Antibody-drug conjugates

Abstract

To provide compounds and compositions that are useful for stimulating an immune response in a subject in need thereof.SOLUTION: The present disclosure provides antibody drug conjugates comprising STING modulators. Also provided are compositions comprising the antibody drug conjugates.SELECTED DRAWING: None

Description

[Technical Field]

[0001] 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. [Background technology]

[0002] Antibody-drug conjugates (ADCs), a rapidly growing class of targeted therapeutics, represent a novel and promising approach to improving drug selectivity and cytotoxic activity. These therapeutic agents are composed of an antibody (or antibody fragment) that can be attached to a payload drug to form an immunoconjugate. The antibody directs the ADC to bind to the targeted cell. The ADC can then be internalized, releasing its payload and resulting in cellular therapy. When the ADC is directed to its targeted cell, the side effects of the conjugated drug may be lower than those seen when the drug is administered systemically.

[0003] The adaptor protein STING (stimulator of interferon genes) has been shown to play a role in the innate immune system. Activating the STING pathway triggers an immune response that leads to the generation of specific killer T cells that shrink tumors, conferring long-lasting immunity and preventing tumor recurrence. An activated STING pathway also contributes to the antiviral response by producing antiviral and proinflammatory cytokines that combat viruses and mobilize both the innate and adaptive immune systems, resulting in long-lasting immunity against pathogenic viruses. The potential therapeutic effect of enhancing both the innate and adaptive immune responses makes STING an attractive target for drug discovery. Cyclic dinucleotides can function as STING agonists and are being tested in clinical trials. However, their anionic properties result in poor membrane permeability, which can limit their ability to engage STING intracellularly, often resulting in unwanted distribution of these compounds in the bloodstream.

[0004] There is a continuing need for novel STING agonists as well as improved methods for delivering the agonists to targeted cells. Summary of the Invention

[0005] In a first aspect, the present disclosure provides a compound of formula (I):

[0006] [ka]

[0007] or a pharmaceutically acceptable salt thereof, wherein

[0008] a is an integer from 1 to 20,

[0009] Ab is an anti-CCR2 antibody, an anti-CCR2 antibody fragment, or an anti-CCR2 antigen-binding fragment;

[0010] D is a modulator of STING activity that contains an amino group on a guanine base, a guanine base derivative, an adenine base, or an adenine base derivative;

[0011] L is a linker that is covalently attached to Ab and also to the amino group on D.

[0012] In a first embodiment of the first aspect, the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein DL is represented by formula (Ia):

[0013] [ka]

[0014] During the ceremony:

[0015] [ka]

[0016] indicates the point of attachment to the Ab,

[0017] b is an integer from 1 to 20;

[0018] m is 0, 1, 2, 3, or 4;

[0019] n is 0 or 1,

[0020] Each R 1 are independently selected from C1-C4 alkyl, O-C1-C4 alkyl, and halogen;

[0021] R 2 is C1-C4 alkyl and -(CH2CH2O) s -CH3, wherein s is an integer of 1 to 10;

[0022] R 3 and R 3’are each independently selected from hydrogen and C1-C3 alkyl;

[0023] L 1 is a cleavable linker fragment.

[0024] In a second embodiment of the first aspect, the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein DL is represented by formula (Ia), wherein:

[0025] a is an integer from 1 to 8,

[0026] b is an integer from 1 to 10,

[0027] m is 0.

[0028] In a third embodiment of the first aspect, the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein DL is represented by formula (Ia), wherein:

[0029] m is 0,

[0030] n is 0,

[0031] R 3 and R 3’ are hydrogen atoms.

[0032] In a third embodiment of the first aspect, the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein DL is represented by formula (Ia): 1 teeth

[0033] [ka]

[0034] and During the ceremony,

[0035] [ka]

[0036] is the point of attachment to the nitrogen atom of formula (Ia),

[0037] [ka]

[0038] is the attachment point to Ab,

[0039] t is an integer from 1 to 10,

[0040] W is absent or a self-immolative group;

[0041] Z is absent or is a peptide of 2 to 5 amino acids;

[0042] U and U' are independently absent or a spacer;

[0043] Q is a heterobifunctional group;

[0044] However, it is not possible for both W and Z to be absent.

[0045] In a fourth embodiment of the first aspect, W is

[0046] [ka]

[0047] is a self-immolative group selected from During the ceremony:

[0048] [ka]

[0049] is the point of attachment to the carbonyl group,

[0050] [ka]

[0051] is the point of attachment to Z.

[0052] In a fifth embodiment of the first aspect, W is

[0053] [ka]

[0054] is.

[0055] In a sixth embodiment of the first aspect, W is

[0056] [ka]

[0057] is.

[0058] In a seventh embodiment of the first aspect Z is an enzymatically cleavable peptide.

[0059] In an eighth embodiment of the first aspect Z is cleavable by a cathepsin.

[0060] In a ninth embodiment of the first aspect Z is a two amino acid peptide selected from Val-Cit, Cit-Val, Val-Ala, Ala-Val, Phe-Lys, and Lys-Phe.

[0061] In a tenth embodiment of the first aspect Z is Ala-Val or Val-Ala.

[0062] In an eleventh embodiment of the first aspect, U′ is absent and U is

[0063] [ka]

[0064] is selected from During the ceremony:

[0065] [ka]

[0066] is the point of attachment to Z,

[0067] [ka]

[0068] is the point of attachment to Q,

[0069] p is an integer from 1 to 6;

[0070] q is an integer from 1 to 20;

[0071] X is O or -CH2-,

[0072] Each r is independently 0 or 1.

[0073] In a twelfth embodiment of the first aspect, U' is absent and U is

[0074] [ka]

[0075] is.

[0076] In a thirteenth embodiment of the first aspect, Q is a heterobifunctional group which is attached to U′ or, if U′ is absent, to Ab by chemical or enzyme-mediated conjugation.

[0077] In a fourteenth embodiment of the first aspect, Q is

[0078] [ka]

[0079] is selected from During the ceremony,

[0080] [ka]

[0081] is the point of attachment to U, or if U is not present, is the point of attachment to Z;

[0082] [ka]

[0083] is the point of attachment to U', or, if U' is absent, to Ab.

[0084] In a fifteenth embodiment of the first aspect, Q is

[0085] [ka]

[0086] is.

[0087] In a sixteenth embodiment of the first aspect, t is 1.

[0088] In a seventeenth embodiment of the first aspect, R 2 is -CH3 and R 3 and R 3’ are hydrogen atoms.

[0089] In an eighteenth embodiment of the first aspect, a is 2-6.

[0090] In a nineteenth embodiment of the first aspect b is 1.

[0091] In a twentieth embodiment of the first aspect, the amino-substituted compound that regulates STING activity is a compound of formula (II):

[0092] [ka]

[0093] During the ceremony:

[0094] X 10 is SH or OH,

[0095] X 20 is SH or OH,

[0096] Y a is O, S, or CH2,

[0097] Y b is O, S, NH, or NR a [In the formula, R a is C1-C4 alkyl;

[0098] R 10 are hydrogen, fluoro, OH, NH2, OR b , or NHR b and

[0099] R 20 is hydrogen or fluoro,

[0100] R 30 is hydrogen and R 40 are hydrogen, fluoro, OH, NH2, OR b , or NHR b or R 30 and R 40 together to form CHO,

[0101] R 50 is hydrogen or fluoro,

[0102] R bis C1-C6 alkyl, halo(C1-C6)alkyl, or C3-C6 cycloalkyl;

[0103] 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 contains at least one N atom in the ring, where Y b is ring A 10 is bonded to a carbon atom of

[0104] 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 contains at least two N atoms in the ring, However, ring A 10 or ring B 10 is attached to "L" in formula (I) via the amino group.

[0105] In a twenty-first embodiment of the first aspect, the amino-substituted compound that regulates STING activity is

[0106] [ka]

[0107] and During the ceremony,

[0108] [ka]

[0109] is the point of attachment to "L" in formula (I).

[0110] In a twenty-second embodiment of the first aspect, the amino-substituted compound that regulates STING activity is a compound of formula (III)

[0111] [ka]

[0112] or a pharmaceutically acceptable salt thereof, wherein:

[0113] X 10 is SH or OH,

[0114] X 20 is SH or OH,

[0115] Y c is O, S, or CH2,

[0116] Y d is O, S, or CH2,

[0117] B 100 is the formula (B 1 -A) or formula (B 1 -B)

[0118] [ka]

[0119] and R 13 , R 14 , R 15 , R 16 , and R 17 are each independently a hydrogen atom or a substituent,

[0120] R 1000 is hydrogen or a bond to the carbonyl group of formula (I),

[0121] Y 11 , Y 12 , Y 13 , Y 14 , Y15 , and Y 16 are each independently N or CR 1a [In the formula, R 1a is hydrogen or a substituent;

[0122] Z 11 , Z 12 , Z 13 , Z 14 , Z 15 , and Z 16 are each independently N or C;

[0123] R 105 is a hydrogen atom or a substituent,

[0124] B 200 is the formula (B 2 -A) or formula (B 2 -B)

[0125] [ka]

[0126] and R 23 , R 24 , R 25 , R 26 , and R 27 are each independently a hydrogen atom or a substituent,

[0127] R 100’ is hydrogen or a bond to the carbonyl group of formula (I),

[0128] Y 21 , Y 22 , Y 23 , Y 24 , Y 25 , and Y 26 are each independently N or CR 2a [In the formula, R 2a is hydrogen or a substituent;

[0129] Z 21 , Z 22 , Z23 , Z 24 , Z 25 , and Z 26 are each independently N or C;

[0130] R 205 is a hydrogen atom or a substituent, and in the formula, R 105 and R 205 are each independently attached to the 2- or 3-position of the 5-membered ring to which they are attached, however,

[0131] B 100 or B 200 is attached to "L" in formula (I) via an amino group.

[0132] In a twenty-third embodiment of the first aspect, the amino-substituted compound that regulates STING activity is a compound of formula (IIIa)

[0133] [ka]

[0134] or a pharmaceutically acceptable salt thereof, wherein: B 100 is the formula (B 1 -A) or formula (B 1 -B)

[0135] [ka]

[0136] and R 13 , R 14 , R 15 , R 16 , and R 17 are each independently a hydrogen atom or a substituent,

[0137] R 1000 is hydrogen or a bond to the carbonyl group of formula (I),

[0138] Y 11 , Y 12 , Y 13 , Y 14 , Y 15 , and Y 16 are each independently N or CR 1a [In the formula, R 1a is hydrogen or a substituent;

[0139] Z 11 , Z 12 , Z 13 , Z 14 , Z 15 , and Z 16 are each independently N or C; R 105 is a hydrogen atom or a substituent,

[0140] B 200 is the formula (B 2 -A) or formula (B 2 -B)

[0141] [ka]

[0142] and R 23 , R 24 , R 25 , R 26 , and R 27 are each independently a hydrogen atom or a substituent,

[0143] R 100’ is hydrogen or a bond to the carbonyl group of formula (I),

[0144] Y 21 , Y 22 , Y 23 , Y 24 , Y 25 , and Y 26 are each independently N or CR 2a [In the formula, R 2a is hydrogen or a substituent;

[0145] Z 21 , Z 22 , Z 23 , Z 24 , Z 25 , and Z 26 are each independently N or C,

[0146] R 205 is a hydrogen atom or a substituent, and in the formula, R 105 and R 205 are each independently attached to the 2- or 3-position of the 5-membered ring to which they are attached, however,

[0147] B 100 or B 200 One of them is

[0148] [ka]

[0149] and During the ceremony:

[0150] R 18 is hydrogen, or C 1-6 is alkyl,

[0151] R 19 is a halogen atom,

[0152] The other is bonded to the "L" group in formula (I) via an --NH-- group.

[0153] In a twenty-fourth embodiment of the first aspect, the amino-substituted compound that regulates STING activity is a compound of formula (IV):

[0154] [ka]

[0155] or a pharmaceutically acceptable salt thereof, wherein:

[0156] R 1 and R 2 are each independently a hydroxy group or a halogen atom,

[0157] B 1 teeth,

[0158] [ka]

[0159] and

[0160] R 18 is hydrogen, or C 1-6 is alkyl,

[0161] R 19 is a halogen atom,

[0162] B 2 teeth,

[0163] [ka]

[0164] and Q 2 and Q 4 are each independently an oxygen atom or a sulfur atom.

[0165] In a twenty-fifth embodiment of the first aspect, the amino-substituted compound that regulates STING activity is

[0166] [ka]

[0167] or a pharmaceutically acceptable salt thereof, wherein:

[0168] [ka]

[0169] is the point of attachment to L.

[0170] In a twenty-sixth embodiment of the first aspect, the present disclosure provides a compound of formula (I) having the structure of formula (VI), or a pharmaceutically acceptable salt thereof:

[0171] [ka]

[0172] In the formula, a is an integer of 1 to 6.

[0173] In a 27th embodiment of the first aspect the 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 CCR2.

[0174] In a 28th embodiment of the first aspect, the antibody is selected from the group consisting of monoclonal antibody 1D9 or an antibody that can compete with 1D9 for binding to human CCR2 or a portion of CCR2; MC-21; STI-B020X; UniTI-101; and 4.40A68G.

[0175] In a 29th embodiment of the first aspect the antibody is monoclonal antibody 1D9 or an antibody capable of competing with 1D9 for binding to human CCR2 or a portion of CCR2.

[0176] In a 30th embodiment of the first aspect 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.

[0177] In a thirty-first embodiment of the first aspect, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment comprises a light chain CDR1 comprising amino acids 24 to 39 of SEQ ID NO: 1; a light chain CDR2 comprising amino acids 55 to 61 of SEQ ID NO: 1; a light chain CDR3 comprising amino acids 94 to 102 of SEQ ID NO: 1; a heavy chain CDR1 comprising amino acids 31 to 35 of SEQ ID NO: 2; a heavy chain CDR2 comprising amino acids 50 to 68 of SEQ ID NO: 2; and a heavy chain CDR3 comprising amino acids 101 to 106 of SEQ ID NO: 2.

[0178] In a thirty-second embodiment of the first aspect the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2.

[0179] In a thirty-third embodiment of the first aspect the antibody, anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:1.

[0180] In a thirty-fourth embodiment of the first aspect, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 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 of SEQ ID NO:2.

[0181] In a thirty-fifth embodiment of the first aspect, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 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 of SEQ ID NO:1.

[0182] In a 36th embodiment of the first aspect, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 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, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 1.

[0183] In a thirty-seventh embodiment of the first aspect, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment further comprises a heavy chain constant region selected from human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 heavy chain constant regions.

[0184] In a thirty-eighth embodiment of the first aspect, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment further comprises a light chain constant region selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions.

[0185] In a thirty-ninth embodiment of the first aspect, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment binds to the same epitope as an antibody comprising the variable heavy chain region of SEQ ID NO:2 and the variable light chain region of SEQ ID NO:1.

[0186] In a fortieth embodiment of the first aspect the anti-CCR2 antibody comprises the heavy chain region of SEQ ID NO:3.

[0187] In a 41st embodiment of the first aspect the anti-CCR2 antibody comprises the light chain region of SEQ ID NO:4.

[0188] In a 42nd embodiment of the first aspect the anti-CCR2 antibody comprises a heavy chain region of SEQ ID NO:3 and a light chain region of SEQ ID NO:4.

[0189] 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.

[0190] In a first embodiment of the second aspect, the pharmaceutical composition comprises a compound of formula (I) and an antibody that binds to programmed cell death 1 (PD-1, CD279, hSLE1, or SLEB2).

[0191] 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 B7H1).

[0192] 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.

[0193] In a first embodiment of the third aspect, a 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.

[0194] In a second embodiment of the third aspect, the method of treating cancer comprises administering to a subject a pharmaceutically acceptable amount of or a pharmaceutically acceptable salt thereof, and an anti-PD-L1 antibody.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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 administered by external beam radiation.

[0200] In a fourth aspect, the present disclosure provides a method of 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. [Brief explanation of the drawings]

[0201] [Figure 1] 1 shows the preparation of Ab-STING agonist conjugates by stochastic cysteine ​​conjugation. [Figure 2] 1 shows the preparation of Ab-STING agonist conjugates by transglutaminase conjugation. [Figure 3] 1 shows the preparation of Ab-STING agonist conjugates by transglutaminase conjugation. [Figure 4] 1 shows the mouse PK profile of antibody drug conjugate B-14. [Figure 5] 1 shows the mouse PK profile of antibody drug conjugate B-15. [Figure 6] 1 shows the mouse PK profile of antibody drug conjugate B-16. [Figure 7] 1 shows the mouse PK profile of antibody drug conjugate B-17. [Figure 8] 1 shows the mouse PK profile of antibody drug conjugate B-18. [Figure 9] 1 shows the change in body weight over time in mice administered with ADC B-17. [Figure 10] 1 shows the change in body weight over time in mice administered with ADC B-20. [Figure 11] 1 shows the antitumor activity of antibody drug conjugate B21 compared to the antitumor activity of the payload alone. [Figure 12] 1 shows changes in CCR2 and CD80 expression on monocytes and MDSCs in non-human primates after administration of antibody drug conjugate B-17. [Figure 13]1 shows changes in serum IL-1RA, IL-6, TNF-α, and IFN-γ in non-human primates after administration of antibody drug conjugate B-17. [Figure 14] 1 shows the non-human primate PK profile of antibody drug conjugate B-17. DETAILED DESCRIPTION OF THE INVENTION

[0202] Unless otherwise defined, 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 publications and patents provided herein are incorporated by reference in their entirety. .

[0203] The singular forms "a," "an," and "the" include plural references unless the context dictates otherwise.

[0204] As used herein, the term "or" refers to a logical or (i.e., and / or) and does not refer to an exclusive or unless expressly indicated by the terms "either," "unless," "or," and words of similar effect.

[0205] As used herein, the term "about" means ±10%.

[0206] Antibody-drug conjugates In some embodiments, the present disclosure provides a compound of formula (I):

[0207] [ka]

[0208] or a pharmaceutically acceptable salt thereof, wherein:

[0209] a is an integer from 1 to 20,

[0210] Ab is an anti-CCR2 antibody, an anti-CCR2 antibody fragment, or an anti-CCR2 antigen-binding fragment;

[0211] D is a modulator of STING activity that contains an amino group on a guanine base, a guanine base derivative, an adenine base, or an adenine base derivative;

[0212] L is a linker that is covalently attached to Ab and also to the amino group on D.

[0213] STING modulator part The present disclosure provides compounds comprising modulators of STING activity. In certain embodiments, the 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, the STING modulator comprises an amino group on a guanine base, a guanine base derivative, an adenine base, or an adenine base derivative. In some embodiments, the STING modulator is a cyclic dinucleotide or a cyclic dinucleotide-like compound (respectively, CDN).

