2019,16,6,2795-2807): transglutaminase (ACTIVA) ^TM Ajinomoto, 50mg of deglycosylated humanized 1D9 solution in PBS (10-20 mg/mL prepared according to the deglycosylation procedure described in example 3) was added to a solution of 50mg of each 1mg antibody in phosphate buffer pH 6.1, followed by 30mM cystamine.2 HCl (50 eq.) solution in phosphate buffer pH 6.1. The reaction mixture was heated to 37 ℃ and gently mixed overnight. The product was purified using a HiTrap Protein A HP column (GE Healthcare, 17-0402-01) by first washing with 20mM phosphate pH 7.0 and then eluting the ADC with 0.1M citric acid pH 4.0. Further purification according to the preparative SEC methods described herein gives humanized 1D9-NH- (CH) ₂ ) ₂ -S-S-(CH ₂ ) ₂ -NH ₂ 。

Maleimide addition: humanized 1D9-NH- (CH) prepared above at 0deg.C ₂ ) ₂ -S-S-(CH ₂ ) ₂ -NH ₂ To the conjugate solution (2-15 mg/mL in 20mM pH 5NaO Ac buffer) was added 5mM TPPTS solution (5 eq.) in water. The resulting solution was incubated overnight at 0 ℃. After removal of the small molecules by dialysis, the solution was incubated at 0 ℃ for a further 24h. The desired linker-payload construct (5 mM solution in DMA, 2.05 eq.) was then slowly added to the mixture. The reaction was incubated at 0℃for 1.5-2h with gentle shaking. The reaction mixture was purified according to the preparative SEC method described herein to give ADC. The ADC concentration, percent aggregation, and DAR were determined by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

A schematic of this procedure is depicted in fig. 2 (rg=sh). Other antibody conjugates were prepared using similar procedures as described above.

Example 6

Preparation of Ab-STING agonist conjugates conjugated via transglutaminase

Antibody drug conjugates listed in table 5 were prepared as described in example 5 using the starter linker-payload constructs shown as starting materials in the table.

TABLE 5

Example 7

Preparation of Ab-STING agonist conjugates conjugated via transglutaminase

To a solution of deglycosylated humanized 1D9 (10-20 mg/mL, prepared according to the deglycosylation procedure described in example 3) in PBS was added 1M Tris, 5M NaCl pH8.0 buffer (10-20% of the total volume) to adjust the pH to 8.0. To the solution was added a 10mM DMSO solution of the primary amine-containing linker-payload construct (20 eq.) followed by the addition of transglutaminase (ACTIVA ^TM Ajinomoto, 100-150mg per 1mg antibody). The reaction mixture was heated to 37 ℃ and gently mixed overnight. The product was purified using a HiTrap Protein A HP column (GE Healthcare, 17-0402-01) by first washing with 20mM phosphate pH 7.0 and then eluting the ADC with 0.1M citric acid pH 4.0. The product was further purified according to the preparative SEC method described herein to give ADC. The ADC concentration, percent aggregation, and DAR were determined by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

A schematic of the procedure is depicted in fig. 3. Other antibody conjugates were prepared using similar procedures as described above.

Example 8

Preparation of Ab-STING agonist conjugates conjugated via transglutaminase

Using the starter linker-payload constructs shown as starting materials in the table, the antibody drug conjugates listed in table 6 were prepared as described in example 7.

TABLE 6

Example 9

Procedure for preparation of mouse Ab-STING agonist conjugates via random cysteine conjugation

To anti-mCCR 2 MC-21 antibodies (Universitaetsklinikum Regen)sburg, regensburg, germany; described in Mack, M.et al J.Immunol.2001,166,4697-4704 and WO 2007/115713) (mIgG 2a with L235A-G237A-E318A mutation in heavy chain) to a solution in 25mM sodium citrate pH 5.5 buffer (3.4 mg/mL) was added 0.5M tris, 25mM EDTA pH 8 solution (10% of total volume) and TCEP (in H) ₂ 10mM solution in O, 20 eq). The reaction mixture was purged with argon and incubated at 37 ℃ for 1.5h with gentle shaking. The reaction mixture was purified according to the preparative SEC method described herein. The purified reduced antibody solution was cooled to 4 ℃. A solution of dehydroascorbic acid (2 mm,3 equivalents relative to the reduced antibody) in DMSO was added and the resulting mixture was stored overnight at 4 ℃. The solution was warmed to rt and then the desired linker-payload construct (5 mM solution in DMA, 7 eq, relative to the reducing antibody) was slowly added. The reaction was incubated at rt for an additional 1.5-2h with gentle shaking. The reaction mixture was purified according to the preparative SEC method described herein to give ADC. The ADC concentration, percent aggregation, and DAR were determined by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

A schematic of this procedure is depicted in fig. 1.

Example 10

Preparation of additional mouse Ab-STING agonist conjugates via random cysteine conjugation

Antibody drug conjugates listed in table 7 were prepared as described in example 9 using linker-payload constructs and antibodies shown as starting materials.

TABLE 7

Example 11

Plasma stability assay conditions

Test compounds were added to 1mL plasma at a concentration of 10 μg/mL, and then 5 equivalent volume aliquots were dispensed into 2mL Eppendorf microcentrifuge tubes (labeled 0, 24, 48, 72, and 96 hours). For the 0h time point, the tube was erectedI.e. stored at-80 ℃ and the remaining tubes incubated at 37 ℃ with moderate shaking. Aliquots were removed from the incubator at their corresponding time points and stored at-80 ℃. After all samples were collected, they were thawed at rt and placed on wet ice. 50 μl of each sample was dispensed in triplicate into 96-well microtiter plates. The samples were quenched with 200 μl ice-cold methanol containing 50nM internal standard. The sample was vortexed for 2min and then centrifuged at 3000rpm for 10min. Transfer 185 μl of supernatant to a clean injection plate, then at 40deg.C, N ₂ Drying under air. The dried sample extracts were reconstituted with 100 μl LCMS grade water and then vortexed for 1min in preparation for LC-MS/MS analysis.

