Antibody-drug conjugates

Abstract

This invention provides an antibody-drug conjugate containing a STING modulator that is useful for stimulating an immune response in subjects requiring immune response stimulation. [Solution] Compound of formula (I): JPEG2026076188000119.jpg3331 or by a pharmaceutically acceptable salt thereof. [In the formula, a is an integer between 1 and 20, Ab is an anti-CCR2 antibody, an anti-CCR2 antibody fragment, or an anti-CCR2 antigen-binding fragment, D is a guanine base, a guanine base derivative, an adenine base, or a STING activity modulator containing an amino group on an adenine base derivative, and L is a linker covalently bonded to Ab and also to the amino group on D.]

Description

[Technical Field]

[0001] This disclosure provides an antibody-drug conjugate comprising a STING modulator. Compositions comprising the antibody-drug conjugate are also provided. The compounds and compositions are useful for stimulating an immune response in subjects requiring stimulation of an immune response. [Background technology]

[0002] Antibody-drug conjugates (ADCs), a rapidly growing class of targeted therapies, represent a novel and promising approach to improving drug selectivity and cytotoxic activity. These therapeutics consist of an antibody (or antibody fragment) capable of binding to a payload drug to form an immune complex. The antibody directs the ADC to bind to targeted cells. The ADC can then internalize and release its payload, delivering therapeutic effects to the cells. Because the ADC is directed to its targeted cells, the side effects of the conjugated drug may be lower than those seen when the drug is administered systemically.

[0003] STING (an interferon gene stimulant), an adapter protein, has been shown to play a role in the innate immune system. Activating the STING pathway triggers an immune response that leads to the production of specific killer T cells that shrink tumors, conferring long-lasting immunity and preventing tumor recurrence. The activated STING pathway also contributes to antiviral responses by producing antiviral and pro-inflammatory cytokines that fight viruses and mobilize both the innate and adaptive immune systems, resulting in long-lasting immunity against pathogenic viruses. Due to its potential therapeutic effects that enhance both innate and adaptive immune responses, STING is an attractive target for drug discovery. Cyclic dinucleotides can function as STING agonists and have been tested in clinical trials. However, due to their anionic properties, cyclic dinucleotides have poor membrane permeability, which can limit their ability to involve STING intracellularly, often resulting in the unwanted distribution of these compounds in the bloodstream.

[0004] Novel STING agonists, as well as improved methods for delivering these agonists to target cells, are still needed. [Overview of the project]

[0005] In the first aspect, this disclosure relates to formula (I):

[0006] [ka]

[0007] or provide a pharmaceutically acceptable salt thereof in the formula

[0008] a is an integer between 1 and 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, containing 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 bonded to Ab and also to the amino group on D.

[0012] In the 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] This indicates the connection point to Ab,

[0017] b is an integer between 1 and 20.

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

[0019] n is either 0 or 1.

[0020] Each R 1 These are independently selected from C1-C4 alkyl, O-C1-C4 alkyl, and halogens.

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

[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 D-L 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 D-L is represented by formula (Ia), wherein:

[0029] m is 0,

[0030] n is 0,

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

[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 D-L is represented by formula (Ia), wherein L 1 is

[0033]

Chemical formula

[0034] and, wherein,

[0035]

Chemical formula

[0036] is the bond point to the nitrogen atom in equation (Ia),

[0037] [ka]

[0038] is a connection point to Ab,

[0039] t is an integer between 1 and 10.

[0040] W either does not exist or is a self-sacrificing element.

[0041] Z is either absent or a peptide of 2-5 amino acids.

[0042] U and U' are either non-existent or spacers, independently.

[0043] Q is a heterodifunctional group.

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

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

[0046] [ka]

[0047] It is a self-sacrificing base selected from, During the ceremony:

[0048] [ka]

[0049] This is a bonding point to the carbonyl group,

[0050] [ka]

[0051] This is a connection point to Z.

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

[0053] [ka]

[0054] That is the case.

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

[0056] [ka]

[0057] That is the case.

[0058] In the seventh embodiment of the first aspect, Z is a peptide that can be cleaved by an enzyme.

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

[0060] In the 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 the tenth embodiment of the first aspect, Z is Ala-Val or Val-Ala.

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

[0063] [ka]

[0064] Selected from, During the ceremony:

[0065] [ka]

[0066] is a connection point to Z,

[0067] [ka]

[0068] is a connection point to Q,

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

[0070] q is an integer between 1 and 20.

[0071] X is either O or -CH2-,

[0072] Each r is independently either 0 or 1.

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

[0074] [ka]

[0075] That is the case.

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

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

[0078] [ka]

[0079] Selected from, During the ceremony,

[0080] [ka]

[0081] It is either a connection point to U, or, if U does not exist, a connection point to Z.

[0082] [ka]

[0083] It is either a connection point to U', or, if U' does not exist, a connection point to Ab.

[0084] In the 15th embodiment of the first aspect, Q is

[0085] [ka]

[0086] That is the case.