[0214] In some embodiments, the STING modulator is a compound of formula (II)

[0215] [ka]

[0216] or a pharmaceutically acceptable salt thereof, wherein:

[0217] X 10 is -SH or -OH,

[0218] X 20 is -SH or -OH,

[0219] Y a is -O-, -S-, or -CH2-,

[0220] Y b is -O-, -S-, -NH-, or NR a -[wherein, R a is C1-C4 alkyl;

[0221] R 10 are hydrogen, fluoro, -OH, -NH2, -OR b , or -NHR b and

[0222] R 20 is hydrogen or fluoro,

[0223] R 30 is hydrogen and R 40 are hydrogen, fluoro, -OH, -NH2, -OR b , or -NHR b or R 30 and R 40 together form -CH2O-,

[0224] R 50 is hydrogen or fluoro,

[0225] R b is C1-C6 alkyl, halo(C1-C6)alkyl, C3-C6 cycloalkyl,

[0226] 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 contains at least one N atom in the ring, where Y b is ring A 10 is bonded to a carbon atom of

[0227] Ring B 10is an optionally substituted 9- or 10-membered bicyclic heteroaryl ring containing 2-5 heteroatoms selected from N, O, or S, wherein ring B 10 contains at least two N atoms in the ring,

[0228] However, ring A 10 or ring B 10 is attached to "L" in formula (I) via the -NH- group.

[0229] As described herein, ring A 10 and Ring B 10 may contain one or more substituents and thus may be optionally substituted. Suitable substituents on the unsaturated carbon atom of the heteroaryl group include -halo, -NO, -CN, -R + , -C(R + )=C(R + )2, -C≡CR + , -OR + , -SR°, -S(O)R°, -SO2R°, -S O3R + , -SO2N(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°, -NR + CO2R + , -NR + SO2R°, -NR + SO2N(R + )2, -OC(O)R + , -O-CO2R + , -OC(O)N(R + )2, -C(O)R +, -C(S)R°, -CO2R + , -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°)=N-OR + , -P(O)(R + )2, -P(O)(OR + )2, -OP(O)-OR + , and -P(O)(NR + )-N(R + )2[wherein, R + are independently hydrogen or an optionally substituted aliphatic, aryl, heteroaryl, alicyclic, or heterocyclyl group, or two independently occurring R + together with the intervening atom(s) form an optionally substituted 5- to 7-membered aryl, heteroaryl, alicyclic, or heterocyclyl. In some embodiments, R + are independently hydrogen, C 1-6 Aliphatic, or C 3-6 Each R° is independently an optionally substituted aliphatic, aryl, heteroaryl, alicyclic, or heterocyclyl group.

[0230] As detailed above, in some embodiments, two independent R +(or any other variable similarly defined in the specification and claims), taken together with their intervening atom(s), form a monocyclic or bicyclic ring selected from a 3-13 membered alicyclic, a 3-12 membered heterocyclyl having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 6-10 membered aryl, or a 5-10 membered heteroaryl having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0231] In some embodiments, the STING modulator is a compound of formula (IIA)

[0232] [ka]

[0233] or a pharmaceutically acceptable salt thereof, wherein R 10 and R 40 are each independently hydrogen, fluoro, -OH, or -OCH2CF3, and ring A 10 and B 10 is as defined for compounds of formula (Ii), with the proviso that ring A 10 or ring B 10 is bonded to "L" via an -NH- group.

[0234] In some embodiments, ring A 10 is an optionally substituted 6-membered monocyclic heteroaryl ring containing 1, 2, or 3 nitrogen atoms.

[0235] In some embodiments, ring B 10 teeth,

[0236] [ka]

[0237] and During the ceremony:

[0238] Z 10 , Z 20 , Z 30 , and Z 40 are each independently N or CR 200 and

[0239] R 210 is hydrogen, or C1-C6 alkyl, halo(C1-C6)alkyl, or C3-C6 cycloalkyl;

[0240] R 230 is hydrogen, or -NH2,

[0241] R 200 , R 220 , and R 240 are each independently hydrogen, halogen, —OH, —NH 2 , —CN, C 1 -C 6 alkyl, halo(C 1 -C 6 )alkyl, or C 3 -C 6 cycloalkyl.

[0242] In some embodiments, the STING modulator comprises:

[0243] [ka]

[0244] or a pharmaceutically acceptable salt thereof, wherein:

[0245] [ka]

[0246] is the point of attachment to the "L" group of the parent molecular moiety.

[0247] In some embodiments, the STING modulator is a compound of formula (III)

[0248] [ka]

[0249] or a pharmaceutically acceptable salt thereof, wherein:

[0250] X 10 is SH or OH,

[0251] X 20 is SH or OH,

[0252] Y c is O, S, or CH2,

[0253] Y d is O, S, or CH2,

[0254] R 105 and R 205 are each independently hydrogen or a substituent, 105 and R 205 are each independently attached to the 2- or 3-position of the 5-membered ring to which they are attached,

[0255] B 100 is the formula (B 1 -A) or formula (B 1 -B)

[0256] [ka]

[0257] and R 13 , R 14 , R 15 , R 16 , and R 17 are each independently a hydrogen atom or a substituent,

[0258] R 1000 is hydrogen or a bond to the carbonyl group of formula (I),

[0259] Y 11 , Y 12 , Y 13 , Y 14 , Y 15, and Y 16 are each independently N or CR 1a and

[0260] Z 11 , Z 12 , Z 13 , Z 14 , Z 15 , and Z 16 are each independently N or C;

[0261] R 1a is a hydrogen atom or a substituent,

[0262] B 200 is the formula (B 2 -A) or formula (B 2 -B)

[0263] [ka]

[0264] and R 23 , R 24 , R 25 , R 26 , and R 27 are each independently a hydrogen atom or a substituent,

[0265] R 100’ is hydrogen or a bond to the carbonyl group of formula (I),

[0266] Y 21 , Y 22 , Y 23 , Y 24 , Y 25 , and Y 26 are each independently N or CR 2a and

[0267] Z 21 , Z 22 , Z 23 , Z 24 , Z 25 , and Z 26 are each independently N or C;

[0268] R 2a is a hydrogen atom or a substituent,

[0269] However, B 100 or B 200 is attached to the carbonyl group of formula (I) via an -NH- group.

[0270] As described herein, the compounds of formula (III) and formula (IIIa) (described below) contain substituents at specific positions. Suitable substituents include halogen atoms, cyano groups, nitro groups, optionally substituted hydrocarbon groups, optionally substituted heterocyclic 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 (SH) groups, and optionally substituted silyl groups, where the optionally substituted groups have one or more substituents selected from the following substituent group A: "Substituent group A": (1) a halogen atom, (2) a nitro group, (3) a cyano group, (4) an oxo group, (5) a hydroxy group, (6) Optionally halogenated C 1-6 alkoxy groups, (7) C 6-14 aryloxy groups (e.g., phenoxy, naphthoxy); (8) C 7-16 aralkyloxy groups (e.g., benzyloxy), (9) 5- to 14-membered aromatic heterocyclyloxy groups (e.g., pyridyloxy), (10) 3- to 14-membered non-aromatic heterocyclyloxy groups (e.g., morpholinyloxy, piperidinyloxy), (11)C 1-6 alkyl-carbonyloxy groups (e.g., acetoxy, propanoyloxy); (12)C 6-14aryl-carbonyloxy groups (e.g., benzoyloxy, 1-naphthoyloxy, 2-naphthoyloxy); (13)C 1-6 alkoxy-carbonyloxy groups (e.g., methoxycarbonyloxy, ethoxycarbonyloxy, propoxycarbonyloxy, butoxycarbonyloxy); (14) Mono- or di-C 1-6 alkyl-carbamoyloxy groups (e.g., methylcarbamoyloxy, ethylcarbamoyloxy, dimethylcarbamoyloxy, diethylcarbamoyloxy); (15)C 6-14 aryl-carbamoyloxy groups (e.g., phenylcarbamoyloxy, naphthylcarbamoyloxy); (16) 5- to 14-membered aromatic heterocyclylcarbonyloxy groups (e.g., nicotinoyloxy), (17) 3- to 14-membered non-aromatic heterocyclylcarbonyloxy groups (e.g., morpholinylcarbonyloxy, piperidinylcarbonyloxy), (18) Optionally halogenated C 1-6 alkylsulfonyloxy groups (e.g., methylsulfonyloxy, trifluoromethylsulfonyloxy); (19)C 1-6 C optionally substituted with alkyl groups 6-14 arylsulfonyloxy groups (e.g., phenylsulfonyloxy, toluenesulfonyloxy); (20) Optionally halogenated C 1-6 alkylthio groups, (21) a 5- to 14-membered aromatic heterocyclic group, (22) a 3- to 14-membered non-aromatic heterocyclic group, (23) a formyl group, (24) a carboxy group, (25) Optionally halogenated C 1-6 alkyl-carbonyl groups, (26)C 6-14 aryl-carbonyl groups, (27) a 5- to 14-membered aromatic heterocyclylcarbonyl group, (28) a 3- to 14-membered non-aromatic heterocyclylcarbonyl group, (29)C 1-6 alkoxy-carbonyl groups, (30)C 6-14 aryloxy-carbonyl groups (e.g., phenyloxycarbonyl, 1-naphthyloxycarbonyl, 2-naphthyloxycarbonyl); (31)C 7-16 aralkyloxy-carbonyl groups (e.g., benzyloxycarbonyl, phenethyloxycarbonyl), (32) a carbamoyl group, (33) a thiocarbamoyl group, (34) Mono- or di-C 1-6 alkyl-carbamoyl groups, (35)C 6-14 aryl-carbamoyl groups (e.g., phenylcarbamoyl); (36) 5- to 14-membered aromatic heterocyclylcarbamoyl groups (e.g., pyridylcarbamoyl, thienylcarbamoyl), (37) 3- to 14-membered non-aromatic heterocyclylcarbamoyl groups (e.g., morpholinylcarbamoyl, piperidinylcarbamoyl), (38) Optionally halogenated C 1~6 alkylsulfonyl groups, (39)C 6-14 arylsulfonyl groups, (40) 5- to 14-membered aromatic heterocyclylsulfonyl groups (e.g., pyridylsulfonyl, thienylsulfonyl), (41) Optionally halogenated C 1-6 alkylsulfinyl groups, (42)C 6-14 arylsulfinyl groups (e.g., phenylsulfinyl, 1-naphthylsulfinyl, 2-naphthylsulfinyl); (43) 5- to 14-membered aromatic heterocyclylsulfinyl groups (e.g., pyridylsulfinyl, thienylsulfinyl), (44) amino group, (45) Mono- or di-C 1-6alkylamino groups (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); (47) 5- to 14-membered aromatic heterocyclylamino groups (e.g., pyridylamino), (48)C 7-16 aralkylamino groups (e.g., benzylamino), (49) formylamino group, (50)C 1-6 alkyl-carbonylamino groups (e.g., acetylamino, propanoylamino, butanoylamino); (51)(C 1-6 Alkyl)(C 1-6 alkyl-carbonyl)amino groups (e.g., N-acetyl-N-methylamino), (52)C 6-14 aryl-carbonylamino groups (e.g., phenylcarbonylamino, naphthylcarbonylamino); (53)C 1-6 alkoxycarbonylamino groups (e.g., methoxycarbonylamino, ethoxycarbonylamino, propoxycarbonylamino, butoxycarbonylamino, tert-butoxycarbonylamino), (54)C 7-16 aralkyloxy-carbonylamino groups (e.g., benzyloxycarbonylamino), (55)C 1-6 alkylsulfonylamino groups (e.g., methylsulfonylamino, ethylsulfonylamino); (56)C 1-6 C optionally substituted with alkyl groups 6-14 arylsulfonylamino groups (e.g., phenylsulfonylamino, toluenesulfonylamino); (57) Optionally halogenated C 1-6 alkyl groups, (58)C 2-6 alkenyl groups, (59)C 2-6 alkynyl groups, (60)C 3-10 cycloalkyl groups, (61)C 3-10 cycloalkenyl groups, and (62)C 6-14 Aryl groups.

[0271] In some embodiments, the STING modulator is a compound of formula (IIIa), or a pharmaceutically acceptable salt thereof:

[0272] [ka]

[0273] or a pharmaceutically acceptable salt thereof, wherein: B 100 is the formula (B 1 -A) or formula (B 1 -B)

[0274] [ka]

[0275] and R 13 , R 14 , R 15 , R 16 , and R 17 are each independently a hydrogen atom or a substituent, R 1000 is hydrogen or a bond to the carbonyl group of formula (I), Y 11 , Y 12 , Y 13 , Y 14 , Y 15 , and Y 16 are each independently N or CR 1a [In the formula, R 1a is hydrogen or a substituent; Z 11 , Z 12 , Z 13 , Z14 , Z 15 , and Z 16 are each independently N or C; R 105 is a hydrogen atom or a substituent, B 200 is the formula (B 2 -A) or formula (B 2 -B)

[0276] [ka]

[0277] and R 23 , R 24 , R 25 , R 26 , and R 27 are each independently a hydrogen atom or a substituent, R 100’ is hydrogen or a bond to the carbonyl group of formula (I), Y 21 , Y 22 , Y 23 , Y 24 , Y 25 , and Y 26 are each independently N or CR 2a [In the formula, R 2a is hydrogen or a substituent; Z 21 , Z 22 , Z 23 , Z 24 , Z 25 , and Z 26 are each independently N or C; R 205 is a hydrogen atom or a substituent, and in the formula, R 105 and R 205 are each independently attached to the 2- or 3-position of the 5-membered ring to which they are attached, however, B 100 or B 200 One of them is

[0278] [ka]

[0279] and During the ceremony: R 18 is hydrogen, or C 1-6 is alkyl, R 19 is a halogen atom, The other is bonded to the carbonyl group of formula (I) via an -NH- group.

[0280] In some embodiments, the STING modulator is a compound of formula (IV), or a pharmaceutically acceptable salt thereof:

[0281] [ka]

[0282] or a pharmaceutically acceptable salt thereof, wherein: R 1 and R 2 are each independently a hydroxy group or a halogen atom, B 1 teeth,

[0283] [ka]

[0284] and; R 18 is hydrogen, or C 1-6 is alkyl, R 19 is a halogen atom, B 2 teeth,

[0285] [ka]

[0286] and; Q 2 and Q 4 are each independently an oxygen atom or a sulfur atom.

[0287] In some embodiments, the cyclic dinucleotide is

[0288] [ka]

[0289] or a pharmaceutically acceptable salt thereof, wherein:

[0290] [ka]

[0291] is the "L" point.

[0292] Linker part The group "L" is a linker. As used herein, the term "linker" refers to any chemical moiety capable of attaching an antibody, antibody fragment, or antigen-binding fragment (Ab) to a drug-containing moiety in the compounds of Formula (I) and (IV). The linker can be branched and substituted with 1 to 20 drug-containing moieties. In some embodiments, the linker can be substituted with 1 to 10 drug-containing moieties. In some embodiments, the linker can be substituted with 1 to 5 drug-containing moieties. In some embodiments, the linker can be substituted with 1 or 2 drug-containing moieties. In some embodiments, the linker can be substituted with 1 drug-containing moiety.

[0293] In some embodiments, the linker "L" is a cleavable linker. In embodiments, the linker is likely to undergo acid-induced cleavage, photo-induced cleavage, enzymatic cleavage, etc. under conditions that allow the drug and / or antibody to remain active. In some embodiments, the cleavable linker can be cleaved by an enzyme. In some embodiments, the cleavable linker can be cleaved by a protease, peptidase, esterase, glycosidase, phosphodiesterase, phosphatase, or lipase. In some embodiments, the cleavable linker can be cleaved by a protease. Examples of proteases include, but are not limited to, cathepsin B, VAGP tetrapeptide, etc.

[0294] In certain embodiments, the linker can be any of those disclosed in PCT Publications WO2018 / 200812, WO2018 / 100558, which are incorporated by reference in their entireties.

[0295] In certain embodiments, "L" is of the formula:

[0296] [ka]

[0297] and During the ceremony:

[0298] [ka]

[0299] is the point of attachment to the nitrogen atom,

[0300] [ka]

[0301] is the point of attachment to the Ab.

[0302] In some embodiments, "L" is of the formula:

[0303] [ka]

[0304] and During the ceremony:

[0305] [ka]

[0306] is the point of attachment to the nitrogen atom,

[0307] [ka]

[0308] is the point of attachment to the antibody.

[0309] The group "W" is absent or a self-immolative group. As used herein, the term "Self-immolative" refers to a group that undergoes an electronic cascade, resulting in the release of the group to which it is attached. In some embodiments, the self-immolative group comprises one or more groups capable of undergoing 1,4-elimination, 1,6-elimination, 1,8-elimination, 1,6-cycloelimination, 1,5-cycloelimination, 1,3-cycloelimination, intramolecular 5-exo-trigger cyclization, and / or 6-exo-trigger cyclization. In certain embodiments, the self-immolative group can be any of those disclosed in PCT Publications WO2018 / 200812, WO2018 / 100558, which are incorporated by reference in their entireties.

[0310] The group "Z" is either absent or is a peptide of 2 to 5 amino acids. In certain embodiments, the peptide is the cleavage site of the linker, thereby facilitating release of the drug upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al. al. (2003) Nat. Biotechnol. 21:778-784). Examples of peptides with two amino acids include alanine-alanine (Ala-Ala), valine-alanine (VA or Val-Ala), valine-citrulline (VC or Val-Cit), alanine-phenylalanine (AF or Ala-Phe); Examples of peptides having three amino acids include, but are not limited to, N-methyl-valine-citrulline (Me-Val-Cit), N-phenylalanine-lysine (FK or Phe-Lys), N-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, N-methyl-valine-citrulline (Me-Val-Cit) and N-methyl-valine-citrulline (Gly ...). The above combinations of amino acids can also occur in the reverse order (i.e., Cit-Val).

[0311] The peptides of the present disclosure can include naturally occurring and / or unnatural 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 not occurring in nature) include, by non-limiting example, homoserine, homoarginine, citrulline, phenylglycine, taurine, iodotyrosine, seleno-cysteine, norleucine ("Nle"), norvaline ("Nva"), beta-alanine, L- or D-naphthalenine, ornithine ("Orn"), and the like. Peptides can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0312] Amino acids also include D-forms of natural and unnatural amino acids. "D-" denotes an amino acid having the "D" (dextrorotatory) configuration, as opposed to the configuration in natural ("L-") amino acids. Natural and unnatural amino acids can be obtained commercially (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

[0313] The groups "U" and "U'" are independently absent or a spacer. As used herein, the term "spacer" refers to a chemical moiety that functions as a connector. In the present disclosure, a spacer can connect an antibody, antibody fragment, or antigen fragment to the heterobifunctional group and / or can connect the heterobifunctional group to the peptide "Z" or, if "Z" is absent, to the group "W." Non-limiting exemplary spacers include -NH-, -S-, -O-, -NHC(=O)CH2CH2-, -S(=O)2-CH2CH2-, -C(=O)NHNH - , -C(=O)O-, -C(=O)NH-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2=CH2-, -C≡C-, -CH=NO-, polyethylene glycol (PEG),

[0314] [ka]

[0315] Examples include:

[0316] In compounds of the present disclosure, when "U" is present, it can be a branched chain group substituted with 1 to 10 "-C(O)-WZ-" groups. In some embodiments, "U" is substituted with 1 to 5 "-C(O)-WZ-" groups. In some embodiments, "U" is substituted with 1 or 2 "-C(O)-WZ-" groups. In some embodiments, "U" is substituted with 1 "-C(O)-WZ-" group. In certain embodiments, the spacer can be any of those disclosed in PCT Publications WO2018 / 200812, WO2018 / 100558, which are incorporated by reference in their entireties.