By reverse phase HPLC, a Synergi 2.5. Mu. Polar-RP 100A C18 column (2.0 mm. Times.30 mm) was usedEach sample was isolated at 40 ℃ using a gradient consisting of 0.1% formic acid in water (solvent a) and 0.1% formic acid in acetonitrile (solvent B). Analytes were detected by positive ion spray in multi-reaction monitoring (MRM) mode using SCIEX API 4500QTRAP instrument. The percent payload loss in human, primate, and mouse plasma at various time points is reported in table 8.

TABLE 8

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Example 12

THP1 double-Lucia reporter gene measurement conditions

THP1-Dual ^TM KI-hSTING-R232 cells (InvivoGen#thpd-R232) were derived from human THP-1 monocytic cells by stable double allele knock-out of the endogenous human HAQ STING gene and knock-in of the R232 variant of human STING. These cells also stably expressed the inducible secreted luciferases reporter under the control of the minimum promoter of ISG54 (interferon stimulatory gene) conjugated to five IFN-stimulatory response elements (ISREs). Expression of the reporter gene allows for assessment of luciferases activity by assessing LuciaTo study the IFN Regulatory Factor (IRF) pathway. In addition to human STING and luciferase, these cells were also engineered to stably express human CCR2, enabling investigation of target-mediated activation of the IRF pathway. THP-1 cells express endogenous human CCR2 at a much lower density than engineered cells that overexpress human CCR2. Thus, empty vector cells were still used as negative controls.

On the day of the experiment, cells were grown in growth medium (RPMI 1640, 2mM L-glutamine, 25mM HEPES, 10% heat-inactivated fetal bovine serum, 100. Mu.g/mL Normocin ^TM 100U/mL-100. Mu.g/mL Pen-Strep, 10. Mu.g/mL blasticidin, 100. Mu.g/mL gecomycin and 1. Mu.g/mL puromycin) were plated in white 384 well plates (Corning 356661) at a density of 15,000 cells/25. Mu.L per well. mu.L of the hCR 2-targeted ADC sample or compound sample was added to the cell plate and then incubated at 37℃for 20 hours. At the end of incubation, 10. Mu.L/well QUANTI-Luc was added ^TM (InvivoGen#rep-qlc 1) and luminescence measured immediately using a LeadSeeker.

For the assay methods described above, the percent luminescence signal induction at various concentrations was calculated for each test ADC or test compound relative to untreated and control treated samples. Fitting a compound concentration versus percent signal induction curve to generate EC ₅₀ Values. Those skilled in the art will appreciate that the production is EC ₅₀ The values of the values are affected by experimental variation. Observed EC ₅₀ And Emax is reported in table 9. The data in table 9 clearly indicate that conjugation of compound No. 14 or compound I-5c to humanized 1D9 or its IgG4 isotype greatly increases in vitro potency in hCCR2 overexpressing THP1 cell lines.

TABLE 9

Example 13

Pharmacokinetic assessment of mice

For in vivo evaluation of ADC in naive Balb/C mice, female Balb/C mice (purchased from Jackson Laboratory) of 6-8 weeks of age were used. Mice were fed a normal diet and housed in an SPF animal facility according to guidelines for laboratory animal care and use, and the institutional care and use committee. Animals were kept at 18-26 ℃ temperature, 50±20% relative humidity, and intermittent light and dark cycles for 12 hours, with food and water ad libitum.

After injection of the ADC into Balb/C mice, the pharmacokinetics of the ADC were studied. Serum samples were taken at different time points and cryopreserved for analysis.

The mouse plasma total antibodies and conjugated payload levels were measured by LC/MS assays based on 2 in 1 immunocapture on a Shimadzu UHPLC system interfaced with a Sciex 6500QTRAP mass spectrometer. Briefly, mouse plasma samples were incubated with anti-human IgG coated magnetic beads for 45min at room temperature, and then non-specifically bound proteins were removed by washing the beads successively with PBST (pH 7.4PBS buffer, containing 0.05% tween 20) and PBS buffer. Thereafter, naked antibody (dar=0) and ADC (dar+.1) were eluted from the beads into 0.1% trifluoroacetic acid. After neutralization of the eluate and addition of the internal standard labeled with stable isotopes, an aliquot of the sample was aspirated and digested with papain at 37 ℃ for 1 hour, then used for LC/MS analysis of the conjugated payload. The remaining samples were subjected to trypsin/lys-C digestion at 70℃for 1 hour and then used for LC/MS analysis of total antibodies.

After plasma protein precipitation, the free payload in the circulation was measured by LC/MS. Briefly, mouse plasma was mixed with 8 volumes of methanol containing stable isotope labeled internal standard, and then the supernatant was evaporated to dryness at 40 ℃ under mild nitrogen flow. Finally, the residue was reconstituted in LC/MS grade water before LC/MS analysis.

The PK profiles for ADC-B14, ADC-B15, ADC-B16, ADC-B17 and ADC-B18 are summarized in Table 10. Graphical representations of plasma PK are shown in figures 4 to 8.

Table 10

Example 14

Tolerance assessment of mice

Tolerability of the ADC was assessed in primary C57BL/6 mice. On study day 0, animals were weighed and then given amounts of ADC (in terms of payload concentration) were administered intravenously. Animals were then weighed periodically for at least 14 days post-dose (no more than 3 days between each measurement), and after each measurement, weight loss was calculated based on the pre-dose starting weight. Any animals that lost more than 20% of weight or were dying or otherwise exhibiting signs of distress beyond the humane endpoint of the study were removed from the study and euthanized according to guidelines within the IACUC protocol. The Maximum Tolerated Dose (MTD) was calculated as the highest dose (in payload concentration) at which no animals died or needed to be removed from the study due to weight loss greater than 20% or otherwise exceeding the humane endpoint. The MTD of ADC-B17 was 200 μg/kg (by payload concentration, FIG. 9), and the MTD of ADC-B20 was 250 μg/kg (by payload concentration, FIG. 10).