[0317] The group "Q" is a heterobifunctional group. In the present disclosure, the term "heterobifunctional group" refers to a chemical moiety that connects a linker to an antibody, antibody fragment, or antigen-binding fragment. See, for example, WO2017 / 191579. Heterobifunctional groups are characterized by having different reactive groups at both ends of the chemical moiety. The heterobifunctional group can be directly attached to an "Ab" or, alternatively, can be attached via a linker "U." Attachment to an "Ab" can be achieved by chemical or enzymatic conjugation, or a combination of both. Chemical conjugation involves the controlled reaction of accessible amino acid residues on the surface of the antibody with reactive handles at "Q" or "U." Examples of chemical conjugation include, but are not limited to, lysine amide coupling, cysteine ​​coupling, and coupling via recombinantly incorporated non-natural amino acids, where non-natural amino acid residues with the desired reactive handles are introduced into the "Ab." In enzymatic conjugation, an enzyme mediates the coupling of a linker to an accessible amino residue on an antibody, antibody fragment, or antigen-binding fragment. Examples of enzymatic conjugation include, but are not limited to, transpeptidation using sortase, transpeptidation using bacterial transglutaminase, and N-glycan recombination. Chemical conjugation and enzymatic conjugation can also be used sequentially. For example, enzymatic conjugation can be used to introduce a unique reactive handle on an "Ab" that is utilized in subsequent chemical conjugation. In certain embodiments, heterologous conjugation can be used to introduce a unique reactive handle on an "Ab" that is utilized in subsequent chemical conjugation. The bifunctional group can be any of those disclosed in PCT Publications WO2018 / 200812, WO2018 / 100558, which are incorporated by reference in their entireties.

[0318] In some embodiments, "Q" is

[0319] [ka]

[0320] is selected from During the ceremony,

[0321] [ka]

[0322] is the point of attachment to U, or if U is not present, is the point of attachment to Z;

[0323] [ka]

[0324] is the point of attachment to U', or, if U' is absent, to Ab.

[0325] In certain embodiments, the present disclosure provides a compound of formula (XX):

[0326] [ka]

[0327] 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 conjugating to an antibody, antibody fragment, or antigen-binding fragment. *Exemplary groups include, but are not limited to, activated carboxylic acid groups such as acid chlorides -C(O)-Cl, acid anhydrides, haloacetamides, maleimides, alkynes, cycloalkynes such as cyclooctynes, oxanorborazines, norbornenes, azides, diaryltetrazines, monoaryltetrazines, aldehydes, ketones, hydroxylamines, vinyl sulfones, and aziridines. In certain embodiments, the reactive functional group can be any of those disclosed in PCT Publications WO2018 / 200812, WO2018 / 100558, which are incorporated by reference in their entireties.

[0328] Anti-CCR2 antibodies, antibody fragments, and antigen-binding fragments The group "Ab" refers to an anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment. An antibody is a protein produced by the immune system that can recognize and bind to a specific antigen. A target antigen generally has multiple binding sites, also called epitopes, recognized by the CDRs of multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have two or more corresponding antibodies. The term "antibody" is used in the broadest sense herein and specifically includes monoclonal antibodies, single-domain antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they exhibit the desired biological activity. Antibodies may be murine, human, humanized, chimeric, or derived from other species. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York).

[0329] Useful anti-CCR2 antibodies, antibody fragments, and antigen-binding fragments include antibodies (immunoglobulins) or functional fragments (e.g., antigen-binding fragments) thereof that bind to mammalian CC-chemokine receptor 2 (also known as CCR2, CKR-2, CD192, MCP-1RA, or MCP-1RB) or a portion of the receptor. In one embodiment, the antibody or fragment has specificity for human or rhesus monkey 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 the receptor and inhibits a function associated with ligand binding to the receptor (e.g., leukocyte trafficking). For example, as described herein, antibodies and fragments thereof useful in the present disclosure can bind to human or rhesus monkey CCR2 or a portion thereof, block the binding of a chemokine (e.g., MCP-1, MCP-2, MCP-3, MCP-4) to the receptor, and inhibit a function associated with chemokine binding to the receptor. In one embodiment, the antibody is monoclonal antibody (mAb) LS132.1D9 (1D9) or an antibody that can compete with 1D9 for binding to human CCR2 or a portion of human CCR2. Functional fragments of the foregoing antibodies are also contemplated.

[0330] In some embodiments, a humanized immunoglobulin or antigen-binding fragment thereof having binding specificity for CCR2 is used, wherein the immunoglobulin comprises 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 regions, human gamma-type constant region). In one embodiment, the humanized immunoglobulin or fragment thereof can compete 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 the light and heavy chain variable regions shown below).

[0331] For example, a humanized immunoglobulin or antigen-binding fragment thereof can comprise an antigen-binding region comprising at least one complementarity-determining region (CDR) of non-human origin, and framework regions (FR) derived from human framework regions. The humanized immunoglobulin with binding specificity comprises a light chain comprising at least one CDR derived from an antibody of non-human origin that binds to CCR2 and FRs derived from a light chain of human origin (e.g., from HF-21 / 28), and a heavy chain comprising a CDR derived from an antibody of non-human origin that binds to CCR2 and FRs derived from a heavy chain of human origin (e.g., from 4B4'CL). In another embodiment, 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.

[0332] In one embodiment, a humanized immunoglobulin with binding specificity for CCR2 comprises CDR1, CDR2, and CDR3 of the light chain of the 1D9 antibody and human light chain FRs, and comprises CDR1, CDR2, and CDR3 of the heavy chain of the 1D9 antibody and human heavy chain FRs. In one embodiment, the humanized immunoglobulin comprises a humanized heavy chain and light chain described herein (e.g., a humanized light chain comprising the light chain variable region shown below, and a humanized heavy chain comprising the heavy chain variable region shown below). Humanized immunoglobulins comprising one or more humanized light chains and / or heavy chains are also encompassed.

[0333] The following shows the amino acid sequence of the kappa light chain variable region (VL) of the humanized 1D9 antibody, with the CDRs highlighted in bold:

[0334] [ka]

[0335] The following shows the amino acid sequence of the heavy chain variable region (VH) of the humanized 1D9 antibody, with the CDRs highlighted in bold:

[0336] [ka]

[0337] In certain embodiments, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment comprises a light chain CDR1 comprising amino acids 24 to 39 of SEQ ID NO: 1; a light chain CDR2 comprising amino acids 55 to 61 of SEQ ID NO: 1; a light chain CDR3 comprising amino acids 94 to 102 of SEQ ID NO: 1; a heavy chain CDR1 comprising amino acids 31 to 35 of SEQ ID NO: 2; a heavy chain CDR2 comprising amino acids 50 to 68 of SEQ ID NO: 2; and a heavy chain CDR3 comprising amino acids 101 to 106 of SEQ ID NO: 2.

[0338] In some embodiments, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2.

[0339] In some embodiments, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:1.

[0340] In some embodiments, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 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 of SEQ ID NO:2. It contains the amino acid sequence.

[0341] In some embodiments, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 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 of SEQ ID NO:1.

[0342] In some embodiments, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 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, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:1.

[0343] In certain embodiments, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment further comprises a heavy chain constant region, hi some embodiments, the heavy chain constant region is selected from human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 heavy chain constant regions.

[0344] In some embodiments, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment further comprises a light chain constant region, hi some embodiments, the light chain constant region is selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions.

[0345] In certain embodiments, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment binds to the same epitope as an antibody comprising the variable heavy chain region of SEQ ID NO:2 and the variable light chain region of SEQ ID NO:1.

[0346] "Percent identity" refers to the degree of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). Percent identity can be measured by aligning two sequences and introducing gaps to maximize the identity between the sequences. Alignments can be generated using programs known in the art. For purposes herein, alignment of nucleotide sequences can be performed using the blastn program set to default parameters, and alignment of amino acid sequences can be performed using the blastp program set to default parameters (see the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov).

[0347] A CCR2 antibody that "binds to the same epitope" as a reference CCR2 antibody means an antibody that binds to the same CCR2 amino acid residues as the reference CCR2 antibody. The ability of a CCR2 antibody to bind to the same epitope as the reference CCR2 antibody is measured by a hydrogen / deuterium exchange assay (see Coales et al. Rapid Commun. Mass Spectrom. 2009;23:639-647).

[0348] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human CCR2 and comprises the six CDRs of the antibody set forth in SEQ ID NO: 1 and SEQ ID NO: 2, and comprises a VH comprising a sequence at least 80% identical to the VH sequence of SEQ ID NO: 2, and a VL comprising 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 and comprises the six CDRs of the antibody set forth in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., the three VH CDRs of the antibody set forth in SEQ ID NO: 2 and the three VL CDRs of SEQ ID NO: 1), and comprises a VH comprising a sequence at least 85% identical to the VH sequence of SEQ ID NO: 2, and a VL comprising a sequence at least 85% identical to the VL sequence of SEQ ID NO: 1.

[0349] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein bind to human CCR 2 and comprises the six CDRs of the antibody 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), with the VH comprising a sequence at least 90% identical to the VH sequence of SEQ ID NO: 2, and the VL comprising a sequence at least 90% identical to the VL sequence of SEQ ID NO: 1. In certain embodiments, the antibody or antigen-binding fragment described herein binds to human CCR2 and comprises the six CDRs of the antibody 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), with the VH comprising a sequence at least 95% identical to the VH sequence of SEQ ID NO: 2, and the VL comprising a sequence at least 95% identical to the VL sequence of SEQ ID NO: 1.

[0350] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human CCR2 and comprises the six CDRs of the antibody 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 comprising a sequence at least 96% identical to the VH sequence of SEQ ID NO: 2, and a VL comprising 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 and comprises the six CDRs of the antibody 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 comprising a sequence at least 97% identical to the VH sequence of SEQ ID NO: 2, and a VL comprising a sequence at least 97% identical to the VL sequence of a SEQ ID NO: 1. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human CCR2 and comprises the six CDRs of the antibody 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 comprising a sequence at least 98% identical to the VH sequence of SEQ ID NO: 2, and a VL comprising 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 and comprises the six CDRs of the antibody 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 comprising a sequence at least 99% identical to the VH sequence of SEQ ID NO: 2, and a VL comprising a sequence at least 99% identical to the VL sequence of SEQ ID NO: 1.

[0351] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human CCR2 and comprises the six CDRs of the antibody 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 comprising a sequence at least 80% identical to the VH sequence of SEQ ID NO: 2, and a VL comprising 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 CCR2. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to CCR2 and comprises the six CDRs of the antibody 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 comprising a sequence at least 85% identical to the VH sequence of SEQ ID NO: 2, and a VL comprising 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 CCR2.

[0352] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein bind to human CCR2 and comprise the six CDRs of the antibodies set forth in SEQ ID NOs: 1 and 2 (i.e., the three VH CDRs of the antibodies and the three VL CDRs of SEQ ID NO: 1), comprising a VH comprising a sequence at least 90% identical to the VH sequence of SEQ ID NO: 2 and a VL comprising a sequence at least 90% identical to the VL sequence of SEQ ID NO: 1, and bind to human, cynomolgus monkey, rat, and / or mouse CCR2. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein bind to human CCR2 and comprise the six CDRs of the antibodies set forth in SEQ ID NOs: 1 and 2 (i.e., the three VH CDRs of SEQ ID NO: 2 and the three VL CDRs of SEQ ID NO: 1), comprising a VH comprising a sequence at least 95% identical to the VH sequence of SEQ ID NO: 2 and a VL comprising a sequence at least 95% identical to the VL sequence of SEQ ID NO: 1, and bind to human, cynomolgus monkey, rat, and / or mouse CCR2. , and / or binds to mouse CCR2.

[0353] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human CCR2 and comprises the six CDRs of the antibody 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 comprising a sequence at least 96% identical to the VH sequence of SEQ ID NO: 2, and a VL comprising 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 CCR2. In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds to human CCR2 and comprises the six CDRs of the antibody 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 comprising a sequence at least 97% identical to the VH sequence of SEQ ID NO: 2, and a VL comprising 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 CCR2. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein bind to human CCR2 and comprise the six CDRs of the antibody 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), comprising a VH comprising a sequence at least 98% identical to the VH sequence of SEQ ID NO:2 and a VL comprising a sequence at least 98% identical to the VL sequence of SEQ ID NO:1, and bind to human, cynomolgus monkey, rat, and / or mouse CCR2. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein bind to human CCR2 and comprise the six CDRs of the antibody 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:2). and three VL CDRs of SEQ ID NO: 1), a VH comprising a sequence at least 99% identical to the VH sequence of SEQ ID NO: 2, and a VL comprising 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 CCR2.

[0354] In certain embodiments, a compound of Formula (I) is combined with an antibody, antibody fragment, or antigen-binding fragment of an antibody that binds to PD-1 and / or an antibody, antibody fragment, and / or antigen-binding fragment of an antibody that binds to 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, regulates the immune system, for example, by promoting apoptosis of antigen-specific T cells and reducing apoptosis of regulatory T cells. PD-L1 is expressed by tumors and can help tumors evade detection and elimination by the immune system. Antagonistic blockade of the PD-1 / PD-L1 interaction advantageously increases T cell activation and improves tumor cell recognition and elimination by the immune system. In certain embodiments, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, pimivalimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, ezabenlimab, INCMGA0012, AMP-224, AMP-514, SYM-021, LZM-009, CS-1003, SYN-125, GNR-051, MW-11, TY-101, BAT-1306, F520, sasanlimab, penprimab, pucotenlimab, CX-188, zimbarelimab, and tebotelimab, or an antibody directed against human PD-1 or PD and / or pembrolizumab, nivolumab, cemiplimab, pimivalimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, ezabenlimab, INCMGA0012, AMP-224, AMP-514, SYM-021, LZM-009, CS-1003, SYN-125, GNR-051, MW-11, TY-101, BAT-1306, F520, sasanlimab, penprimab, pucotenlimab, CX-188, zimbarelimab, or tebotelimab for binding to a portion of IL-1.

[0355] In some embodiments, the anti-PD-1 antibody is pembrolizumab.

[0356] In certain embodiments, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, durvalumab, cosibelimab, MSB-2311, ZKAB-001, FAZ-053, MDX-1105, CBT-502, IMC-001, RC-98, KL-A167, GR-1405, lodapolimab, sugemalimab, embafolimab, opucolimab, and galibrimab, or a human PD-L1 or PD-L1 antibody. -An antibody that can compete with atezolizumab, avelumab, durvalumab, cosibelimab, MSB-2311, ZKAB-001, FAZ-053, MDX-1105, CBT-502, IMC-001, RC-98, KL-A167, GR-1405, rodapolimab, sugemalimab, embafolimab, opcolimab, or galibrimab for binding to a portion of L1.

[0357] In some embodiments, the anti-PD-1 antibody is atezolizumab.

[0358] Further anti-PD-1 antibodies useful in combination with the compounds of Formula (I) include NAT105 (abcam ab5287); CAL20 (abcam ab237728); EPR20665 (abcam ab214421); NAT105-chimera (abcam ab216352); EPR4877(2) (abcam ab137132); EP23119-111 (abcam ab 243644); SP269 (abcam ab227681); PDCD1 / 1410R (abcam ab218475); EH12.22H7 (abcam ab 223562); PDCD1 / 922 (abcam ab216037); J43 (abcam ab95789); J43.1 (abcam ab 218768); SPM597 (abcam ab218474); J116 (abcam ab171267); RMP1-14 (abcam ab171265); EPR18017-203(abcam ab242810);EPR18017-253(abcam ab242562); ab259656);EPR22234-42(abcam ab259655);MAB10861(R&D Systems);MAB10864(R&D Systems);MAB1086(R&D Systems);MAB10863(R&D MAB8578(R&D Systems);MAB77381(R&D Systems);MAB7738(R&D Systems);MAB10866(R&D Systems);MAB10865(R&D Systems);MAB10867(R&D Systems) PD-1 blocking Ab (HUABIO); J43 (MyBioSource); RMP1-30 (MyBioSource); 8A1 (BIOSS Inc.); BSR1 (Abeomics); PDCD1 / 922 (Abeomics); PD1.3.1.3 (Miltenyi Biotec); Intl.);J116(United States Biological);BSR1(Nordic BioSite); PDCD1 (BosterBio); 10B3 (ProSci Inc.); 4C7 (ProSci Inc.); mhT28 blocking (Sino Biological Inc.); HF06 neutralizing (Sino Biological Inc.); or TK12-02 (Creative Diagnostics), or an antibody that can compete with any one of the foregoing antibodies for binding to PD-1 or a portion of PD-1.

[0359] Further anti-PD-L1 antibodies useful in combination with the compounds of Formula (I) include 28-8 (abcam ab205921); EPR19759 (abcam ab213524); CAL10 (abcam ab237726); 73-10 (abcam ab228415); EPR20529 (abcam ab213480); SP142 (abcam ab228462);BLR020E(abcam ab243877);RM1012(abcam ab282458);EPR23546-160(abcam ab2 52436);ABM4E54(abcam ab210931);PDL1 / 2744(abcam ab269674);MIH5(abcam ab269253);29E.2A3(abcam ab259283);MIH6(abcam ab80276);BMS-5-28(abcam ab278010);EPR23939-25(abcam ab278009);MAB1561(R&D Systems);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&D Systems);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 can compete with any of the foregoing antibodies for binding to PD-L1 or a portion of PD-L1;

[0360] As used herein, the term "antibody" also refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule containing an antigen-binding site that immunospecifically binds to a target antigen or portion thereof of interest, including, but not limited to, cancer cell(s) 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 IgA2), or subclass of immunoglobulin molecule. The immunoglobulin can be derived from any species. However, in one aspect, the immunoglobulin is of human, murine, or rabbit origin.

[0361] The term "single domain antibody", also known as nanobody, refers to an antibody fragment consisting of a single monomeric variable antibody domain with a molecular weight of about 12 kDa to about 15 kDa. Single body antibodies can be based on the heavy chain variable region or the light chain. Examples of single domain antibodies include V H H fragment and V fragment NAR Examples of such fragments include, but are not limited to, those described in, for example, Harmsen MM et al. Applied Microbiology and Biotechnology 77(1):13-22.

[0362] An "antibody fragment" is a portion of an intact antibody, generally the antigen-binding region or region thereof. Examples of antibody fragments include Fab, Fab', F(ab').sub2, and Fv fragments; diabodies; linear antibodies; fragments produced by an Fab expression library, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity-determining regions), and epitope-binding fragments of any of the above that immunospecifically bind to a cancer cell antigen, a viral antigen, or a bacterial antigen, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0363] An "intact antibody" is one that comprises an antigen-binding variable region, as well as a light chain constant domain (CL) and heavy chain constant domains (CH1, CH2, and CH3). The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0364] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present disclosure may be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol., 222:581-597.

[0365] The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to 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.

[0366] Various methods have been used to generate monoclonal antibodies (MAbs). "Hybridoma technology," which refers to a cloned cell line that produces a single type of antibody, uses cells from various species, including mice (murine), hamsters, rats, and humans. Another method for preparing MAbs uses genetic engineering, including recombinant DNA technology. Monoclonal antibodies generated from these techniques include chimeric and humanized antibodies, among others. Chimeric antibodies combine DNA encoding regions from two or more species. For example, a chimeric antibody can be derived from a murine variable region and a human constant region. Humanized antibodies, while primarily human, also contain non-human portions. Like chimeric antibodies, humanized antibodies can contain fully human constant regions. However, unlike chimeric antibodies, the variable regions can be partially derived from humans. The non-human, synthetic portions of humanized antibodies are often derived from the CDRs of mouse antibodies. In either case, these regions are crucial for enabling the antibody to recognize and bind to specific antigens. While useful for diagnosis and short-term therapy, mouse antibodies cannot be administered long-term to humans without increasing the risk of a harmful immunogenic response. This response, called human anti-mouse antibody (HAMA), occurs when the human immune system recognizes murine antibodies as foreign and attacks them. The HAMA response can lead to toxic shock or even death.

[0367] Chimeric and humanized antibodies reduce the likelihood of a HAMA response by minimizing the non-human portion of the administered antibody. Furthermore, chimeric and humanized antibodies can have the added benefit of activating a secondary human immune response, such as antibody-dependent cellular cytotoxicity.

[0368] An intact antibody may possess one or more "effector functions," which refer to biological activities attributable to the Fc region of an antibody (a native-sequence Fc region or an 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, and down-regulation of cell surface receptors (e.g., B cell receptor; BCR).

[0369] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0370] Useful non-immunoreactive protein, polypeptide, or peptide antibodies include, but are not limited to, transferrin, epidermal growth factor ("EGF"), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factors ("TGF") (e.g., TGF-α and TGF-β), varicella growth factor ("VGF"), insulin and insulin-like growth factors I and II, lectins, and apoproteins derived from low-density lipoproteins.

[0371] Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Various procedures well known in the art can be used to generate polyclonal antibodies to an antigen of interest. For example, for the production of polyclonal antibodies, various host animals, including but not limited to rabbits, mice, rats, and guinea pigs, can be immunized by injection with the antigen of interest or its derivatives. Various adjuvants can be used to increase the immune response, depending on the host species. These include, but are not limited to, Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as bacille Calmette-Guerin (BCG) and corynebacterium parvum. Such adjuvants are also well known in the art.

[0372] Useful monoclonal antibodies can be directed against specific antigenic determinants (e.g., cancer cell antigens, viral antigens, bacterial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids, or fragments thereof). Monoclonal antibodies (mAbs) to an antigen of interest can be prepared using any technique known in the art that provides for the production of antibody molecules by continuous cell lines in culture. These 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., pp. 77-96). Such antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof. The hybridoma producing the mAb used in this disclosure may be cultivated in vitro or in vivo.

[0373] 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 prepared using various techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79; and Olsson, J. Med. Chem. Soc. 1999, 103:101-103). It can be prepared by either of the methods described above (E. et al., 1982, Meth. Enzymol. 92, 3-16).

[0374] The antibody can also be a bispecific antibody. Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Milstein et al., 1983, Nature 305:537-539). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) can produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, usually performed using affinity chromatography steps, is somewhat cumbersome and results in low product yields. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J. 10:3655-3659 (1991).

[0375] According to a different approach, antibody variable domains (antibody-antigen combining sites) with the desired binding specificities are fused to immunoglobulin constant domain sequences. Fusions can be with immunoglobulin heavy chain constant domains, including at least part of the hinge, C.sub.H2, and C.sub.H3 regions. The first heavy chain constant region (C.sub.H1) can contain the site necessary for light chain binding, present in at least one of the fusions. Nucleic acids containing sequences encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the relative proportions of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used in the construction result in optimal yields. However, it is possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when expression of at least two polypeptide chains in equal ratios results in higher yields, or when their ratio is not particularly critical.

[0376] Bispecific antibodies consist of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain (providing a second binding specificity) in the other arm. The asymmetric structure facilitates separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for facile separation methods (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 / Technology 10:163-167; 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 as ADCs in the treatment or prevention of disease as defined herein.

[0377] Hybrid or bifunctional antibodies can be derived biologically, i.e., by cell fusion techniques, or chemically, using, inter alia, cross-linking agents or disulfide bridge-forming reagents, and can include whole antibodies or fragments thereof (EP 105360; WO 83 / 03679; EP 217577).

[0378] The antibody can be a functionally active fragment, derivative, or analog of an antibody that immunospecifically binds to a cancer cell antigen, a viral antigen, or a bacterial antigen, or other antibody that binds to tumor cells or matrix. In this context, "functionally active" means that the fragment, derivative, or analog is capable of eliciting anti-anti-idiotypic antibodies that recognize the same antigen recognized by the antibody from which the fragment, derivative, or analog is derived. Specifically, in exemplary embodiments, the idiotypic antigenicity of an immunoglobulin molecule can be improved by deleting framework and CDR sequences that are C-terminal to the CDR sequences that specifically recognize the antigen. To determine which CDR sequences bind to an antigen, synthetic peptides containing the CDR sequences can be used in a binding assay together with the antigen by any binding assay method known in the art (e.g., BIAcore assay) (see, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md.; Kabat E et al., 1980, J. of Immunology 125(3):961-969).

[0379] Other useful antibodies include, but are not limited to, antibody fragments such as F(ab')2 fragments containing the variable region, light chain constant region, and heavy chain CH1 domain, which can be produced by pepsin digestion of an antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of F(ab')2 fragments. Other useful antibodies include antibodies such as FV or single-chain antibodies (SCAs), or any minimal fragment thereof, heavy and light chain dimers (e.g., those 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 with the same specificity as an antibody.

[0380] Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, containing both human and non-human portions, which can be made using standard recombinant DNA techniques, are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal and a human immunoglobulin constant region (see, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,567). (See U.S. Patent No. 4,816,397 to Gill, et al., Humanized antibodies are antibody molecules from non-human species having one or more complementarity-determining regions (CDRs) from the 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 are described, for example, in WO 87 / 02671; EP 184,187; EP 171496; EP 173494; WO 86 / 01533; U.S. Pat. No. 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. 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. al.,1986,BioTechniques 4:214;USPat.No.5,225,539;Jones et al.,1986,Nature 321:552-525;Verhoeyan et al.(1988)Science 239:1534;and Beidler et al. It can be produced by recombinant DNA techniques known in the art using the methods described in [PubMed], [Chemical], [Immunol.

[0381] 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. The transgenic mice are immunized in the usual manner with a selected antigen, e.g., all or a portion of a polypeptide of the present disclosure. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice can rearrange during B-cell differentiation and subsequently undergo class switching and somatic mutation. Thus, such technology can be used to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Patent Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806. Other human antibodies are commercially available from, for example, Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).

[0382] Fully human antibodies that recognize a selected epitope can be generated using a technique called "guided selection." In this approach, a selected non-human monoclonal antibody, e.g., a murine antibody, can be used to guide the selection of a fully human antibody that recognizes the same epitope. (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)).

[0383] An antibody can be, for example, a fusion protein of an antibody, or a functionally active fragment thereof, in which the antibody is fused via a covalent bond (e.g., a peptide bond) at the N-terminus or C-terminus of the amino acid sequence of another protein (or portion thereof, e.g., at least a 10, 20, or 50 amino acid portion of the protein) that is not an antibody. The antibody or fragment thereof can be covalently linked to the other protein at the N-terminus of the constant domain.

[0384] Antibodies include analogs and derivatives that have been modified in any way (i.e., by the covalent attachment of any type of molecule), so long as such covalent attachment allows the antibody to retain its antigen-binding immunospecificity. For example, without limitation, antibody derivatives and analogs include those that have been further modified by, for example, glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to cellular antibody units or other proteins, etc. Any of a number of chemical modifications can be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Additionally, analogs or derivatives can contain one or more unnatural amino acids.

[0385] The antibody in the antibody-drug conjugate includes an antibody having a modification (e.g., substitution, deletion, or addition) in an amino acid residue that interacts with an Fc receptor. Specifically, the antibody includes an antibody having a modification in an amino acid residue identified as being involved in the interaction between an anti-Fc domain and an FcRn receptor (see, e.g., WO97 / 34631). Antibodies immunospecific for cancer cell antigens can be commercially obtained, for example, from Genentech (San Francisco, Calif.), or can be produced by any method known to those skilled in the art, such as, for example, 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 a similar database, literature publications, or by routine cloning and sequencing.

[0386] The antibody of the ADC can be a monoclonal antibody, e.g., a murine monoclonal antibody, a chimeric antibody, or a humanized antibody. The antibody can be an antibody fragment, e.g., a Fab fragment.

[0387] Known anti-CCR2 antibodies for cancer treatment or prevention can be conjugated as ADCs. Antibodies immunospecific for cancer cell antigens can be commercially available or produced by any method known to those skilled in the art, such as 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 routine cloning and sequencing. Examples of antibodies that can 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 those described in WO97 / 31949, which is incorporated herein by reference.

[0388] The term "amino acid sequence variant" refers to a polypeptide that differs to some extent from a native sequence polypeptide. "A" refers to a polypeptide having an amino acid sequence that is identical to at least one receptor-binding domain of a native antibody or at least one ligand-binding domain of a native receptor. Typically, an amino acid sequence variant will have at least about 70% sequence identity with at least one receptor-binding domain of a native antibody, and typically will have at least about 80%, more typically about 90%, sequence identity with 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, one-letter and three-letter codes.

[0389] "Sequence identity" is defined as the percentage of residues of amino acid sequence variants that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment methods and computer programs for alignment are well known in the art. One such computer program is "Align 2," developed by Genentech, Inc., which, together with user documentation, is registered with the United States Copyright Office. Filed December 10, 1991, in the Federal Circuit Court of Appeals (Fed. Cir. Office), Washington, DC 20559.

[0390] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. Illustratively, the FcR is a native-sequence human FcR. Furthermore, an FcR may bind an IgG antibody (a gamma receptor), and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, with the RIII subclass including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See reviews in M. in Daeron, Annu. Rev. Immunol., 15:203-234 (1997).) FcRs are also described by Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991); Capel et al. al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs, including those yet to be identified, are encompassed by the term "FcR" herein. This term also encompasses FcRn, the neonatal receptor responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., 117:587 (1976) and Kim et al., J. Immunol., 24:249 (1994)).

[0391] " Complement-dependent cytotoxicity " or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., antibody) complexed with a cognate antigen. To assess complement activation, for example, a CDC assay such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996) can be performed.

[0392] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain (VL) at the other end. Each heavy-chain antibody has a constant domain, the light-chain constant domain of which is aligned with the first constant domain of the heavy chain, and the light-chain variable domain of which is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain.

[0393] The term "variable" refers to the fact that certain portions of the variable domains vary widely in sequence among antibodies and are responsible for the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy and light chain variable domains each contain four FRs in a primarily beta-sheet configuration connected by three hypervariable regions that form loops that connect, and in some cases form part of, the beta-sheet structure. The hypervariable regions in each chain are held in close proximity to the hypervariable regions of the other chain by the FRs, contributing to the formation of the antigen-binding site of antibodies (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participating in antibody-dependent cellular cytotoxicity (ADCC).

[0394] When used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from 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 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., supra), and / or residues from the "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 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 those variable domain residues other than the hypervariable region residues as herein defined.

[0395] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0396] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific hypervariable regions) has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site.

[0397] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments in that they have a few additional residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH, as used herein, refers to fragments in which the cysteine ​​residue(s) of the constant domains are / are not present. is the designation for Fab' which has at least one free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0398] The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0399] "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. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). Anti-ErbB2 antibody scFv fragments are described in International Publication No. WO 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458.

[0400] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, which fragment comprises a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains can be paired with complementary domains on another chain to generate two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0401] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. Humanization is a method for transferring mouse antigen-binding information to non-immunogenic human antibody acceptors, resulting in a number of therapeutically useful drugs. The humanization process generally begins by transferring all six mouse complementarity-determining regions (CDRs) into a human antibody framework (Jones et al., (1986) Nature 321:522-525). Antibodies with these CDRs grafted generally do not retain their original affinity for antigen binding; in fact, affinity is often severely impaired. In addition to the CDRs, selected non-human antibody framework residues must also be incorporated to maintain proper CDR configuration (Chothia et al., (1989) Nature 342:877). Transferring key murine framework residues into the human acceptor to support the structural alignment of the grafted CDRs has been shown to restore antigen binding and affinity (Riechmann et al., (1992) J. Mol. Biol. 224, 487-499; Foote and Winter, (1992) J. Mol. Biol. 224:487-499; Presta et al., (1993) J. Immunol. 151, 2623-2632; Werther et al., (1996) J. Immunol. Methods 157:4986-4995, and Presta et al. (2001) Thromb. Haemost. 85:379-389). For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding In addition, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications are 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 FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region (Fc). For further details, see 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.

[0402] A "parent antibody" is an antibody comprising an amino acid sequence derived from a sequence in which one or more amino acid residues have been replaced with one or more cysteine ​​residues. A parent antibody may comprise a native or wild-type sequence. A parent antibody may have pre-existing amino acid sequence modifications (such as additions, deletions, and / or substitutions) relative to other native, wild-type, or modified forms of the antibody. A parent antibody is directed against a 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.

[0403] An "isolated" antibody is one that has been identified and separated and / or recovered from components of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and these may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody is purified to (1) greater than 95% by weight, or greater than 99% by weight, as determined by the Lowry method, (2) sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence by gas-phase protein sequencing, or (3) homogeneous by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver stain. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibodies will be prepared by at least one purification step.

[0404] An antibody that "binds" a molecular target or antigen of interest is one that is able to bind to that antigen with sufficient affinity so that the antibody is useful for targeting cells that express the antigen.

[0405] The term "treat" or "treatment" refers to both therapeutic and prophylactic or preventative treatment, the purpose of which is to prevent or slow (alleviate) an undesired physiological change or disorder (such as the onset or spread of cancer). For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, attenuation of the extent of the disease, a stabilized (i.e., not worsening) disease state, delay or slowing of disease progression, alleviation or remission of the disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in whom the condition or disorder is to be prevented.

[0406] "Phage display" is a technique in which variant polypeptides are displayed as fusion proteins with coat proteins on the surface of phage, e.g., filamentous phage, particles. One utility of phage display is to rapidly and efficiently sort large libraries of random protein variants for sequences that bind with high affinity to a target molecule. The key to successful monovalent phage display lies in the fact that it can be used to screen millions of polypeptides for those with specific binding properties. Display of peptide and protein libraries on phage has been used to screen millions of polypeptides for those with specific binding properties. Polyvalent phage display methods have been used to display small random peptides and small proteins, typically through fusion to either PIII or PVIII of filamentous phage. Wells and Lowman, Curr. Opin. Struct. Biol., 3:355-362 (1992) and references cited therein. In monovalent phage display, protein or peptide libraries are fused to a phage coat protein or a portion thereof and expressed at low levels in the presence of wild-type protein. Avidity effects are reduced for polyvalent phage so that selection is based on intrinsic ligand affinity, and phagemid vectors are used, simplifying DNA manipulation. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991). Phage display includes a technique for producing antibody-like molecules (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., Garland Publishing, New York, p627-628).

[0407] A "phagemid" is a plasmid vector containing a bacterial origin of replication, e.g., Co1E1, and a copy of the intergenic region of a bacteriophage. Phagemids can be used with any known bacteriophage, including filamentous and lambdoid bacteriophages. Plasmids will also generally contain a selectable marker for antibiotic resistance. DNA segments cloned into these vectors can be propagated as plasmids. Providing all the genes necessary for phage particle production in cells harboring these vectors changes the replication mode of the plasmid to rolling circle replication, producing single-stranded copies of the plasmid DNA and packaged phage particles. Phagemids can form infectious or non-infectious phage particles. This term includes phagemids containing a phage coat protein gene or fragment thereof linked to a heterologous polypeptide gene as a gene fusion, such that the heterologous polypeptide is displayed on the surface of the phage particle. The compounds described herein can be in the form of pharmaceutical salts or pharmaceutically acceptable salts. In some embodiments, such salts are derived from inorganic or organic acids or bases. For reviews of suitable salts, see, e.g., Berge et al., J. Pharm. Sci., 1977, 66, 1-19 and Remington: The Science and Practice of Pharmacy, 20th Ed., A. Gennaro (ed.), Lippincott Williams & Wilkins (2000).

[0408] In the present disclosure, the group "Ab" (i.e., antibody, antibody fragment, and / or antigen fragment) can be conjugated to two or more drug-containing moieties. In some embodiments, an "Ab" can be conjugated to 1 to 20 drug-containing moieties. In some embodiments, an "Ab" can be conjugated to 1 to 10 drug-containing moieties. In some embodiments, an "Ab" can be conjugated to 1 to 5 drug-containing moieties. In some embodiments, an "Ab" can be conjugated to 1 or 2 drug-containing moieties. In some embodiments, an "Ab" can be conjugated to 1 drug-containing moiety.

[0409] In some embodiments of the disclosure, the ADC is combined with an antibody that binds to PD-1 and / or an antibody that binds to PDL-1.

[0410] The compounds described herein are in the form of pharmaceutical salts 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 See Science and Practice of Pharmacy, 20th Ed., A. Gennaro (ed.), Lippincott Williams & Wilkins (2000).

[0411] Examples of suitable acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate salts.

[0412] 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, dicyclohexylamine salts, salts with organic bases such as N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and the like.