Example 15

Evaluation of antitumor Activity in mice

The efficacy of ADC-B21 compared to compound No. 14 was evaluated in a MC38 (murine colon adenocarcinoma) tumor-bearing C57BL/6 mouse model. For tumor implantation, 1x10 ⁶ Individual MC38 cells were subcutaneously injected into C57BL/6 mice and the mice were subsequently monitored for tumor growth. When the tumor volume reached an average of about 100mm ³ At this time, animals were randomized to tumor volume and given 100. Mu.L of vehicle, 2000. Mu.g/kg of compound No. 14 or 50. Mu.g/kg of ADC-B21 intravenously. The first day of dosing was considered study day 0. Compound 14 and vehicle were again administered on study day 3 and day 6, while ADC-B21 was administered as a single administration on study day 0. Tumor volume and body weight measurements were taken at least twice weekly until the end of the study and removal of greater than 20% weight loss or tumor volume exceeding 2000mm compared to the starting body weight ³ Is a natural animal. By study day 63, 1 out of 6 animals treated with compound 14 had complete responses, as compared to 4 out of 6 of the total of ADC-B21 treatments.

A graphical representation of the observed anti-tumor activity is shown in fig. 11, which demonstrates that anti-CCR 2 ADC significantly enhances efficacy at much lower dose levels compared to the payload alone.

Example 16

Toxicity/pharmacodynamics assessment of non-human primate

2 ADC variants were evaluated in toxicity studies in cynomolgus macaque.

Single dose studies were performed with intravenous administration of 0.15, 0.5, 1.5 or 5mg/kg ADC-B2 (2 monkeys/sex/group) (protein dose). Administration of 5mg/kg ADC-B2 was associated with early mortality in both animals at day 2 due to similar pulmonary toxicity (clinical signs of pale mucosa reduction and heart sound reduction in the body; and histological findings of mild pulmonary vascular congestion and acute alveolar hemorrhage, associated with macroscopic redness, intra-alveolar edema and fibrin; increased alveolar macrophage and neutrophil infiltration; and in one animal, pleural and pericardial effusions) as compared to previous studies with unconjugated payload (compound No. 14). Other findings specific to these early dead animals are found in bone marrow (decreased hematopoietic cell necrosis, increased single cell necrosis and histiocytes), liver (multifocal random necrosis lesions) and lymphoid tissue (decreased cellular and/or center necrosis of hair growth in spleen and tonsils) and single cell necrosis in thymus. One clinical pathology and cytokine analysis of an early dead animal (where the samples were available) demonstrated an elevated level of pro-inflammatory/acute phase responses, IP-10, IL-6, MCP 1 and TNF-alpha cytokines (similar to animals that survived to final euthanasia). Histological findings in animals living to final euthanasia were limited to increased cytology of lymph nodes at ≡1.5mg/kg (due to increased lymphocytes and tissue cells) and central necrosis of lymph node germinal sites in the next animal at 5 mg/kg. The pharmacological endpoint added to the study consisted of flow cytometry evaluating the monocyte population and identified on day 1: dose-dependent reduction of the relative percentages of typical, intermediate and non-classical monocytes and Myeloid Derived Suppressor Cells (MDSCs) by day 1, 6 and 24 hours post-dose: partial recovery was achieved 48 hours after dosing.

Repeated dose studies were performed in which it was planned to administer ADC-B17 intravenously every 2 weeks at 0.3, 1 or 3mg/kg (protein dose), for a total of 3 doses (2 monkeys/sex/group); however, since 2 animals in the 3mg/kg dose group died early after the second dose on day 15, the remaining 2 animals in group 4 received a reduced dose of 2mg/kg on day 29 (third/final dose). Repeated administration ≡0.3mg/kg ADC-B17 was associated with 10 of the 12 animals developing anti-drug antibodies (ADA) at 1 or more time points after day 15 (signal to noise ratio increased by 1 to 3 orders of magnitude) mainly against the immunostimulatory payload of ADC, with some ADA towards the antibody component of ADC also observed at the end of the time course. In most ADA positive animals, these ADAs are associated with a decrease in exposure (Cmax) after the third dose. At > 1mg/kg, ADC-B17 related early death was observed. On day 29, approximately 7 hours after dosing, one moribund animal at 1mg/kg was euthanized. At 3mg/kg, 1 animal was found to die about 6 hours after the 15 th day of dosing, and 1 moribund animal was euthanized about 7 hours after the 15 th day of dosing. The ADC-B17 related clinical signs of these animals before death include redness of the skin (face), reduced activity, humpback posture, weight loss, excessive salivation, partial eye closure, depressed eyeballs, elevated body temperature, heart murmurs, and/or elevated heart and/or respiratory rates. The cause of death is due to immune related effects that are thought to be likely due to immunogenic/allergic reactions, but the direct effects of ADC-B17 cannot be excluded. Serum chemistry of all 3 early dead subjects was found to be substantially similar to animals that survived to final euthanasia and consistent with systemic pro-inflammatory responses and muscle and/or hepatocyte damage. Hematology and coagulation parameters were assessed in animals moribund and euthanized on day 29, but animals on day 15 were not assessed; lymphocytes and eosinophils were minimal due to stress and there was no change in coagulation parameters. The observed immunophenotyping changes were similar to those of surviving animals, as described below. Most subtle findings of early dead animals on day 15 were similar to but more severe than those of animals living to final euthanasia and consisted of minimal stem cell necrosis and systemic findings consistent with immune-mediated effects (immune cell infiltration in the liver sinuses, adrenal glands, pulmonary interstitium and spleen, thrombosis of pulmonary capillaries, necrosis/fibrin deposition of the spleen, myocardial degeneration, and microhemorrhages of adrenal and epicardial fat). Other findings specific to early dead persons are believed to be secondary to stress or dying states (reduced thymus cytopenia and pancreatic acinar cell degeneration associated with reduced thymus weight). Of the animals dying and euthanized on day 29, the only finding was minimal adrenal hemorrhage.