[0413] For example, Berge lists the following commercially available FDA-approved salts: acetate, besylate (benzenesulfonate), benzoate, bicarbonate, bitartrate, bromide, calcium edetate (ethylenediaminetetraacetate), camsylate (camphorsulfonate), carbonate, chloride, citrate, dihydrochloride, edetate (ethylenediaminetetraacetate), edisylate (1,2-ethanedisulfonate), and ethylenediaminetetraacetate. Straight (lauryl sulfate), esylate (ethanesulfonate), fumarate, gluceptate (glucoheptonate), gluconate, glutamate, glycolyl arsanilate (glycolamidophenylarsonate), hexylresorcinate, hydrabamine (N,N'-di(dehydroabietyl)ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, iodosulfonate The following compounds are also known: cethionate (2-hydroxyethanesulfonate), lactate, lactobionate, malate, maleate, mandelate, mesylate (methanesulfonate), methyl bromide, methyl nitrate, methyl sulfate, mucate, napsylate (2-naphthalenesulfonate), nitrate, pamoate (embonate), pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, diacetate, succinate, sulfate, tannate, tartrate, teoclate (8-chlorotheophyllinate), and triethiodide; the organic cations benzathine (N,N'-dibenzylethylenediamine), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine; and the metal cations aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.

[0414] Berge further lists the following salts commercially available (outside the United States) that are not FDA approved: the anions adipate, alginate, aminosalicylate, anhydromethylene citrate, arecoline, aspartate, bisulfate, butyl bromide, camphorate, digluconate, dihydrobromide, disuccinate, glycerophosphate, hemisulfate, hydrofluoride, hydroiodide, methylenebis(salicylate), napadisilate (1,5-naphthalenedisulfonate), oxalate, pectinate, persulfate, and fluoride. phenylethyl barbiturate, picrate, propionate, thiocyanate, tosylate, and undecanoate, the organic cations benethamine (N-benzylphenethylamine), clemizole (1-p-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole), diethylamine, piperazine, and tromethamine (tris(hydroxymethyl)aminomethane), and the metal cations barium and bismuth.

[0415] The compounds described herein can also include suitable carriers, excipients, and adjuvants, which can vary depending on the method of administration.

[0416] In some embodiments, pharmaceutical compositions can be formulated into suitable parenteral dosage forms.Suitable formulations can be prepared by various methods known in the art.Pharmaceutical compositions can be administered directly into the bloodstream, muscle, or organ.Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous.Suitable devices for parenteral administration include needle syringes, needleless syringes, and infusion techniques.

[0417] Parenteral compositions are typically aqueous solutions, which may contain excipients such as salts, carbohydrates, and buffers. However, the compositions can also be formulated as sterile nonaqueous solutions or a dry form that can be used in combination with a suitable vehicle, such as sterile, pyrogen-free water.

[0418] The preparation of parenteral compositions under sterile conditions, for example, by lyophilization, may be readily accomplished using standard techniques well known to those skilled in the art.

[0419] Compositions for parenteral administration can be formulated for immediate release and / or modified release. Modified release formulations include delayed-release, sustained-release, pulsed-release, sustained-release, targeted-release, and programmed-release. Thus, compositions can be formulated as solids, semi-solids, or thixotropic liquids for administration as implanted depots that provide modified release of the active compound.

[0420] The parenteral preparations may be mixed with other suitable pharmaceutically acceptable excipients used in parenteral dosage forms, including, but not limited to, preservatives.

[0421] In another embodiment, the pharmaceutical composition can be formulated as a suitable oral dosage form, for example, a tablet, capsule, powder, pellet, suspension, solution, emulsion, etc. Other suitable carriers can be present, for example, disintegrants, diluents, chelating agents, binders, glidants, lubricants, fillers, bulking agents, anti-adherents, etc.

[0422] Orally administered formulations may also contain other suitable pharmaceutical excipients, such as sweeteners, vehicles / humectants, colorants, flavoring agents, preservatives, viscosity enhancing / thickening agents, and the like.

[0423] Doses of the pharmaceutical compositions of the present disclosure can be titrated to the individual patient.

[0424] The term "radiation" refers to photon radiation or particle radiation. In some embodiments, the radiation can be photon radiation (x-rays and gamma rays). In such embodiments, the photons can be generated as a high-energy photon beam from a radioactive source such as a cobalt or linear accelerator. In some embodiments, the radiation can be particle radiation (such as electrons, protons, neutrons, carbon ions, alpha particles, and beta particles). Particle radiation can be produced by a linear accelerator. In some embodiments, the radiation can be electrons. In some embodiments, the radiation can be a proton beam. In some embodiments, the radiation can be a neutron beam.

[0425] In some embodiments, the radiation can be delivered by external beam radiation. In some embodiments, the external beam radiation can be three-dimensional conformal radiation therapy (3D-CRT). In some embodiments, the external beam radiation can be intensity-modulated radiation therapy (IMRT). In some embodiments, the external beam radiation can be image-guided radiation therapy (IGRT). In some embodiments, the external beam radiation can be intensity-modulated proton beam therapy (IMPT). In some embodiments, the external beam radiation can be stereotactic radiosurgery (SRS). In some embodiments, the external beam radiation therapy can be fractionated stereotactic radiation therapy. In some embodiments, the external beam radiation can be stereotactic body radiation therapy (SBRT). Examples of machines that perform SBRT are the Gamma Knife®, X-Knife®, CyberKnife®, and Clinac®. In some embodiments, the radiation can be administered using three-dimensional conformal or stereotactic body radiation therapy delivery.

[0426] In some embodiments, radiation can be delivered by internal radiation therapy (brachytherapy). In such embodiments, internal radiation therapy can be, for example, interstitial radiation using small pellets, seeds, wires, or tubes placed near the cancer or tumor site. In such embodiments, internal radiation therapy can be, for example, intracavitary radiation using a container of radioactive material placed within a body cavity.

[0427] 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. The compositions can be used in the treatment of cancer.

[0428] The compounds of the present disclosure exhibit STING modulatory / agonist activity. Certain compounds of the present disclosure may be useful as pharmaceuticals because they may be superior in terms of efficacy, pharmacokinetics (e.g., absorption, distribution, metabolism, excretion), solubility (e.g., water solubility), interactions with other pharmaceuticals (e.g., drug-metabolizing enzyme inhibitory activity), safety (e.g., acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, carcinogenicity, central nervous system toxicity), and / or stability (e.g., chemical stability, enzymatic stability).

[0429] Thus, the compounds of the present disclosure can be used to increase STING activity in mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, humans).

[0430] The compounds of the present disclosure can be used to treat diseases that can be affected by STING (sometimes abbreviated herein as "STING-associated diseases"), such as cancer, for example, colon 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, 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, mucinous adenocarcinoma, adenosquamous carcinoma), duodenal cancer, small intestinal cancer, breast cancer (e.g., invasive ductal carcinoma, ductal carcinoma in situ), , inflammatory breast cancer), ovarian cancer (e.g., epithelial ovarian cancer, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian low malignant potential tumor), testicular tumor, prostate cancer (e.g., hormone-dependent prostate cancer, hormone-independent prostate cancer, castration-resistant prostate cancer), liver cancer (e.g., hepatocellular carcinoma, primary liver cancer, extrahepatic bile duct cancer), thyroid cancer (e.g., medullary thyroid carcinoma), kidney cancer (e.g., renal cell carcinoma (e.g., clear cell renal cell carcinoma), transitional cell carcinoma of the renal pelvis and ureter), uterine cancer (e.g., cervical cancer, uterine corpus carcinoma, uterine sarcoma), gestational choriocarcinoma, brain tumor (e.g., medulloblastoma, glioma, pine The compounds may be used as pharmaceuticals such as drugs for preventing or treating cancer of unknown primary origin, cancer growth inhibitors, cancer metastasis inhibitors, apoptosis promoters, drugs for treating precancerous lesions (e.g., myelodysplastic syndromes), and the like.

[0431] In certain embodiments, the compounds of the present disclosure may be used as pharmaceuticals for colon cancer, breast cancer, skin cancer, malignant lymphoma, or lung cancer.

[0432] In certain embodiments, compounds of the present disclosure can be used concomitantly 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.

[0433] In certain embodiments, compounds of the present disclosure can be used simultaneously with antibody therapy and radiation therapy. In some embodiments, the radiation therapy can be photon radiation therapy. In some embodiments, the radiation therapy can be particle radiation therapy.

[0434] Furthermore, the compounds of the present disclosure can be used simultaneously with non-drug therapies. Specifically, the compounds of the present disclosure or the combination agents of the present disclosure can 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 ablation, and (7) radiation therapy.

[0435] For example, by using the compounds of the present disclosure before or after the above-mentioned surgery or before or after a combination therapy of two or three of these, effects such as preventing the development of resistance, extending disease-free survival, suppressing cancer metastasis or recurrence, and prolonging life can be obtained.

[0436] In some embodiments, the present disclosure relates to a method of treating cancer in a patient by administering to a patient in need thereof a combination of a compound of formula (I) or a pharmaceutically acceptable salt thereof and radiation.

[0437] In some embodiments, the disclosure relates to a method of treating cancer in a patient by administering to a patient in need thereof 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.

[0438] 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 the treatment of cancer in a patient.

[0439] In some embodiments, the present disclosure relates to a composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof, for use in treating cancer in a patient, wherein the patient is also being 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 use in 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) is combined with a checkpoint inhibitor, radiation, and / or a combination thereof. In some embodiments, the present disclosure relates to a method 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.

[0440] In some embodiments, radiation can be administered at least 5 hours prior to administration of the checkpoint inhibitor and / or compound of Formula (I). In some embodiments, radiation can be administered at least 10 hours prior to administration of the checkpoint inhibitor and / or compound of Formula (I). In some embodiments, radiation can be administered at least 20 hours prior to administration of the checkpoint inhibitor and / or compound of Formula (I). In some embodiments, radiation can be administered at least 40 hours prior to administration of the checkpoint inhibitor and / or compound of Formula (I). In some embodiments, radiation can be administered at least 80 hours prior to administration of the checkpoint inhibitor and / or compound of Formula (I).

[0441] In some embodiments, radiation is administered on days 1-5 of each week and can be repeated for 2-8 weeks. In some embodiments, radiation is administered on days 1-5 of each week and can be repeated for 6-8 weeks. In some embodiments, radiation is administered on days 1-5 of each week and can be repeated for 2 weeks. In some embodiments, radiation is administered on days 1-5 of each week and can be repeated for 3 weeks. In some embodiments, radiation is administered on days 1-5 of each week and can be repeated for 4 weeks. In some embodiments, radiation is administered on days 1-5 of each week and can be repeated for 5 weeks. In some embodiments, radiation is administered on days 1-5 of each week and can be repeated for 6 weeks. In some embodiments, radiation is administered on days 1-5 of each week and can be repeated for 7 weeks. In some embodiments, radiation is administered on days 1-5 of each week and can be repeated for 8 weeks.

[0442] In some embodiments, radiation is administered on any two days from days 1 to 5 each week and can be repeated for five to eight weeks. In some embodiments, radiation is administered on any two days from days 1 to 5 each week and can be repeated for six to eight weeks. In some embodiments, radiation is administered on any two days from days 1 to 5 each week and can be repeated for five weeks. In some embodiments, radiation is administered on any two days from days 1 to 5 each week and can be repeated for six weeks. In some embodiments, radiation is administered on any two days from days 1 to 5 each week and can be repeated for seven weeks. In some embodiments, radiation is administered on any two days from days 1 to 5 each week and can be repeated for eight weeks.

[0443] In some embodiments, the checkpoint inhibitor can be administered once every 12 weeks, once every 4 weeks, once every 3 weeks, once every 2 weeks, once a week, twice a week, three times a week, or daily. In some embodiments, the checkpoint inhibitor can be administered once every 2 weeks. In some embodiments, the checkpoint inhibitor can be administered once every 3 weeks. In some embodiments, the checkpoint inhibitor can be administered once every 4 weeks. In some embodiments, the checkpoint inhibitor can be administered once every 12 weeks.

[0444] In certain embodiments, radiation is administered at least 40 hours prior to administration of the checkpoint inhibitor and / or compound of Formula (I). In certain embodiments, radiation is administered at least 30 hours prior to administration of the checkpoint inhibitor and / or compound of Formula (I). In certain embodiments, radiation is administered at least 20 hours prior to administration of the checkpoint inhibitor and / or compound of Formula (I). In certain embodiments, radiation is administered at least 40 hours prior to administration of the checkpoint inhibitor and / or compound of Formula (I). In certain embodiments, radiation is administered at least 30 hours prior to administration of the checkpoint inhibitor and / or compound of Formula (I). In certain embodiments, radiation is administered at least 20 hours prior to administration of the checkpoint inhibitor and / or compound of Formula (I). In certain embodiments, radiation is administered at least 10 hours before the administration of the checkpoint inhibitor and / or the compound of Formula (I). In certain embodiments, radiation is administered at least 5 hours before the administration of the checkpoint inhibitor and / or the compound of Formula (I). In certain embodiments, radiation is administered at least 1 hour before the administration of the checkpoint inhibitor and / or the compound of Formula (I).

[0445] In some embodiments, the compound of Formula (I) and / or checkpoint inhibitor can be administered to a patient 1 day to 3 months after the patient receives radiation therapy. In some embodiments, the compound of Formula (I) and / or checkpoint inhibitor can be administered to a patient 1 day to 2 months after the patient receives radiation therapy. In some embodiments, the compound of Formula (I) and / or checkpoint inhibitor can be administered to a patient 1 day to 1 month after the patient receives radiation therapy. In some embodiments, the compound of Formula (I) and / or checkpoint inhibitor can be administered to a patient 1 day to 15 days after the patient receives radiation therapy. In some embodiments, the compound of Formula (I) and / or checkpoint inhibitor can be administered to a patient 1 day to 7 days after the patient receives radiation therapy.

[0446] In some embodiments, radiation can be administered in fractionated doses of about 1 Gy to about 100 Gy. In some embodiments, radiation can be administered in fractionated doses of about 1 Gy to about 50 Gy. In some embodiments, radiation can be administered in fractionated doses of about 1 Gy to about 20 Gy. In some embodiments, radiation can be administered in fractionated doses of about 5 Gy to about 20 Gy. In some embodiments, radiation can be administered in fractionated doses of about 6 Gy to about 18 Gy. In some embodiments, radiation can be administered in fractionated doses of about 8 Gy to about 16 Gy. In some embodiments, radiation can be administered in fractionated doses of about 5 Gy to about 10 Gy. In some embodiments, radiation can be administered in fractionated doses of about 10 Gy to about 15 Gy. In some embodiments, radiation can be administered in fractionated doses of about 15 Gy to about 20 Gy. In some embodiments, radiation can be administered in fractionated doses of about 8 Gy or about 16 Gy.

[0447] In some embodiments, radiation can be administered in fractionated doses of about 1 Gy. In some embodiments, radiation can be administered in fractionated doses of about 2 Gy. In some embodiments, radiation can be administered in fractionated doses of about 3 Gy. In some embodiments, radiation can be administered in fractionated doses of about 4 Gy. In some embodiments, radiation can be administered in fractionated doses of about 5 Gy. In some embodiments, radiation can be administered in fractionated doses of about 6 Gy. In some embodiments, radiation can be administered in fractionated doses of about 7 Gy. In some embodiments, radiation can be administered in fractionated doses of about 8 Gy. In some embodiments, radiation can be administered in fractionated doses of about 9 Gy. In some embodiments, radiation can be administered in fractionated doses of about 10 Gy. In some embodiments, radiation can be administered in fractionated doses of about 11 Gy. In some embodiments, radiation can be administered in fractionated doses of about 12 Gy. In some embodiments, radiation can be administered in fractionated doses of about 13 Gy. In some embodiments, radiation can be administered in fractionated doses of about 14 Gy. In some embodiments, radiation can be administered in fractionated doses of about 15 Gy. In some embodiments, radiation can be administered in fractionated doses of about 16 Gy. In some embodiments, radiation can be administered in fractionated doses of about 17 Gy. In some embodiments, radiation can be administered in fractionated doses of about 18 Gy. In some embodiments, radiation can be administered in fractionated doses of about 19 Gy. In some embodiments, radiation can be administered in fractionated doses of about 20 Gy.

[0448] In some embodiments, radiation can be administered in fractions. In some embodiments, radiation can be administered in 1 to 10 fractions. In some embodiments, radiation can be administered in 1 to 5 fractions. In some embodiments, radiation can be administered in one fraction, or in two fractions, or in three fractions, or in four fractions, or in five fractions. In some embodiments, radiation can be administered in one fraction, or in three fractions.

[0449] In some embodiments, radiation can be administered in one to three fractionated doses of about 1-5 Gy. In some embodiments, radiation can be administered in one to three fractionated doses of about 5-10 Gy. In some embodiments, radiation can be administered in one to three fractionated doses of about 10-15 Gy. In some embodiments, radiation can be administered in one to three fractionated doses of about 15-20 Gy. In some embodiments, radiation can be administered in one to three fractionated doses of about 5-10 Gy, or in one to three fractionated doses of about 15-20 Gy. In some embodiments, radiation can be administered in a single fractionated dose of about 8 Gy. In some embodiments, radiation can be administered in three fractionated doses of about 8 Gy. In some embodiments, radiation can be administered in a single fractionated dose of about 16 Gy. In some embodiments, radiation can be administered in a single fractionated dose of about 8 Gy, or in three fractionated doses of about 8 Gy, or in a single fractionated dose of about 16 Gy.

[0450] Furthermore, treatment with a compound of the present disclosure or a combination drug of the present disclosure can be combined with supportive care such as: (i) administration of antibiotics (e.g., β-lactams such as pansporin, macrolides such as clarithromycin) for various concurrent infections; (ii) administration of high-calorie infusions, amino acid preparations, or multivitamins to improve malnutrition; (iii) administration of morphine to relieve pain; (iv) administration of drugs to improve side effects such as nausea, vomiting, loss of appetite, diarrhea, leukopenia, thrombocytopenia, decreased hemoglobin concentration, hair loss, liver damage, kidney damage, DIC, fever, etc.; and (v) administration of drugs to suppress multidrug resistance in cancer. [Example]

[0451] definition Ab antibody ACN Acetonitrile ADA anti-drug antibodies ADC Antibody Drug Conjugate BLQ lower limit of quantitation C Celsius CCR2 CC motif chemokine receptor 2 CR complete remission CD Cluster of Differentiation DAR Drug-antibody ratio DMA N,N-dimethylacetamide DMSO dimethyl sulfoxide DTT Dithiothreitol ε extinction coefficient E0.1% Absorption coefficient of 0.1% solution EC 50 Median effective concentration EDTA Ethylenediaminetetraacetic acid h time HIC Hydrophobic Interaction Chromatography hIgG human immunoglobulin G HPLC High Pressure Liquid Chromatography IACUC Animal Care and Use Committee IFN Interferon IgG immunoglobulin G IgM immunoglobulin M IL Interleukin IP interferon-γ-inducible protein LC liquid chromatography LCMS Liquid Chromatography Mass Spectrometry μM micromolar MCP Monocyte chemotactic protein MDSC myeloid-derived immunosuppressive cells mL milliliter MS mass spectrum MTD maximum capacity NA Not available OAc acetate PBS Phosphate-buffered saline PEG polyethylene glycol QTOF quadrupole flight time rt room temperature SEC size exclusion chromatography STING Stimulator of interferon genes TCEP (tris(2-carboxyethyl)phosphine) TNF tumor necrosis factor TPPTS 3,3',3''-phosphanetriyltris(benzenesulfonic acid) trisodium salt Tris Tris(hydroxymethyl)aminomethane UFLC Ultra High Performance Liquid Chromatograph UV ultraviolet light

[0452] Analysis method Analysis SEC conditions: SEC spectra were recorded at 280 nm on a Hewlett-Packard HP1100 or Agilent 1100 Series LC system equipped with a diode array detector using an SEC column (typically a Tosoh Biosep TSK Gel, G3000SWxl; P / N 8541; 250A; 5 μm; 7.8 mm × 300 mm). The mobile phase was 100 mM sodium phosphate, 300 mM sodium chloride, pH 6.8, 10% acetonitrile (v / v), or 1× PBS. A typical run was isocratic for 20 minutes at a flow rate of 1 mL / min.

[0453] Analysis HIC conditions: HIC spectra were recorded at 280 nm on a Hewlett-Packard HP1100 or Agilent 1100 Series LC system equipped with a diode array detector using an HIC column (typically a Tosoh Butyl-NPR, 4.6 x 35 mm, 2.5 um, P / N: 14947). Mobile phase A was 25 mM sodium phosphate, 1.5 M ammonium sulfate (pH 7), and mobile phase B was 75% 25 mM sodium phosphate (pH 7), 25% isopropanol. For a 20 min run, a 12 min linear gradient of 95% / 5% A / B to 100% B is used between the initial and final intervals of isocratic flow.

[0454] LC-QTOF conditions: LCMS spectra were recorded on an Agilent 1260 Bioinert Series LC system connected to an Agilent 6545 QTOF mass spectrometer using a reversed-phase column (typically an Agilent PLRP-S, 5 μm, 1000 Å, 2.1 mm × 50 mm) heated to 80 °C. Various gradients and run times were selected to optimize compound characterization. The mobile phase was based on an ACN / water gradient containing 0.1% formic acid. An example of the solvent gradient used was from 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), as shown in Table 1.

[0455] [Table 1]

[0456] Samples were either intact or reduced (20 μL of 1-5 mg / mL ADC solution treated with 4 μL of 0.5 M DTT solution for 30 min at 37°C). Raw data were deconvoluted to the appropriate mass range using Agilent BioConfirm software to obtain the protein molecular weight(s), and DARs were calculated using the Agilent DAR Calculator.

[0457] LC / MS / MS conditions: LC / MS / MS analysis was performed using a Shimadzu UFLC LC-20AD XR dual pump and SIL-30AC MP autosampler system and an AB SCIEX Triple Quad 4500 ESI mass spectrometer.

[0458] Typically, a 5uL sample aliquot was injected into the LC / MS / MS after passing it through a Waters Xselect C18 CSH 3.5u 2.1mm ID x 30mm column. Mobile phase A contained 0.1% formic acid in water, and mobile phase B contained 0.1% formic acid in water (5%) and acetonitrile (95%). The total run time was 3 minutes at a flow rate of 1.5mL / min with a linear gradient from 100% A to 100% B in 1.5 minutes. The instrument was first run with 100% aqueous mobile phase solvent for 0.5 minutes, then increased to 100% organic solvent over the next 1.5 minutes.

[0459] Preparative SEC: SEC column (typically GE Superdex 200 Increase 1 Preparative SEC purification was performed on a Gilson preparative HPLC system equipped with a UV detector using a UV filter (0 / 300 GL). The mobile phase was 1x PBS (pH 7.4). A typical run was performed isocratically for 30 min at a flow rate of 1 mL / min. Fraction collection was triggered based on UV thresholds (214 and 280 nm).

[0460] ADC concentration: ADC concentrations were calculated from UV absorbance at 280 nm measured by NanoDrop (2000c; Fisher Scientific) coefficients after subtracting the UV absorbance from the corresponding linker-payload construct.

[0461] Table 2 lists the linker-payload constructs used in the ADC preparation. The compounds contain either compound No. 14 (described in WO2018 / 100558A2) or compound I-5c (described in WO2019 / 092660) as the payload. The synthesis of the linker-payload constructs was described in PCT Application No. PCT / IB2020 / 054400.

[0462] [Table 2-1]

[0463] [Table 2-2]

[0464] [Table 2-3]

[0465] [Table 2-4]

[0466] [Table 2-5]

[0467] An anti-human CCR2 monoclonal antibody composed of humanized variable domains of the heavy and light chains of the 1D9 mouse monoclonal antibody and the constant domains of a human IgG1 heavy chain and a human kappa light chain (humanized 1D9, also known as TAK-202, and hereinafter also referred to as the hIgG1 isotype) was produced as described in US Pat. No. 7,473,421 B2. The hIgG4 isotype of humanized 1D9 was prepared in a manner similar to that described in Anticancer Research March-April 2006, vol. 26, no. 2A, pp. 1057-1063. Sequence of humanized 1D9 Heavy chain: EVQLVESGGG LVKPGGSLRL SCAASGFTFS AYAMNWVRQA PGKGLEWVGR IRTKNNNYAT YYADSVKDRF TISRDDSKNT LYLQMNSLKT EDTAVYYCTT FYGNGVWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKKVEPKSC DKTHTCPPCP APELAGAPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 3) Light chain: DVVMTQSPLS LPVTLGQPAS ISCKSSQSLL DSDGKTFLNW FQQRPGQSPR RLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP YTFGQGTRLE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC (SEQ ID NO: 4) Humanized 1D9 hIgG4 isotype sequence Heavy chain: EVQLVESGGGLVKPGGSLRLSCAASGFTFSAYAMNWVRQAPGKGLEWVGRIRTKNNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTFYGNGVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 5) Light chain: DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTFLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 6)

[0468] Example 1 Procedure for the preparation of Ab-STING agonist conjugates by stochastic cysteine ​​conjugation

[0469] To a solution of anti-CCR2 antibody (humanized 1D9, 10 mg / mL) in 50 mM histidine, 125 mM arginine, and pH 6.1 buffer, TCEP (1 mM aqueous solution, 2–3 equivalents) was added. The reaction mixture was purged with argon and incubated at room temperature or 37°C for 1–3 hours with gentle shaking. The desired linker-payload construct (5 mM DMA solution, 6–9 equivalents) was then slowly added to the mixture. The reaction mixture was purged with argon and incubated at room temperature or 37°C for an additional 1–2 hours with gentle shaking. The reaction mixture was purified according to the preparative SEC method described herein to obtain the ADC. The ADC concentration, aggregation rate, and DAR were measured by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

[0470] A schematic diagram of this procedure is shown in FIG.

[0471] Other antibody conjugates were prepared using procedures similar to those described above.

[0472] Example 2 Preparation of additional Ab-STING agonist conjugates by stochastic cysteine ​​conjugation The antibody drug conjugates listed in Table 3 were prepared as described in Example 1 using the linker-payload constructs and the indicated antibodies as starting materials.

[0473] [Table 3]

[0474] Example 3 Procedure for preparation of Ab-STING agonist conjugates by transglutaminase conjugation Deglycosylation: A solution of anti-CCR2 antibody (humanized 1D9, produced as described in US Pat. No. 7,473,421B2, 60 mg / mL) in a buffer solution of 50 mM histidine, 125 mM arginine, pH 6.1 was diluted with an equal volume of PBS, pH 7.2. N-glycosylase F (New England Biolabs, P0704S, 500,000 units / mL, 300 units per mg of antibody) was added to the solution, and the reaction mixture was heated to 37° C. overnight with gentle stirring. The resulting deglycosylated humanized 1D9 was buffer-exchanged with PBS (pH 7.2).

[0475] Transglutaminase conjugation: To a PBS solution (10-20 mg / mL) of deglycosylated humanized 1D9, prepared as described above, was added a 0.1 M DMSO solution of amine-PEG-azide (40 equivalents), followed by transglutaminase (ACTIVA™, Ajinomoto, 5-10 mg per mg of antibody). The reaction mixture was heated to 37°C overnight with gentle stirring. The product was purified according to the preparative SEC method described herein to yield humanized 1D9-NH-PEG-azide.

[0476] Strain-promoted azide-alkyne cycloaddition: To a solution of humanized 1D9-NH-PEG-azide (2-15 mg / mL in PBS), prepared as described above, was added the linker-payload. A 4-10 mM DMSO solution of the strained alkyne containing construct (3-5 equivalents, DMSO <10% of the total solvent volume) was added. The resulting solution was gently stirred overnight at room temperature. The product was purified according to the preparative SEC method described herein to obtain the ADC. The ADC concentration, aggregation rate, and DAR were measured by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

[0477] A schematic of this procedure is shown in Figure 2 (RG=N3). Other antibody conjugates were prepared using procedures similar to those described above.

[0478] Example 4 Preparation of Ab-STING agonist conjugates by transglutaminase conjugation The antibody drug conjugates listed in Table 4 were prepared as described in Example 3 using the initial linker-payload constructs shown in the table as starting materials.

[0479] [Table 4]

[0480] Example 5 Procedure for preparation of Ab-STING agonist conjugates by transglutaminase conjugation Transglutaminase conjugation (following the procedure described in Tumey, L. et al., Mol. Pharmaceutics 2019, 16, 6, 2795-2807): To a solution of transglutaminase (ACTIVA™, Ajinomoto, 50 mg per 1 mg of antibody) in pH 6.1 phosphate buffer was added a solution of deglycosylated humanized 1D9 in PBS (10–20 mg / mL, prepared according to the deglycosylation procedure described in Example 3), followed by a solution of 30 mM cystamine·2HCl (50 equiv.) in pH 6.1 phosphate buffer. The reaction mixture was heated to 37°C overnight with gentle stirring. The product was purified using a HiTrap Protein A HP column (GE Healthcare, 17-0402-01) by first washing with 20 mM phosphate (pH 7.0) and then eluting the ADC with 0.1 M citric acid (pH 4.0). Further purification by the preparative SEC method described herein gave humanized 1D9-NH—(CH 2 ) 2 —SS—(CH 2 ) 2 —NH 2 .

[0481] Maleimide addition: To a solution of humanized 1D9-NH—(CH2)2-SS—(CH2)2-NH2 conjugate (2–15 mg / mL in 20 mM pH 5 NaOAc buffer), prepared as described above, was added a 5 mM aqueous solution of TPPTS (5 equivalents) at 0°C. The resulting solution was incubated overnight at 0°C. After removal of the small molecule by dialysis, the mixture was further incubated at 0°C for 24 hours. Next, the desired linker-payload construct (5 mM DMA solution, 2.05 equivalents) was slowly added to the mixture. The reaction was incubated at 0°C for 1.5–2 hours with gentle shaking. The reaction mixture was purified according to the preparative SEC method described herein to obtain the ADC. The ADC concentration, aggregation rate, and DAR were measured by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

[0482] A schematic diagram of this procedure is shown in Figure 2 (RG = SH). Other antibody conjugates were prepared using procedures similar to those described above.

[0483] Example 6 Preparation of Ab-STING agonist conjugates by transglutaminase conjugation The antibody drug conjugates listed in Table 5 were prepared as described in Example 5 using the initial linker-payload constructs shown in the table as starting materials.

[0484] [Table 5]

[0485] Example 7 Preparation of Ab-STING agonist conjugates by transglutaminase conjugation To a solution of deglycosylated humanized 1D9 in PBS (10-20 mg / mL, prepared according to the deglycosylation procedure described in Example 3), 1 M Tris, 5 M NaCl, pH 8.0 buffer (10-20% of the total volume) was added to adjust the pH to 8.0. To the solution was added a 10 mM DMSO solution of a primary amine-containing linker-payload construct (20 equivalents), followed by transglutaminase (ACTIVA™, Ajinomoto, 100-150 mg per mg of antibody). The reaction mixture was heated to 37°C overnight with gentle stirring. The mixture was first washed with 20 mM phosphate (pH 7.0), then with 0. The product was purified using a HiTrap Protein A HP column (GE Healthcare, 17-0402-01) by eluting the ADC with 1 M citric acid (pH 4.0). The product was further purified according to the preparative SEC method described herein to obtain the ADC. The ADC concentration, aggregation rate, and DAR were measured by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

[0486] A schematic diagram of the procedure is shown in Figure 3. Other antibody conjugates were prepared using procedures similar to those described above.

[0487] Example 8 Preparation of Ab-STING agonist conjugates by transglutaminase conjugation The antibody drug conjugates listed in Table 6 were prepared as described in Example 7 using the initial linker-payload constructs shown in the table as starting materials.

[0488] [Table 6]

[0489] Example 9 Procedure for the preparation of mouse Ab-STING agonist conjugates by stochastic cysteine ​​conjugation To a solution of anti-mCCR2 MC-21 antibody (Universitaetsklinikum Regensburg, Regensburg, Germany; described in Mack, M. et al. J. Immunol. 2001, 166, 4697-4704 and WO 2007 / 115713) (mIgG2a with L235A-G237A-E318A mutations in the heavy chain) in 25 mM sodium citrate (pH 5.5 buffer) (3.4 mg / mL) was added 0.5 M tris, 25 mM EDTA (pH 8 solution, 10% of the total volume), and TCEP (10 mM aqueous solution, 20 equivalents). The reaction mixture was purged with argon and incubated at 37°C for 1.5 hours 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°C. A solution of dehydroascorbic acid in DMSO (2 mM, 3 equivalents relative to reduced antibody) was added, and the resulting mixture was stored at 4 °C overnight. After warming to room temperature, the desired linker-payload construct (5 mM in DMA, 7 equivalents relative to reduced antibody) was slowly added. The reaction was incubated at room temperature for an additional 1.5–2 h with gentle agitation. The reaction mixture was purified according to the preparative SEC method described herein to obtain the ADC. The ADC concentration, aggregation rate, and DAR were measured by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

[0490] A schematic diagram of this procedure is shown in Figure 1.

[0491] Example 10 Further mouse Ab-STING antibody binding via stochastic cysteine ​​conjugation Preparation of Nist conjugates The antibody drug conjugates listed in Table 7 were prepared as described in Example 9 using the linker-payload constructs and the indicated antibodies as starting materials.

[0492] [Table 7]

[0493] Example 11 Plasma stability assay conditions Test compounds were spiked into 1 mL of plasma at a concentration of 10 μg / mL, and five equal-volume aliquots were distributed into 2 mL Eppendorf microcentrifuge tubes (0, 24, 48, 72, and 96 h). At time 0, the tube was immediately stored at -80°C, and the remaining tubes were incubated at 37°C with gentle shaking. Aliquots were removed from the incubator at their corresponding time points and stored at -80°C. After all samples were collected, they were thawed at room temperature and placed on wet ice. 50 μL of each sample was distributed in triplicate into a 96-well microtiter plate. Samples were quenched with 200 μL of ice-cold methanol containing 50 nM internal standard. Samples were vortexed for 2 minutes and then centrifuged at 3000 rpm for 10 minutes. 185 μL of the supernatant was transferred to a clean injection plate and then dried at 40°C under N2 gas. The dried sample extracts were reconstituted with 100 μL of LCMS-grade water and then vortexed for 1 min in preparation for LC-MS / MS analysis.

[0494] A gradient consisting of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) was used at 40°C on a Synergi 2.5μ Polar-RP. Each sample was separated by reversed-phase HPLC using a 100A C18 column (2.0 mm x 30 mm) (Phenomenex®). Analytes were analyzed by positive ion spray in multiple reaction monitoring (MRM) mode using a SCIEX API 4500 QTRAP instrument. The percentage payload loss in human, primate, and mouse plasma at various time points is reported in Table 8.

[0495] [Table 8]

[0496] Example 12 THP1 Dual Lucia reporter gene assay conditions THP1-Dual(TM) KI-hSTING-R232 cells (InvivoGen The THP-1 cell line (#thpd-r232) was derived from the human THP-1 monocytic cell line by a stable biallelic knockout of the endogenous human HAQ STING gene and knock-in of the R232 variant of human STING. These cells also stably express an inducible secreted Lucia luciferase reporter gene under the control of the ISG54 (interferon-stimulated gene) minimal promoter combined with five IFN-stimulated response elements (ISREs). Reporter gene expression allows for the study of the IFN-regulated factor (IRF) pathway by assessing Lucia luciferase activity. In addition to human STING and luciferase, these cells were engineered to stably express human CCR2, allowing target-mediated activation of the IRF pathway to be studied. THP-1 cells express endogenous human CCR2 at a much lower density than that of cells engineered to overexpress human CCR2. Therefore, empty vector cells could still be used as a negative control.

[0497] On the day of the experiment, cells were plated in growth medium (RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 100 μg / mL Normocin™, 100 U / mL-100 μg / mL Pen-Strep, 10 μg / mL blasticidin, 100 μg / mL Zeocin, and 1 μg / mL puromycin) at a density of 15,000 cells / 25 μL per well in a white 384-well plate. Cells were plated onto a Corning 356661 plate. Cell plates were dosed with 5 μL of hCCR2-targeting ADC or compound samples and then incubated at 37° C. for 20 hours. At the end of the incubation, 10 μL / well of QUANTI-Luc™ (InvivoGen #rep-qlc1) was added, and luminescence was immediately measured using a LeadSeeker.

[0498] For the assay described above, the percent luminescence signal induction for each test ADC or test compound at various concentrations was calculated relative to untreated and control-treated samples. Compound concentration vs. percent signal induction curves were fitted to determine the EC 50 Those skilled in the art will appreciate that EC 50 It will be understood that the values ​​generated as observed EC 50 and Emax are reported in Table 9. The data in Table 9 clearly demonstrate that conjugation of either Compound No. 14 or Compound I-5c to humanized 1D9 or its IgG4 isotype dramatically increases in vitro potency in the hCCR2-overexpressing THP1 cell line.

[0499] [Table 9]

[0500] Example 13 Pharmacokinetic evaluation in mice For in vivo evaluation of ADCs in naive Balb / C mice, 6-8 weeks of age Female Balb / c mice (purchased from Jackson Laboratory) were used. Mice were fed a normal diet and maintained in accordance with the Guide for the Care and Use of Laboratory Animals (Guide). Animals were housed in a SPF animal facility in accordance with regulations of the United Nations for Care and Use of Laboratory Animals (UNCIL) and the Animal Care and Use Committee. Animals were maintained on a 12-h intermittent light / dark cycle at a temperature of 18–26°C, a relative humidity of 50 ± 20%, and with free access to food and water.