In animals living to final euthanasia, clinical pathology findings were observed at > 0.3mg/kg on day 3, consisting of mild to moderate increases of 1 or more of: aspartate aminotransferase, alanine aminotransferase, glutamate dehydrogenase and creatine kinase. These findings are consistent with muscle and/or hepatocyte sources and lack a clear histological correlation. Other findings on day 3 are consistent with the following systemic pro-inflammatory/acute phase responses (minimal to mild increases in globulin and c-reactive protein and minimal to mild decreases in total protein, albumin and albumin/globulin ratio) which are histologically associated with inflammatory cell infiltration in multiple tissues or dehydration (mild increases in urea, creatinine and phosphorus) and not histologically associated. Each of these changes was partially to fully recovered by day 30. The additional serum chemistry changes in males at day 14 and/or day 30 consisted of only a slight increase in globulin and a slight increase in total bilirubin, both consistent with ongoing acute phase inflammatory responses. At final euthanasia on day 30, the hematological and clotting findings of individual animals at > 0.3mg/kg consisted of: white blood count, neutrophil count, fibrinogen and activated partial thromboplastin time were slightly increased and red blood cell count, hemoglobin, hematocrit were slightly decreased. These findings are consistent with systemic pro-inflammatory/acute phase responses.

Changes in monocytes and MDSCs in plasma samples were assessed using flow cytometry panels designed to assess monocyte and MDSC counts and CCR2, CD80 and CD86 expression on monocytes. The findings of this assessment are consistent with the expected pharmacology ≡0.3mg/kg ADC-B17 and consist of: absolute counts of classical monocytes, non-classical monocytes and Myeloid Derived Suppressor Cells (MDSCs) after each dose were reduced slightly to moderately dose-responsive, which returned to or above baseline prior to each subsequent dose, as measured by flow cytometry. After dosing, CCR2 expression was reduced in classical monocytes and all doses returned to near baseline prior to the subsequent dose (fig. 12, upper panel). In addition, CD80 expression in classical monocytes and MDSCs was found to increase after each dose and then to revert to baseline or below baseline levels (fig. 12, middle and bottom, respectively) prior to subsequent dosing.

The change in cytokines in plasma samples was also assessed and shown to consist of the following at ≡0.3mg/kg ADC-B17: the plasma IP-10 and MCP-1 concentrations (potential biomarkers of pharmacology) increased dramatically in dose independence, peaking at 6 hours post-dose and returning or tending to return to baseline values 24 hours post-dose. Additional elevations in serum IL-1RA, IL-6, TNF- α and IFN- γ were observed, peaking at 6 hours post-dose and returning or tending to return to baseline values 24 hours post-dose or prior to the next dose (FIG. 13).

At the final euthanasia, the histological findings of the animals consisted of: multifocal hepatocyte necrosis at > 0.3mg/kg, irrespective of clinical pathology. At ≡1mg/kg, there was minimal to mild reduction in erythrocyte and myeloid precursor in the bone marrow (associated with hematological findings of mild reduction in erythrocytes and significant reduction in lymphocytes in 1 animal), mixed cell infiltration was sporadically observed in the adrenal gland and hepatic sinus, an increase in the cytopenia of the spleen red marrow (mixed cells) associated with a mild increase in spleen weight, and minimal focal bleeding of the duodenum or heart. These organs with inflammatory cell infiltration/hemorrhage are considered likely to be part of the systemic pro-inflammatory response and are not considered direct target organ toxicity. Immunohistochemistry of human IgG, monkey IgG and IgM, C3 and/or C9 was performed to determine if immune complex formation and tissue deposition were present in immune cell infiltration and/or tissue injury areas. No particle deposit indicative of immune complex formation was detected.

Example 17

Pharmacokinetic assessment of non-human primate

Serum samples were taken from non-human primates dosed with ADC-B17 as described in example 16 at different time points and stored frozen for analysis. Monkey plasma total antibodies and conjugated payload levels were measured by LC/MS assays based on 2 in 1 immunocapture on Shimadzu UHPLC system interfaced with Sciex 6500+qtrap mass spectrometer. Briefly, monkey plasma samples were incubated with anti-idiotype antibody coated magnetic beads for 60min at room temperature, then the non-specifically bound proteins were removed by washing the magnetic beads three times with PBS buffer. Thereafter, naked antibody (dar=0) and ADC (dar+.1) were eluted from the beads into 0.1% trifluoroacetic acid. After neutralization of the eluate and incorporation of the stable isotope-labeled internal standard, an aliquot of the sample was aspirated and digested with trypsin/lys-C at 60 ℃ for 1 hour, then used for LC/MS analysis of total antibodies. The remaining samples were subjected to papain digestion at 37 ℃ for 1 hour and then used for LC/MS analysis of the conjugated payloads.

After plasma protein precipitation, the free payload in the circulation was measured by LC/MS. Briefly, stable isotope labeled compound No. 14 was first incorporated into monkey plasma, followed by protein precipitation using methanol, and then the supernatant was evaporated to dryness under a gentle stream of nitrogen. Finally, the residue was reconstituted with ammonium acetate solution before LC/MS analysis.

The PK profile of ADC-B17 is summarized in Table 11. A graphical representation of plasma PK is shown in figure 14.

TABLE 11

Example 18 (prophetic)

Combination therapy with PD-1/PD-L1 antibodies

Tolerability of the ADC in combination with anti-PD-1 and/or anti-PD-L1 antibodies can be assessed in naive C57BL/6 mice.

The ADC and anti-PD-1/anti-PD-L1 combinations that can be used are shown in Table 12.