[0501] The pharmacokinetics of the ADC was investigated after injection into Balb / C mice. Serum samples were collected at various time points and stored frozen for analysis.

[0502] Mouse plasma levels of total antibody and conjugated payload were measured using a 2-in-1 immunocapture-based LC / MS assay on a Shimadzu UHPLC system interfaced with a Sciex 6500 QTRAP mass spectrometer. Briefly, mouse plasma samples were incubated with anti-human IgG-coated magnetic beads for 45 minutes at room temperature. Nonspecifically bound proteins were then removed by washing the magnetic beads sequentially with PBST (PBS buffer, pH 7.4, containing 0.05% Tween 20) and PBS buffer. Both naked antibody (DAR = 0) and ADC (DAR ≥ 1) were then eluted from the magnetic beads with 0.1% trifluoroacetic acid. After neutralizing the eluate and spiking it with a stable isotope-labeled internal standard, one aliquot of the sample was pipetted and used for LC / MS analysis of the conjugated payload after papain digestion at 37°C for 1 hour. The remaining sample was subjected to trypsin / lys-C digestion at 70°C for 1 hour and then used for LC / MS analysis of total antibody.

[0503] Circulating free payload was also measured by LC / MS after plasma protein precipitation. Briefly, mouse plasma was mixed with 8 volumes of methanol containing stable isotope-labeled internal standards, and the supernatant was then evaporated to dryness at 40°C under a gentle stream of nitrogen. Finally, the residue was reconstituted in LC / MS-grade water prior to LC / MS analysis.

[0504] The PK profiles of 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 4-8.

[0505] [Table 10]

[0506] Example 14 Tolerability evaluation in mice The tolerability of the ADC was evaluated in naive C57BL / 6 mice. On day 0 of the study, animals were weighed and then intravenously administered the indicated amount of ADC (according to payload concentration). Animals were then measured regularly (with no more than 3 days between measurements) for at least 14 days after dosing, and after each measurement, weight loss was calculated based on the starting weight before dosing. Any animals that lost more than 20% of their body weight or that became moribund or otherwise exhibited distress in excess of the study's humane endpoint were removed from the study and euthanized according to the guidelines of the IACUC protocol. The maximum tolerated dose (MTD) was calculated as the highest dose (according to payload concentration) at which no animals were found dead or required removal from the study due to greater than 20% weight loss or otherwise exceeding the humane endpoint. The MTD of ADC-B17 was 200 μg / kg (depending on payload concentration, FIG. 9), and the MTD of ADC-B20 was 250 μg / kg (depending on payload concentration, FIG. 10).

[0507] Example 15 Antitumor activity evaluation in mice The efficacy of ADC-B21 compared to Compound No. 14 was evaluated in a C57BL / 6 mouse model bearing MC38 (murine colon adenocarcinoma) tumors. For tumor implantation, 1×10 6 MC38 cells were subcutaneously injected into C57BL / 6 mice, and the mice were then monitored for tumor growth. 3 When tumor volume reached a mean of 2000 mm, animals were randomized by tumor volume and administered 100 μL of either vehicle, Compound No. 14 (2000 μg / kg), or ADC-B21 (50 μg / kg) intravenously. The first day of administration was considered day 0 of the study. Compound No. 14 and vehicle were administered again on days 3 and 6 of the study, while ADC-B21 was administered as a single dose on study day 0. Tumor volume and body weight measurements were taken at least twice weekly until the end of the study, and tumor volume and body weight measurements were taken at least twice weekly until the end of the study. Tumor volume and body weight measurements were taken at least twice weekly until the end of the study, and tumors were discontinued if there was a weight loss of more than 20% from the starting weight or a weight loss of 2000 mm. 3 Animals with tumor volumes exceeding 100 mg / kg were removed. By study day 63, animals treated with Compound #14 had a total of 4 complete responses compared to ADC-B21 treatment. Compared with the previous treatment, there was a total of 1 complete remission out of 6.

[0508] A graphical representation of the observed anti-tumor activity is shown in Figure 11, demonstrating the significantly improved efficacy of the anti-CCR2 ADC at much lower dose levels compared to the payload alone.

[0509] Example 16 Toxicity / pharmacodynamic evaluation in non-human primates The two ADC variants were evaluated in a toxicity study in cynomolgus monkeys.

[0510] A single-dose study was conducted using ADC-B2 administered intravenously at 0.15, 0.5, 1.5, or 5 mg / kg (2 monkeys / sex / group) (protein dose). Administration of 5 mg / kg ADC-B2 was associated with early death in two animals on day 2, attributed to pulmonary toxicity similar to previous studies with unconjugated payload (Compound No. 14) (clinical signs were macroscopic red discoloration, intra-alveolar edema and fibrin, increased alveolar macrophage and neutrophil infiltration, and decreased corporal mucosal pallor and depressed heart sounds, correlating with pleural and pericardial effusion in some animals, as well as histological findings of mild pulmonary vascular congestion and acute alveolar hemorrhage). Other findings unique to these early-death animals were present in the bone marrow (decreased hematopoietic cellularity, single-cell necrosis, and increased histiocytes), liver (multifocal random foci of necrosis), and lymphoid tissues (decreased germinal center cellularity and / or necrosis in the spleen and tonsils, and single-cell necrosis in the thymus). Clinical pathology and cytokine analysis of one of the early-death animals for which a sample was available revealed a proinflammatory / acute-phase response and elevated IP-10, IL-6, MCP1, and TNF-α cytokine levels similar to those of animals that survived to terminal euthanasia. Histologic findings in animals that survived to terminal euthanasia were limited to increased lymph node cellularity (due to increases in lymphocytes and histiocytes) at ≥1.5 mg / kg and, in one animal, lymph node germinal center necrosis at 5 mg / kg. Pharmacological endpoints included in the study consisted of flow cytometry to assess monocyte populations and identified a dose-dependent decrease in the relative proportions of classical, intermediate, and non-classical monocytes, as well as myeloid-derived immunosuppressive cells (MDSCs) at 6 and 24 hours post-dose on Day 1, with partial recovery by 48 hours post-dose.

[0511] A repeat-dose study was conducted with intravenous administration of ADC-B17 at 0.3, 1, or 3 mg / kg (protein dose) (two monkeys / sex / group), scheduled every two weeks for a total of three doses. However, due to early death of two animals from the 3 mg / kg dose group after the second dose on Day 15, the remaining two animals in Group 4 received a 2 mg / kg dose reduction on Day 29 (third / final dose). Repeated administration of ADC-B17 at ≥0.3 mg / kg was associated with the development of anti-drug antibodies (ADAs) (signal-to-noise ratio increases of 1-3 grades) in 10 of 12 animals at one or more time points after Day 15, most of which were directed against the immunostimulatory payload of the ADC; some ADAs were observed at the end of the time course, directed against the antibody component of the ADC. These ADAs were associated with a decrease in exposure (Cmax) after the third dose in the majority of ADA-positive animals. Early ADC-B17-related mortality was observed at doses ≥ 1 mg / kg. One animal at 1 mg / kg was euthanized in moribund conditions approximately 7 hours after dosing on Day 29. At 3 mg / kg, one animal was found dead approximately 6 hours after dosing on Day 15, and one animal was euthanized in moribund conditions approximately 7 hours after dosing on Day 15. ADC-B17-related clinical signs in these animals preceding death included red skin (face), decreased activity, hunched posture, weight loss, excessive salivation, partially closed eyes, sunken eyes, increased body temperature, heart murmur, and / or increased heart and / or respiratory rate. The cause of death was immune-related, likely due to an immunogenic / hypersensitivity reaction. Although stress-related effects were attributable to the effects of ADC-B17, a direct effect of ADC-B17 could not be discounted. Serum chemistry findings from all three prematurely deceased animals were generally similar to those that survived to terminal euthanasia, consistent with a systemic proinflammatory response and muscle and / or hepatocyte injury. Hematological and coagulation parameters were assessed in animals euthanized at moribund day 29, but not in animals euthanized at day 15. Stress-related lymphocyte and eosinophil depletion was minimal, and there were no changes in coagulation parameters. The immunophenotypic changes observed were similar to those in surviving animals, as described below. Most microscopic findings in animals that died early on day 15 were similar to, but more severe than, those in animals that survived to terminal euthanasia, consisting of minimal hepatocyte necrosis and physical findings consistent with immune-mediated effects (immune cell infiltrates in the liver sinusoids, adrenal glands, pulmonary interstitium, and spleen; thrombosis in pulmonary capillaries; necrosis / fibrin deposits in the spleen; myocardial degeneration; and microhemorrhages in the adrenal glands and epicardial fat). Additional findings unique to animals that died early were considered secondary to stress or moribundity (decreased thymic cellularity, correlating with decreased thymus weight and pancreatic acinar cell breakdown). In animals euthanized at day 29 due to moribundity, the only finding was minimal adrenal hemorrhage.

[0512] In animals that survived to terminal euthanasia, clinical pathology findings were observed at ≥0.3 mg / kg on day 3 and consisted of mild to moderate increases in one or more of aspartate aminotransferase, alanine aminotransferase, glutamate dehydrogenase, and creatine kinase. These findings were consistent with muscle and / or hepatocyte origin and lacked clear histologic correlation. Other findings on day 3 were consistent with a systemic proinflammatory / acute-phase response (minimal to mild increases in globulin and c-reactive protein, and minimal to mild decreases in total protein, albumin, and albumin / globulin ratio) histologically correlated with inflammatory cell infiltration in multiple tissues or dehydration (mild increases in urea, creatinine, and phosphorus) without histologic correlation. Each of these changes partially to completely resolved by day 30. In males, further serum chemistry changes on days 14 and / or 30 consisted only of modest increases in globulins and modest elevations in total bilirubin, both consistent with an ongoing acute-phase inflammatory response. At terminal euthanasia on day 30, hematological and coagulation findings in individual animals at ≥0.3 mg / kg consisted of modest increases in white blood cell count, neutrophil count, fibrinogen, and activated partial thromboplastin time, and modest decreases in red blood cell count, hemoglobin, and hematocrit. These findings were consistent with a systemic proinflammatory / acute-phase response.

[0513] Changes in monocytes and MDSCs in plasma samples were assessed using a flow cytometry panel designed to assess monocyte and MDSC enumeration, as well as CCR2, CD80, and CD86 expression on monocytes. Findings from this assessment were consistent with the expected pharmacology of ADC-B17 at doses ≥0.3 mg / kg and consisted of a dose-responsive, moderate to profound decrease in absolute numbers of classical monocytes, non-classical monocytes, and myeloid-derived immunosuppressive cells (MDSCs) as measured by flow cytometry after each dose, with recovery toward or above baseline before each subsequent dose. CCR2 expression on classical monocytes declined after each dose, with recovery to near baseline at all doses before subsequent doses (Figure 12, top). Furthermore, CD80 expression on both classical monocytes and MDSCs was found to increase after each dose and then recover to near or below baseline levels before subsequent doses (Figure 12, middle and bottom, respectively).

[0514] Changes in cytokines were also assessed in plasma samples and showed a dose-independent large increase in serum IP-10 and MCP-1 concentrations, potential biomarkers of pharmacology, with ADC-B17 at ≥ 0.3 mg / kg, which peaked 6 hours after dosing and returned or trended back to baseline values ​​24 hours after dosing. Further increases in IL-1RA, IL-6, TNF-α, and IFN-γ were observed, peaking 6 hours after dosing and returning or trending back to baseline values ​​24 hours after dosing or before subsequent doses (Figure 13).

[0515] Histologic findings in animals at terminal euthanasia consisted of multifocal hepatocellular necrosis without clinical pathologic correlation at doses ≥0.3 mg / kg. At doses ≥1 mg / kg, there was minimal to mild reduction in bone marrow cellularity, both erythroid and myeloid precursors (correlating with mildly decreased erythrocytes in hematology and in one animal with markedly decreased lymphocytes), scattered mixed cell infiltrates in the adrenal glands and liver sinusoids, increased cellularity (mixed cells) in the splenic red pulp correlating with mildly increased spleen weight, and minimal focal hemorrhage in the duodenum or heart. These organs with inflammatory cell infiltration / hemorrhage were considered likely part of a systemic proinflammatory response and were not considered direct target organ toxicity. Immunohistochemistry for human IgG, monkey IgG and IgM, C3, and / or C9 was performed to determine whether immune complex formation and tissue adhesion were present in areas of immune cell infiltration and / or tissue damage. Granule adhesion, which indicates immune complex formation, was not detected.

[0516] Example 17 Pharmacokinetic evaluation in non-human primates Serum samples were collected at various time points from non-human primates administered ADC-B17 as described in Example 16 and stored frozen for analysis. Monkey plasma levels of total antibody and conjugated payload were measured using a 2-in-1 immunocapture-based LC / MS assay on a Shimadzu UHPLC system interfaced with a Sciex 6500+ QTRAP mass spectrometer. Briefly, monkey plasma samples were incubated with anti-idiotypic antibody-coated magnetic beads for 60 minutes at room temperature, after which nonspecifically bound proteins were removed by washing the magnetic beads three times with PBS buffer. Both the naked antibody (DAR = 0) and the ADC (DAR ≥ 1) were then eluted from the magnetic beads with 0.1% trifluoroacetic acid. After neutralizing the eluate and spiking it with a stable isotope-labeled internal standard, one aliquot of the sample was pipetted and digested with trypsin / lys-C at 60°C for 1 hour before use in LC / MS analysis of the total antibody. The remaining sample was subjected to papain digestion at 37°C for 1 hour and then used for LC / MS analysis of the conjugated payload.

[0517] Circulating free payload was also measured by LC / MS after plasma protein precipitation. Briefly, monkey plasma was first spiked with stable isotope-labeled Compound 14, followed by protein precipitation using methanol and evaporation to dryness under a gentle stream of nitrogen. Finally, the residue was reconstituted in ammonium acetate solution and then analyzed by LC / MS.

[0518] The PK profile of ADC-B17 is summarized in Table 11. A graphical representation of the plasma PK is shown in Figure 14.

[0519] [Table 11]

[0520] Example 18 (Prophetic) Combination therapy with PD-1 / PD-L1 antibody The tolerability of ADCs in combination with anti-PD-1 and / or anti-PD-L1 antibodies can be assessed in naive C57BL / 6 mice.

[0521] Available ADC and anti-PD-1 / anti-PD-L1 combinations are shown in Table 12.

[0522] [Table 12]

[0523] For tolerability studies, the ADCs shown in Table 12 can be administered at 0.05 mg / kg, and the anti-PD-1 and anti-PD-L1 antibodies can be administered at 0.5, 5, or 50 mg / kg. Because the anti-PD-1 antibody pembrolizumab is not cross-reactive with rodent PD-1, mice will receive the rat anti-mouse PD-1 antibody J43 and the rat anti-mouse PD-L1 antibody MIH5 at 0.5, 5, and 50 mg / kg, respectively.

[0524] On study day 0, animals can be weighed and then intravenously administered the labeled amount of ADC in combination with the labeled amount of anti-PD-1 and / or anti-PD-L1 antibody. Animals can then be weighed regularly (with no more than 3 days between measurements) for at least 14 days after dosing, and weight loss can be calculated after each measurement based on the starting weight before dosing. Any animals that lose more than 20% of their body weight or that appear moribund or otherwise exhibit distress that exceeds the humane endpoint of the study can be removed from the study and euthanized according to the guidelines of the IACUC protocol. The maximum tolerated dose (MTD) can be calculated as the highest dose (by payload concentration + PD-1 / PD-L1 antibody concentration) at which no animals were detectably dead or required removal from the study due to greater than 20% weight loss or otherwise exceeding the humane endpoint. If satisfactory tolerability is achieved with the ADC and either the anti-PD-1 or anti-PD-L1 antibody, the combination of the ADC and the anti-PD-1 and anti-PD-L1 antibodies will be considered. Combination therapy with PD-L1 antibodies can be carried out in a similar manner.

[0525] Efficacy studies for combination therapy in mice The efficacy of the ADCs shown in Table 12 in combination with the anti-PD-1 antibody J43 or the anti-PD-L1 antibody MIH5 can be tested in a C57BL / 6 mouse model bearing MC38 (murine colon adenocarcinoma) tumors. For tumor implantation, 1 x 10 6 MC38 cells can be injected subcutaneously into C57BL / 6 mice, and the mice can then be monitored for tumor growth. 3 When tumor volume reaches a mean of 100 μL, animals can be randomized by tumor volume and administered intravenously with 100 μL of vehicle, the corresponding ADC from Table 12 (50 μg / kg), and either J43 (0.5, 5, or 50 mg / kg), or MIH5 (0.5, 5, or 50 mg / kg). The first day of administration can be considered day 0 of the study. Tumor volume and body weight measurements can be taken at least twice weekly until the end of the study, and tumor volume and body weight measurements can be taken at least twice weekly until the end ... 3 Animals with tumor volumes exceeding 63 days can be removed from the study. Animals can be evaluated for complete and partial response on day 63 of the study. If a satisfactory reduction in tumor volume is not achieved by combining the ADC with either the anti-PD-1 or anti-PD-L1 antibody, combination therapy with the ADC and anti-PD-1 and anti-PD-L1 antibodies can be performed in a similar manner.

[0526] Efficacy studies of combination therapy in non-human primates The ADCs, in combination with the anti-PD-1 antibody pembrolizumab or the anti-PD-L1 antibody atezolizumab, can be administered intravenously to cynomolgus monkeys every 2 weeks (Days 1-29) at 0.3, 0.5, or 1 mg / kg (protein dose) (2 monkeys / sex / group) for a total of 3 doses. Pembrolizumab can be administered at 0.5 or 15 mg / kg, and atezolizumab can be administered at 0.5 or 15 mg / kg. Animals can be evaluated for hematological and coagulation parameters, overall serum chemistries, and histological evaluation at the end of the study.

[0527] At various time points, blood samples can be collected from the non-human primates, and monkey plasma levels of total antibody and conjugated payload can be measured by a 2-in-1 immunocapture-based LC / MS assay on a Shimadzu UHPLC system interfaced to a Sciex 6500+ QTRAP mass spectrometer, as described above. Circulating free payload can also be measured by LC / MS after plasma protein precipitation, as described above.

[0528] Example 19 (Prophetic) Combined therapy with radiation Tolerability studies The tolerability of ADCs in combination with anti-PD-1 and / or anti-PD-L1 antibodies and radiation can be assessed in naive C57BL / 6 mice.

[0529] The available combinations of ADC, anti-PD-1 and / or anti-PD-L1 antibody, and radiation are shown in Table 13.

[0530] [Table 13]

[0531] For tolerability studies, the ADC can be administered at 0.05 mg / kg, the anti-PD-1 and anti-PD-L1 antibodies can be administered at 0.5, 5, or 50 mg / kg, and radiation can be administered at 0.5 Gy and 1 Gy.