Table 12

For tolerability studies, ADCs as shown in Table 12 can be administered at 0.05mg/kg, and anti-PD-1 and anti-PD-L1 antibodies can be administered at 0.5, 5 or 50 mg/kg. Since the anti-PD-1 antibody palbociclib did not cross-react with rodent PD-1, mice will receive the rat anti-mouse PD-1 antibody J43 and the rat anti-mouse PD-L1 antibody MIH5, which were administered at 0.5, 5 and 50mg/kg, respectively.

On study day 0, animals may be weighed and then given amounts of ADC in combination with given amounts of anti-PD-1 and/or anti-PD-L1 antibodies administered intravenously. Animals will then be weighed periodically for at least 14 days after dosing (no more than 3 days between each measurement), and after each measurement, weight loss can be calculated based on the pre-dosing initial weight. Any animals that lost more than 20% of weight or that appeared to be dying or otherwise exhibited signs of distress beyond the humane endpoint of the study could be removed from the study and euthanized according to guidelines within the IACUC protocol. The Maximum Tolerated Dose (MTD) can be calculated as the highest dose (in terms of payload concentration + PD-1/PD-L1 antibody concentration) at which no animals die or need to be removed from the study due to weight loss greater than 20% or otherwise exceeding the humane end point. If satisfactory tolerability is achieved in the case of ADC and anti-PD-1 or anti-PD-L1 antibodies, then combination therapy with ADC and anti-PD-1 and anti-PD-L1 antibodies can be performed in a similar manner.

Efficacy study of combination therapies in mice

The efficacy of the combination of ADC with anti-PD-1 antibody J43 or anti-PD-L1 antibody MIH5 as shown in Table 12 can be tested in MC38 (murine colon adenocarcinoma) tumor-bearing C57BL/6 mouse model. For tumor implantation, 1x10 can be used ⁶ Individual MC38 cells were subcutaneously injected into C57BL/6 mice and the mice could then be monitored for tumor growth. When the tumor volume reached an average of about 100mm ³ At this point, animals may be randomized to tumor volume and given 100. Mu.L of vehicle, 50. Mu.g/kg of the corresponding ADC of Table 12, and 0.5, 5 or 50mg/kg of J43 or 0.5, 5 or 50mg/kg of MIH5 intravenously. The first day of dosing can be considered the study day 0. Tumor volume and body weight measurements can be taken at least twice weekly until the end of the study, and body weight loss greater than 20% or tumor volume greater than 2000mm compared to the starting body weight can be removed from the study ³ Is a natural animal. Animals may be assessed for complete and partial response on study day 63. If a satisfactory reduction in tumor volume is not achieved in the case of a combination of ADC with anti-PD-1 or anti-PD-L1 antibodies, then the combination therapy with ADC with anti-PD-1 and anti-PD-L1 antibodies can be performed in a similar manner.

Efficacy studies of combination therapies in non-human primates

The combination of ADC and anti-PD-1 antibody palbociclizumab or anti-PD-L1 antibody alemtuzumab can be administered intravenously to cynomolgus macaques every 2 weeks (day 1 to day 29) at 0.3, 0.5 or 1mg/kg (protein dose) for a total of 3 doses (2 monkeys/sex/group). Palbociclib may be administered at 0.5 or 15mg/kg and alemtuzumab may be administered at 0.5 or 15 mg/kg. Animals can be assessed for hematology and coagulation parameters, general serum chemistry, and histological assessment can be performed at the end of the study.

Blood samples can be taken from non-human primates at different time points, as described above, and monkey plasma total antibodies and conjugated payload levels can be measured by performing LC/MS assays based on 2 in 1 immunocapture on a Shimadzu UHPLC system interfaced with a Sciex 6500+qtrap mass spectrometer. As described above, the free payload in the circulation can be measured by LC/MS after plasma protein precipitation.

Example 19 (prophetic)

Combination therapy with radiation

Tolerability study

Combinations of ADCs, anti-PD-1 and/or anti-PD-L1 with radiation that may be used are shown in table 13.

TABLE 13

For tolerability studies, ADC may be administered at 0.05mg/kg, anti-PD-1 and anti-PD-L1 antibodies may be administered at 0.5, 5 or 50mg/kg, and radiation may be administered at 0.5Gy and 1 Gy.

On study day 0, animals may be weighed, irradiated for about 5 hours, followed by intravenous administration of a specified amount of ADC in combination with a specified amount of anti-PD-1J 43 and/or anti-PD-L1 MIH5 antibody. Animals will then be weighed periodically for at least 14 days after dosing (no more than 3 days between each measurement), and after each measurement, weight loss can be calculated based on the pre-dosing initial weight. Any animals that lost more than 20% of weight or that appeared to be dying or otherwise exhibited signs of distress beyond the humane endpoint of the study could be removed from the study and euthanized according to guidelines within the IACUC protocol. The Maximum Tolerated Dose (MTD) can be calculated as the highest dose of the combination of radiation and treatment regimen when no animal is dead or needed to be removed from the study due to weight loss greater than 20% or otherwise exceeding the humane endpoint. If satisfactory tolerability is achieved in the case of ADC, anti-PD-1 or anti-PD-L1 antibodies and radiation, then combination therapy with ADC and anti-PD-1 and anti-PD-L1 antibodies with radiation can be performed in a similar manner.