[0532] On study day 0, animals can be weighed and radiation administered with the indicated dose of anti-PD-1 The indicated doses of ADC were administered in combination with J43 and / or anti-PD-L1 MIH5 antibody. The dose can be administered approximately 5 hours before intravenous administration. Animals are then weighed regularly (with no more than 3 days between each measurement) for at least 14 days after dosing, and after each measurement, weight loss can be calculated based on the starting weight before dosing. Any animals that lose more than 20% of their body weight or that appear moribund or otherwise exhibit distress that exceeds the humane endpoint of the study can be removed from the study and euthanized according to the guidelines of the IACUC protocol. The maximum tolerated dose (MTD) can be calculated as the highest dose of radiation in the combination therapy regimen at which no animals are detectably dead or require removal from the study due to either a greater than 20% weight loss or otherwise exceeding the humane endpoint. If satisfactory tolerability is achieved with the ADC, anti-PD-1 or anti-PD-L1 antibody, and radiation, combination therapy of the ADC, anti-PD-1 and anti-PD-L1 antibody, and radiation can be performed in a similar manner.

[0533] Efficacy studies in mice for combination with radiation therapy The efficacy of the ADCs shown in Table 13 in combination with anti-PD-1 and / or anti-PD-L1 antibodies and radiation can be tested in a C57BL / 6 mouse model bearing MC38 (murine colon adenocarcinoma) tumors. For tumor implantation, 1 x 10 6 MC38 cells can be injected subcutaneously into C57BL / 6 mice, and the mice can then be monitored for tumor growth. 3When tumor volume reaches a mean of 1000 mm, animals are randomized by tumor volume and irradiated with either 0.5 Gy or 1 Gy of radiation, and administered intravenously with 100 μL of vehicle, the corresponding ADC from Table 13 (50 μg / kg), and either the anti-PD1 antibody J43 (0.5, 5, or 50 mg / kg), or the anti-PD-L1 antibody MIH5 (0.5, 5, or 50 mg / kg). The first day of administration can be considered day 0 of the study. Tumor volume and body weight measurements can be performed at least twice weekly until the end of the study, and a tumor volume of 1000 mm can be determined if the tumor volume decreases by more than 20% from the starting weight or if the tumor volume exceeds 2000 mm. 3 Animals with tumor volumes exceeding 63 days can be removed from the study. Animals can be evaluated for complete and partial response on day 63 of the study. If a satisfactory reduction in tumor volume is not achieved with the combination of the ADC, radiation, and either the anti-PD-1 or anti-PD-L1 antibody, combination therapy with the ADC, radiation, and anti-PD-1 and anti-PD-L1 antibody can be performed in a similar manner.

[0534] Efficacy studies in non-human primates for combination with radiation therapy Before administering the ADC and anti-PD-1 and / or anti-PD-L1 antibodies, cynomolgus monkeys can be treated with 0.8 Gy and 1.2 Gy. Following radiation therapy, the ADC can be administered intravenously to cynomolgus monkeys at 0.3, 0.5, or 1 mg / kg (protein dose) (two monkeys / sex / group) for a total of three doses every two weeks (Days 1-29). Pembrolizumab can be administered at 0.5 or 15 mg / kg, and atezolizumab can be administered at 0.5 or 15 mg / kg. Animals can be evaluated for hematological and coagulation parameters, overall serum chemistry, and histological evaluation at the end of the study.

[0535] At various time points, blood samples can be collected from non-human primates, and monkey plasma levels of total antibody and conjugated payload can be measured by a 2-in-1 immunocapture-based LC / MS assay on a Shimadzu UHPLC system interfaced to a Sciex 6500+ QTRAP mass spectrometer, as described above. Circulating free payload can also be measured by LC / MS after plasma protein precipitation, as described above. Post-radiation hematologic recovery and extramedullary toxicity can be assessed in the animals.

[0536] It is understood that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more, but not all, example embodiments of the present disclosure as contemplated by the inventor(s) and are not intended to limit the scope of the disclosure and the appended claims in any way.

[0537] The present disclosure has been described above using functional building blocks that illustrate the implementation and relationship of specific functions thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of description. Other boundaries may be defined as long as the specific functions and relationships thereof are appropriately performed.

[0538] The foregoing description of specific embodiments will sufficiently disclose the general features of the present disclosure that others, by applying knowledge within the purview of those skilled in the art, may readily modify and / or adapt such specific embodiments for various applications without undue experimentation and without departing from the general concepts 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 used herein is for the purpose of description and not of limitation, as would be understood by one of ordinary skill in the art in light of the teaching and guidance.

[0539] 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. According to the present disclosure, the following aspects and embodiments are provided: [1] Compounds of formula (I): [ka] or a pharmaceutically acceptable salt thereof. [In the formula, a is an integer from 1 to 20, Ab is an anti-CCR2 antibody, an anti-CCR2 antibody fragment, or an anti-CCR2 antigen-binding fragment; D is a modulator of STING activity that contains an amino group on a guanine base, a guanine base derivative, an adenine base, or an adenine base derivative; L is a linker that is covalently attached to Ab and also covalently attached to the amino group on D. [2] The compound according to [1], wherein DL is represented by formula (Ia), or a pharmaceutically acceptable salt thereof. [ka] [In formula: [ka] indicates the point of attachment to the 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 is C 1 -C 4 Alkyl, OC 1 -C 4 independently selected from alkyl, and halogen; R 2 is C 1 -C 4 Alkyl and -(CH 2 CH 2 O) s -CH 3 [wherein s is an integer of 1 to 10], R 3 and R 3’ are hydrogen and C, respectively. 1 -C 3 independently selected from alkyl, L 1 is a cleavable linker fragment. [3] a is an integer from 1 to 8, b is an integer from 1 to 10; The compound according to [2], wherein m is 0, or a pharmaceutically acceptable salt thereof. [4] m is 0, n is 0, R 3 and R 3’

[0023] The compound according to [2] or [3], or a pharmaceutically acceptable salt thereof, wherein each of [5] L 1 but

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

[11] The compound according to any one of [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 according to any one of [5] to

[11] , wherein Z is Ala-Val or Val-Ala, or a pharmaceutically acceptable salt thereof.

[13] U' does not exist and U is

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[12] , or a pharmaceutically acceptable salt thereof, selected from: [In formula:

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change

[14] U' does not exist and U is

change

[13] , or a pharmaceutically acceptable salt thereof, wherein

[15] The compound according to any one of [5] to

[14] , or a pharmaceutically acceptable salt thereof, wherein Q is a heterobifunctional group and is bound to U', or if U' is absent, is bound to Ab by chemical or enzyme-mediated conjugation.

[16] Q is

change

[15] , or a pharmaceutically acceptable salt thereof, selected from: [In the ceremony

change

change

[17] Q is

change

[16] , or a pharmaceutically acceptable salt thereof, wherein

[18] The compound according to any one of [5] to

[17] , wherein t is 1, or a pharmaceutically acceptable salt thereof.

[19] R 2 Ga-CH 3 and R 3 and R 3’

[0033] The compound according to any one of [2] to

[18] , or a pharmaceutically acceptable salt thereof, wherein each of

[20] The compound according to any one of [1] to

[19] , wherein a is 2 to 6, or a pharmaceutically acceptable salt thereof.

[21] The compound according to any one of [1] to

[20] , wherein b is 1, or a pharmaceutically acceptable salt thereof.

[22] The amino-substituted compound that regulates STING activity is a compound of formula (II):

change

[21] , or a pharmaceutically acceptable salt thereof, wherein [In formula: X 10 is SH or OH, X 20 is SH or OH, Y a is O, S, or CH 2 and Y b is O, S, NH, or NR a [In the formula, Ra is C 1 -C 4 alkyl. R 10 are hydrogen, fluoro, OH, NH 2 , OR b , or NHR b and R 20 is hydrogen or fluoro, R 30 is hydrogen and R 40 are hydrogen, fluoro, OH, NH 2 , OR b , or NHR b or R 30 and R 40 Both are CH 2 Forming O R 50 is hydrogen or fluoro, R b is C 1 -C 6 Alkyl, halo(C 1 -C 6 ) alkyl, or C 3 -C 6 is 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 contains at least one N atom in the ring, where Y b is ring A 10 is bonded to a carbon atom of 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 contains at least two N atoms in the ring, However, ring A 10 or ring B 10 is bonded to "L" in formula (I) via the amino group.

[23] The amino-substituted compound that regulates STING activity is

change

[22] , or a pharmaceutically acceptable salt thereof, wherein [In the formula,

change

[24] The amino-substituted compound that regulates STING activity is a compound of formula (III):

change

[21] , 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 and Y d is O, S, or CH 2 and B 100 is the formula (B 1 -A) or formula (B 1 -B)

change

change

[25] The amino-substituted compound that regulates STING activity is a compound of formula (IIIa):

change

[21] and

[24] , or a pharmaceutically acceptable salt thereof. B 100 is the formula (B 1 -A) or formula (B 1 -B)

change

change

change

[26] The amino-substituted compound that regulates STING activity is a compound of formula (IV):

change

[21] and

[24] , or a pharmaceutically acceptable salt thereof. [In the ceremony R 1 and R 2 are each independently a hydroxy group or a halogen atom, B 1 teeth,

change

change

[27] The amino-substituted compound that regulates STING activity is

change

change

[28] A compound according to any one of [1] to

[21] and

[24] to

[26] of formula (VI), or a pharmaceutically acceptable salt thereof:

change

[29] The compound according to any one of [1] to

[28] , or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody or a 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 CCR2.

[30] The compound according to

[29] , or a pharmaceutically acceptable salt thereof, wherein the antibody is selected from the group consisting of monoclonal antibody 1D9 or antibodies capable of competing with 1D9 for binding to human CCR2 or a portion of CCR2; MC-21; STI-B020X; UniTI-101; and 4.40A68G.

[31] The compound according to

[30] , or a pharmaceutically acceptable salt thereof, wherein the antibody is monoclonal antibody 1D9 or an antibody capable of competing with 1D9 for binding to human CCR2 or a portion of CCR2.

[32] The compound according to any one of [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 according to

[31] , or a pharmaceutically acceptable salt thereof, wherein the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment comprises a light chain CDR1 comprising amino acids 24 to 39 of SEQ ID NO: 1; a light chain CDR2 comprising amino acids 55 to 61 of SEQ ID NO: 1; a light chain CDR3 comprising amino acids 94 to 102 of SEQ ID NO: 1; a heavy chain CDR1 comprising amino acids 31 to 35 of SEQ ID NO: 2; a heavy chain CDR2 comprising amino acids 50 to 68 of SEQ ID NO: 2; and a heavy chain CDR3 comprising amino acids 101 to 106 of SEQ ID NO: 2.

[34] The compound according to

[31] , or a pharmaceutically acceptable salt thereof, wherein the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2.

[35] The compound according to

[31] , or a pharmaceutically acceptable salt thereof, wherein the antibody, the anti-CCR2 antibody, the anti-CCR2 antibody fragment, or the anti-CCR2 antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1.

[36] The compound according to

[31] , or a pharmaceutically acceptable salt thereof, wherein the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 2.

[37] The compound according to

[31] , or a pharmaceutically acceptable salt thereof, wherein the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 1.

[38] The compound according to

[31] , or a pharmaceutically acceptable salt thereof, wherein the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 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, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 1.

[39] the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment is human immunoglobulin IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 The compound according to any one of

[31] to

[38] , or a pharmaceutically acceptable salt thereof, further comprising a heavy chain constant region selected from heavy chain constant regions.

[40] The compound according to any one of

[31] to

[39] , or a pharmaceutically acceptable salt thereof, wherein the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment further comprises a light chain constant region selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions.

[41] The compound according to

[31] , or a pharmaceutically acceptable salt thereof, wherein the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment binds to the same epitope as an antibody comprising the variable heavy chain region of SEQ ID NO: 2 and the variable light chain region of SEQ ID NO: 1.

[42] The compound according to

[31] , or a pharmaceutically acceptable salt thereof, wherein the anti-CCR2 antibody comprises a heavy chain region of SEQ ID NO: 3.

[43] The compound according to

[31] , or a pharmaceutically acceptable salt thereof, wherein the anti-CCR2 antibody comprises a light chain region of SEQ ID NO: 4.

[44] The compound according to

[31] , or a pharmaceutically acceptable salt thereof, wherein the anti-CCR2 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 the compound according to any one of [1] to

[44] or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

[46] The pharmaceutical composition according to

[45] , further comprising an anti-PD-1 antibody.

[47]

[46] The pharmaceutical composition according to

[46] , wherein the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, pimivalimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, ezabenlimab, INCMGA0012, AMP-224, AMP-514, SYM-021, LZM-009, CS-1003, SYN-125, GNR-051, MW-11, TY-101, BAT-1306, F520, sasanlimab, penprimab, pucotenlimab, CX-188, zimbarelimab, and tebotelimab.

[48] The pharmaceutical composition according to

[45] , further comprising an anti-PD-L1 antibody.

[49]

[48] ​​The pharmaceutical composition of

[48] , wherein the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, durvalumab, cosibelimab, MSB-2311, ZKAB-001, FAZ-053, MDX-1105, CBT-502, IMC-001, RC-98, KL-A167, GR-1405, lodapolimab, sugemalimab, embafolimab, opucolimab, and galibrimab.

[50] A method for treating cancer in a subject in need of cancer treatment, the method comprising administering to the subject a pharmaceutically acceptable amount of a compound according to any one of [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 according to any one of [1] to

[44] .

[52] The method of

[50] or

[51] , further comprising administering an anti-PD-1 antibody to the subject.

[53] The method of

[50] or

[51] , further comprising administering to the subject an anti-PD-L1 antibody.

[54]

[52] The method of

[52] , wherein the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, pimivalimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, ezabenlimab, INCMGA0012, AMP-224, AMP-514, SYM-021, LZM-009, CS-1003, SYN-125, GNR-051, MW-11, TY-101, BAT-1306, F520, sasanlimab, penprimab, pucotenlimab, CX-188, zimbarelimab, and tebotelimab.

[55]

[53] The method of

[53] , wherein the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, durvalumab, cosibelimab, MSB-2311, ZKAB-001, FAZ-053, MDX-1105, CBT-502, IMC-001, RC-98, KL-A167, GR-1405, lodapolimab, sugemalimab, embafolimab, opucolimab, and galibrimab.

[56] The method according to any one of

[52] to

[55] , wherein the anti-PD-1 antibody or the anti-PD-L1 antibody is administered simultaneously with the compound according to any one of [1] to

[44] .

[57] The method according to any one of

[52] to

[55] , wherein the anti-PD-1 antibody or the anti-PD-L1 antibody is administered consecutively with the compound according to any one of [1] to

[44] .

[58] The method according to any one of

[50] to

[57] , further comprising administering radiation to the subject.

[59] The method according to

[58] , wherein the radiation is particle radiation.

[60] The method of

[58] or

[59] , wherein the radiation is administered by external beam radiation.

Claims

1. Formula: 【Chemistry 1】 or a pharmaceutically acceptable salt thereof. [In the formula, a is an integer from 1 to 8, Ab is an anti-CCR2 antibody or anti-CCR2 antigen-binding fragment; R 2′ is C 1 -C 4 alkyl; W is 【Chemistry 2】 [In the formula, 【Transformation 3】 is the point of attachment to the carbonyl group, 【Chemistry 4】 is the point of attachment to Z. is selected from Z is Ala-Val or Val-Ala; U is 【Transformation 5】 [In the formula, p is an integer from 1 to 6; q is an integer from 1 to 20; 【Transformation 6】 is the point of attachment to Z, 【Transformation 7】 is the point of attachment to Q. and Q is 【Transformation 8】 [In the formula, 【Chemistry 9】 is the point of attachment to U, 【Chemistry 10】 is the point of attachment to the Ab. is selected from D is 【Chemistry 11】 [In the formula, R 1 and R 2 each independently represent a hydroxy group or a halogen atom; B 1 is 【Chemistry 12】 [In the formula, R 18 is hydrogen or C 1-6 alkyl; R 19 is a halogen atom; 【Chemistry 13】 is the point of attachment to D. and B2 is 【Chemistry 14】 [In the formula, 【Chemistry 15】 is the point of attachment to D, 【Chemistry 16】 is the point of attachment to the parent molecular moiety. and Q 2 and Q 4 each independently represent an oxygen atom or a sulfur atom.] It is.]

2. Q is 【Chemistry 17】 The drug according to claim 1, wherein

3. W is [Chemistry 18] The drug according to claim 2, wherein

4. R ２ Ga-CH ３ The drug according to claim 3, wherein

5. The drug according to claim 4, wherein a is an integer of 2 to 6.

6. D is 【Chemistry 19】 or a pharmaceutically acceptable salt thereof, wherein: 【Chemistry 20】 is the point of attachment to the parent molecular moiety.

7. The method of claim 1, wherein the compound is a compound of formula (VI), or a pharmaceutically acceptable salt thereof: 【Chemistry 21】 [In the formula, a is an integer of 1 to 8.]

8. The agent of claim 7, wherein the antibody is monoclonal antibody 1D9 or an antibody capable of competing with 1D9 for binding to human CCR2 or a portion of CCR2.

9. The agent of claim 7, wherein the anti-CCR2 antibody or anti-CCR2 antigen-binding fragment comprises a light chain CDR1 comprising amino acids 24 to 39 of SEQ ID NO: 1; a light chain CDR2 comprising amino acids 55 to 61 of SEQ ID NO: 1; a light chain CDR3 comprising amino acids 94 to 102 of SEQ ID NO: 1; a heavy chain CDR1 comprising amino acids 31 to 35 of SEQ ID NO: 2; a heavy chain CDR2 comprising amino acids 50 to 68 of SEQ ID NO: 2; and a heavy chain CDR3 comprising amino acids 101 to 106 of SEQ ID NO:

2.

10. The agent of claim 7, wherein the anti-CCR2 antibody or anti-CCR2 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, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

1.

11. the anti-CCR2 antibody or anti-CCR2 antigen-binding fragment is a human immunoglobulin IgG １ , IgG ２ , IgG ３ , IgG ４ , IgA １ , and IgA ２ 10. The method of claim 9, further comprising a heavy chain constant region selected from the group consisting of heavy chain constant regions and a light chain constant region selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions.

12. 11. The agent of claim 10, wherein the anti-CCR2 antibody or anti-CCR2 antigen-binding fragment further comprises a heavy chain constant region selected from human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 heavy chain constant regions, and a light chain constant region selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions.

13. The method of claim 7, wherein the anti-CCR2 antibody comprises a heavy chain region of SEQ ID NO: 3 and a light chain region of SEQ ID NO:

4.

14. The drug described in claim 1, wherein the cancer is selected from colon cancer, breast cancer, skin cancer, malignant lymphoma, and lung cancer.

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