Efficacy study in mice in combination with radiation therapy

The efficacy of the combination of ADC with anti-PD-1 and/or anti-PD-L1 with radiation as shown in table 13 can be tested in a MC38 (murine colon adenocarcinoma) tumor-bearing C57BL/6 mouse model. For tumor implantation, 1x10 can be used ⁶ Subcutaneous injection of MC38 cellsInto C57BL/6 mice, and then tumor growth of the mice can be monitored. When the tumor volume reached an average of about 100mm ³ At this time, animals may be randomized to tumor volume and irradiated with 0.5Gy or 1Gy radiation and administered intravenously 100 μl of vehicle, 50 μg/kg of the corresponding ADC of table 13, and 0.5, 5 or 50mg/kg of anti-PD 1 antibody J43 or 0.5, 5 or 50mg/kg of anti-PD-L1 antibody MIH5. The first day of dosing can be considered the study day 0. Tumor volume and body weight measurements can be taken at least twice weekly until the end of the study, and body weight loss greater than 20% or tumor volume greater than 2000mm compared to the starting body weight can be removed from the study ³ Is a natural animal. Animals may be assessed for complete and partial response on study day 63. If a satisfactory reduction in tumor volume is not achieved in the case of combination of ADC with radiation and anti-PD-1 or anti-PD-L1 antibodies, then combination therapy with ADC with radiation and anti-PD-1 and anti-PD-L1 antibodies can be performed in a similar manner.

Efficacy studies in combination with radiation therapy in non-human primates

Cynomolgus macaque can be treated with 0.8Gy and 1.2Gy prior to administration of ADC and anti-PD-1 and/or anti-PD-L1 antibodies. The combination of ADC and anti-PD-1 antibody pamglizumab or anti-PD-L1 antibody alemtuzumab can be administered intravenously to cynomolgus macaques every 2 weeks (day 1 to day 29) at 0.3, 0.5 or 1mg/kg (protein dose) after radiation therapy for a total of 3 doses (2 monkeys/sex/group). Palbociclib may be administered at 0.5 or 15mg/kg and alemtuzumab may be administered at 0.5 or 15 mg/kg. Animals can be assessed for hematology and coagulation parameters, general serum chemistry, and histological assessment can be performed at the end of the study.

Blood samples can be taken from non-human primates at different time points, as described above, and monkey plasma total antibodies and conjugated payload levels can be measured by performing LC/MS assays based on 2 in 1 immunocapture on a Shimadzu UHPLC system interfaced with a Sciex 6500+qtrap mass spectrometer. As described above, the free payload in the circulation can be measured by LC/MS after plasma protein precipitation. Hematological recovery after radiation and extramedullary toxicity can be assessed in animals.

It is to be understood that the detailed description section is intended to explain the claims, and the summary and abstract sections are not intended to be. The summary and abstract sections may set forth one or more, but not all, exemplary embodiments of the present disclosure as contemplated by the inventors, and thus are not intended to limit the disclosure and appended claims in any way.

The present disclosure has been described above with the aid of functional elements illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional components have been arbitrarily defined herein for the convenience of description. Alternate boundaries may be defined so long as the specified functions and relationships thereof are appropriately performed.

The foregoing description of the specific embodiments will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the general concept of the present disclosure. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

1. A compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

a is an integer from 1 to 20;

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein D-L is represented by formula (Ia):

wherein:

represents the point of attachment to Ab;

b is an integer from 1 to 20;

m is 0, 1, 2, 3 or 4;

n is 0 or 1;

R ² selected from C ₁ -C ₄ Alkyl and- (CH) ₂ CH ₂ O) _s -CH ₃ The method comprises the steps of carrying out a first treatment on the surface of the Wherein s is an integer of 1 to 10;

R ³ and R is ³ ' each independently selected from hydrogen and C ₁ -C ₃ An alkyl group; and is also provided with

L ¹ Is a cleavable linker fragment.

3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein:

a is an integer from 1 to 8;

b is an integer from 1 to 10; and is also provided with

m is 0.

4. A compound according to claim 2 or 3, or a pharmaceutically acceptable salt thereof, wherein:

m is 0;

n is 0; and is also provided with

R ³ And R is ³ ' each is hydrogen.

5. The compound of any one of claims 2 to 4, or a pharmaceutically acceptable salt thereof, wherein L ¹ Is that

Wherein:

is the point of attachment to the nitrogen atom of formula (Ia);

is the point of attachment to Ab;

t is an integer from 1 to 10;

w is absent or a self-degrading group;

z is absent or is a peptide of 2 to 5 amino acids;

u and U' are independently absent or a spacer; and is also provided with

Q is a heterobifunctional group;

provided that neither W nor Z are present.

6. The compound of claim 5, or a pharmaceutically acceptable salt thereof, wherein W is a self-degrading group selected from the group consisting of

Wherein:

is the point of attachment to the carbonyl group; and is also provided with

Is the point of attachment to Z.

7. The compound of claim 5 or 6, or a pharmaceutically acceptable salt thereof, wherein W is

8. The compound of any one of claims 5 to 7, or a pharmaceutically acceptable salt thereof, wherein W is

9. The compound of any one of claims 5 to 8, or a pharmaceutically acceptable salt thereof, wherein Z is a peptide capable of being enzymatically cleaved.

10. The compound of any one of claims 5 to 9, or a pharmaceutically acceptable salt thereof, wherein Z is cathepsin cleavable.

11. The compound of any one of claims 5 to 10, or a pharmaceutically acceptable salt thereof, wherein Z is a two amino acid peptide selected from: val-Cit, cit-Val, val-Ala, ala-Val, phe-Lys and Lys-Phe.

12. The compound of any one of claims 5 to 11, or a pharmaceutically acceptable salt thereof, wherein Z is Ala-Val or Val-Ala.

13. The compound of any one of claims 5 to 12, or a pharmaceutically acceptable salt thereof, wherein U' is absent and U is selected from the group consisting of

Wherein:

is the point of attachment to Z;

is the point of attachment to Q; p is an integer from 1 to 6;

q is an integer from 1 to 20; x is O or-CH ₂ -; and each r is independently 0 or 1.

14. The compound of any one of claims 5 to 13, or a pharmaceutically acceptable salt thereof, wherein U' is absent and U is:

15. the compound of any one of claims 5 to 14, or a pharmaceutically acceptable salt thereof, wherein Q is a heterobifunctional group that is linked to U 'or to Ab by chemical or enzyme-mediated conjugation when U' is absent.

16. The compound of any one of claims 5 to 15, or a pharmaceutically acceptable salt thereof, wherein Q is selected from

Wherein the method comprises the steps of

Is the point of attachment to U 'or to Ab when U' is absent.

17. The compound of any one of claims 5 to 16, or a pharmaceutically acceptable salt thereof, wherein Q is:

18. the compound of any one of claims 5 to 17, or a pharmaceutically acceptable salt thereof, wherein t is 1.

19. The compound of any one of claims 2 to 18, or a pharmaceutically acceptable salt thereof, wherein R ² is-CH ₃ And R is ³ And R is ³ ' each is hydrogen.

20. The compound of any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, wherein a is 2 to 6.

21. The compound of any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof, wherein b is 1.

22. The compound of any one of claims 1 to 21, or a pharmaceutically acceptable salt thereof, wherein the amino substituted compound that modulates STING activity is a compound of formula (II):

wherein:

X ¹⁰ is SH or OH;

X ²⁰ is SH or OH;

Y ^a is O, S or CH ₂ ；

Y ^b Is O, S, NH or NR ^a Wherein R is ^a Is C ₁ -C ₄ An alkyl group;

R ¹⁰ is hydrogen, fluorine, OH, NH ₂ 、OR ^b Or NHR ^b ；

R ²⁰ Is hydrogen or fluorine;

R ⁵⁰ Is hydrogen or fluorine;

23. A compound according to any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, wherein the amino substituted compound that modulates STING activity is:

24. A compound according to any one of claims 1 to 21 wherein the amino substituted compound that modulates STING activity is a compound of formula (III):

X ¹⁰ Is SH or OH;

X ²⁰ is SH or OH;

Y ^c is O, S or CH ₂ ；

Y ^D Is O, S or CH ₂ ；

R ¹⁰⁰⁰ Is hydrogen or a bond to a carbonyl group of formula (I);

R ¹⁰⁵ is a hydrogen atom or a substituent;

R ¹⁰⁰ ' is hydrogen or a bond to a carbonyl group of formula (I);

the limiting conditions are:

B ¹⁰⁰ or B is a ²⁰⁰ One of which is linked to 'L' in formula (I) via an amino group.

25. The compound of any one of claims 1 to 21 and 24, or a pharmaceutically acceptable salt thereof, wherein the amino substituted compound that modulates STING activity is a compound of formula (IIIa):

R ¹⁰⁰⁰ is hydrogen or a bond to a carbonyl group of formula (I);

R ¹⁰⁵ is a hydrogen atom or a substituent;

R ¹⁰⁰ ' is hydrogen or a bond to a carbonyl group of formula (I);

the limiting conditions are:

B ¹⁰⁰ or B is a ²⁰⁰ One of them is:

wherein:

R ¹⁸ is hydrogen or C _1-6 An alkyl group; and is also provided with

R ¹⁹ Is a halogen atom;

and the other is linked to the 'L' group in formula (I) via an-NH-group.

26. The compound of any one of claims 1 to 21 and 24, or a pharmaceutically acceptable salt thereof, wherein the amino substituted compound that modulates STING activity is a compound of formula (IV):

or a pharmaceutically acceptable salt thereof, wherein:

R ¹ and R is ² Each independently is a hydroxyl group or a halogen atom;

B ¹ the method comprises the following steps:

R ¹⁸ is hydrogen or C _1-6 An alkyl group;

R ¹⁹ is a halogen atom;

B ² the method comprises the following steps:

and is also provided with

Q ² And Q ⁴ Each independently is an oxygen atom or a sulfur atom.

27. The compound of any one of claims 1 to 21 and 24 to 26, or a pharmaceutically acceptable salt thereof, wherein the amino substituted compound that modulates STING activity is:

or a pharmaceutically acceptable salt thereof, wherein Is the point of attachment to L.

28. The compound of any one of claims 1 to 21 and 24 to 26, or a pharmaceutically acceptable salt thereof, which is a compound of formula (VI):

wherein a is an integer of 1 to 6.

29. The compound of any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody or fragment thereof that binds to human CCR2 or a portion thereof and is capable of blocking the binding of a chemokine to CCR2 and inhibiting the function of CCR 2.

30. The compound of claim 29, or a pharmaceutically acceptable salt thereof, wherein the antibody is selected from the group consisting of: monoclonal antibody 1D9 or an antibody that competes with 1D9 for binding to human CCR2 or a portion of CCR 2; MC-21; STI-B020X; uniTI-101 and 4.40A68G.

31. The compound of claim 30, or a pharmaceutically acceptable salt thereof, wherein the antibody is monoclonal antibody 1D9 or an antibody that competes with 1D9 for binding to human CCR2 or a portion of CCR 2.

32. The compound of any one of claims 1 to 31, or a pharmaceutically acceptable salt thereof, wherein the antibody is a chimeric antibody, a humanized antibody, a human antibody, a mouse antibody, a rat antibody, a goat antibody, or a rabbit antibody.

33. The compound of claim 31, or a pharmaceutically acceptable salt thereof, wherein the anti-CCR 2 antibody, anti-CCR 2 antibody fragment, or anti-CCR 2 antigen binding fragment comprises: a light chain CDR1 comprising amino acids 24-39 of SEQ ID NO. 1; a light chain CDR2 comprising amino acids 55-61 of SEQ ID NO. 1; a light chain CDR3 comprising amino acids 94-102 of SEQ ID No. 1; a heavy chain CDR1 comprising amino acids 31-35 of SEQ ID NO. 2; a heavy chain CDR2 comprising amino acids 50-68 of SEQ ID NO. 2; and a heavy chain CDR3 comprising amino acids 101-106 of SEQ ID NO. 2.

34. The compound of claim 31, or a pharmaceutically acceptable salt thereof, wherein the anti-CCR 2 antibody, anti-CCR 2 antibody fragment, or anti-CCR 2 antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 2.

35. The compound of claim 31, or a pharmaceutically acceptable salt thereof, wherein the antibody, the anti-CCR 2 antibody, anti-CCR 2 antibody fragment, or anti-CCR 2 antigen binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID No. 1.

36. The compound of claim 31, or a pharmaceutically acceptable salt thereof, wherein the anti-CCR 2 antibody, anti-CCR 2 antibody fragment, or anti-CCR 2 antigen binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence SEQ ID No. 2.

37. The compound of claim 31, or a pharmaceutically acceptable salt thereof, wherein the anti-CCR 2 antibody, anti-CCR 2 antibody fragment, or anti-CCR 2 antigen binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises the amino acid sequence SEQ ID No. 1.

38. The compound of claim 31, or a pharmaceutically acceptable salt thereof, wherein the anti-CCR 2 antibody, anti-CCR 2 antibody fragment, or anti-CCR 2 antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 2 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 1.

39. The compound of any one of claims 31 to 38, or a pharmaceutically acceptable salt thereof, wherein the anti-CCR 2 antibody, anti-CCR 2 antibody fragment, or anti-CCR 2 antigen binding fragment further comprises a polypeptide selected from the group consisting of human immunoglobulin IgG ₁ 、IgG ₂ 、IgG ₃ 、IgG ₄ 、IgA ₁ And IgA ₂ Heavy chain constant region of heavy chain constant region.

40. The compound of any one of claims 31-39, or a pharmaceutically acceptable salt thereof, wherein the anti-CCR 2 antibody, anti-CCR 2 antibody fragment, or anti-CCR 2 antigen binding fragment further comprises a light chain constant region selected from the group consisting of human immunoglobulin iggk and iggλ light chain constant regions.

41. The compound of claim 31, or a pharmaceutically acceptable salt thereof, wherein the anti-CCR 2 antibody, anti-CCR 2 antibody fragment, or anti-CCR 2 antigen binding fragment binds the same epitope as an antibody comprising the heavy chain variable region of SEQ ID No. 2 and the light chain variable region of SEQ ID No. 1.

42. The compound of claim 31, or a pharmaceutically acceptable salt thereof, wherein the anti-CCR 2 antibody comprises the heavy chain region of SEQ ID No. 3.

43. The compound of claim 31, or a pharmaceutically acceptable salt thereof, wherein the anti-CCR 2 antibody comprises the light chain region of SEQ ID No. 4.

44. The compound of claim 31, or a pharmaceutically acceptable salt thereof, wherein the anti-CCR 2 antibody comprises a heavy chain region of SEQ ID No. 3 and a light chain region of SEQ ID No. 4.

45. A pharmaceutical composition comprising a compound of any one of claims 1 to 44, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

46. The pharmaceutical composition of claim 45, further comprising an anti-PD-1 antibody.

47. The pharmaceutical composition of claim 46, wherein the anti-PD-1 antibody is selected from the group consisting of: palbociclib, nivolumab, cimetidine Li Shan, picomab, swadarizumab, carrilizumab, singdi Li Shan, tirelib, terlipressin Li Shan, polystataliab, ependymab, incmsga 0012, AMP-224, AMP-514, SYM-021, LZM-009, CS-1003, SYN-125, GNR-051, MW-11, TY-101, BAT-1306, F520, sartan Li Shan, pe An Puli mab, putirib, CX-188, sirolimab, and teporizumab.

48. The pharmaceutical composition of claim 45, further comprising an anti-PD-L1 antibody.

49. The pharmaceutical composition of claim 48, wherein the anti-PD-L1 antibody is selected from the group consisting of: abutilizumab, ablutuzumab, divali You Shan, ke Xili mab, MSB-2311, ZKAB-001, FAZ-053, MDX-1105, CBT-502, IMC-001, RC-98, KL-A167, GR-1405, lodalimab, shu Geli mab, en Wo Lishan antibody, euclidean mab and glipizumab.

50. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutically acceptable amount of a compound of any one of claims 1 to 44.

51. A method for stimulating an immune response in a subject in need thereof, the method comprising administering to the subject a pharmaceutically acceptable amount of a compound of any one of claims 1 to 44.

52. The method of claim 50 or 51, further comprising administering to the subject an anti-PD-1 antibody.

53. The method of claim 50 or 51, further comprising administering to the subject an anti-PD-Ll antibody.

54. The method of claim 52, wherein the anti-PD-1 antibody is selected from the group consisting of: palbociclib, nivolumab, cimetidine Li Shan, picomab, swadarizumab, carrilizumab, singdi Li Shan, tirelib, terlipressin Li Shan, polystataliab, ependymab, incmsga 0012, AMP-224, AMP-514, SYM-021, LZM-009, CS-1003, SYN-125, GNR-051, MW-11, TY-101, BAT-1306, F520, sartan Li Shan, pe An Puli mab, putirib, CX-188, sirolimab, and teporizumab.

55. The method of claim 53, wherein the anti-PD-L1 antibody is selected from the group consisting of: abutilizumab, ablutuzumab, divali You Shan, ke Xili mab, MSB-2311, ZKAB-001, FAZ-053, MDX-1105, CBT-502, IMC-001, RC-98, KL-A167, GR-1405, lodalimab, shu Geli mab, en Wo Lishan antibody, euclidean mab and glipizumab.

56. The method of any one of claims 52-55, wherein the anti-PD-1 antibody or the anti-PD-L1 antibody is administered concurrently with the compound of any one of claims 1-44.

57. The method of any one of claims 52-55, wherein the anti-PD-1 antibody or the anti-PD-L1 antibody is administered sequentially with the compound of any one of claims 1-44.

58. The method of any one of claims 50-57, further comprising administering radiation to the subject.

59. The method of claim 58, wherein the radiation is particle radiation.

60. The method of claim 58 or 59, wherein said radiation is applied by external beam radiation.