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

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

[0089] In the 18th embodiment of the first aspect, a is 2 to 6.

[0090] In the 19th embodiment of the first aspect, b is 1.

[0091] In the 20th embodiment of the first aspect, the amino-substituted compound that controls the STING activity is the 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 It is a C1-C4 alkyl group.

[0098] R 10 is hydrogen, fluoro, OH, NH2, OR b , or NHR b And,

[0099] R 20 is hydrogen or fluoro,

[0100] R 30 is hydrogen, R 40 is hydrogen, fluoro, OH, NH2, OR b , or NHR b is or, R 30 and R 40 Together they form CH2O,

[0101] R 50 is hydrogen or fluoro,

[0102] R bThese are 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, where ring A 10 It contains at least one N atom in the ring, and in the formula, Y b is ring A 10 It is bonded to the carbon atom,

[0104] Ring B 10 is an optionally substituted 9 or 10-membered bicyclic heteroaryl ring containing 2 to 5 heteroatoms selected from N, O, or S, where ring B 10 It contains at least two N atoms in the ring, However, ring A 10 or ring B 10 One of them is bonded to "L" in formula (I) via an amino group.

[0105] In the 21st embodiment of the first aspect, the amino-substituted compound that controls STING activity is:

[0106] [ka]

[0107] And, During the ceremony,

[0108] [ka]

[0109] is the connection point to "L" in equation (I).

[0110] In the 22nd embodiment of the first aspect, the amino-substituted compound that controls STING activity is a compound of formula (III)

[0111]

Chemical formula

[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 a group represented by formula (B 1 -A) or formula (B 1 -B)

[0118]

Chemical formula

[0119] where 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 Each of these can be 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 Each is independently either N or C,

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

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

[0125] [ka]

[0126] It is a group represented by R 23 , R 24 , R 25 , R 26 , and R 27 Each of these is independently a hydrogen atom or a substituent.

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

[0128] Y 21 , Y 22 , Y 23 , Y 24 , Y 25 , and Y 26 Each of these can be 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 is each independently N or C,

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

[0131] B 100 or B 200 one of which is bonded to "L" of formula (I) via an amino group.

[0132] In the 23rd embodiment of the first aspect, the amino-substituted compound that controls STING activity is a compound of formula (IIIa)

[0133] [Chemical formula]

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

[0135] [Chemical formula]

[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 Each of these can be 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 Each is independently either N or C, R 105 is a hydrogen atom or substituent,

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

[0141] [ka]

[0142] It is a group represented by R 23 , R 24 , R 25 , R 26 , and R 27 Each of these is independently a hydrogen atom or a substituent.

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

[0144] Y 21 , Y 22 , Y 23 , Y 24 , Y 25 , and Y 26 Each of these can be 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 Each of them is independent of N or C is,

[0146] R 205 R is a hydrogen atom or substituent, where R is a hydrogen atom or substituent. 105 and R 205 Each of them is independently bonded to the 2nd or 3rd position of the 5-membered ring to which they are joined. 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 It is alkyl,

[0151] R 19 It is a halogen atom,

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

[0153] In the 24th embodiment of the first aspect, the amino-substituted compound that controls the STING activity is a compound of formula (IV).

[0154] [ka]

[0155] or a pharmaceutically acceptable salt thereof, in the formula,

[0156] R 1 and R 2 Each of these is independently a hydroxyl group or a halogen atom.

[0157] B 1 teeth,

[0158] [ka]

[0159] And,

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

[0161] R 19 It is a halogen atom,

[0162] B 2 teeth,

[0163] [ka]

[0164] And, Q 2 and Q 4 Each of these is independently either an oxygen atom or a sulfur atom.

[0165] In the 25th embodiment of the first aspect, the amino-substituted compound that controls STING activity is:

[0166] [ka]

[0167] or a pharmaceutically acceptable salt thereof, in the formula,

[0168] [ka]

[0169] This is the connection point to L.

[0170] In a 26th 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 between 1 and 6.

[0173] In the 27th embodiment of the first aspect, Ab is an antibody or fragment thereof that binds to human CCR2 or a part thereof, and is capable of blocking the binding of chemokines to CCR2 and inhibiting the function of CCR2.

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

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

[0176] In the 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 the 31st embodiment of the first aspect, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment comprises a light chain CDR1 containing amino acids 24-39 of SEQ ID NO: 1; a light chain CDR2 containing amino acids 55-61 of SEQ ID NO: 1; a light chain CDR3 containing amino acids 94-102 of SEQ ID NO: 1; a heavy chain CDR1 containing amino acids 31-35 of SEQ ID NO: 2; a heavy chain CDR2 containing amino acids 50-68 of SEQ ID NO: 2; and a heavy chain CDR3 containing amino acids 101-106 of SEQ ID NO: 2.

[0178] In the 32nd embodiment of the first aspect, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment includes a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2.

[0179] In the 33rd embodiment of the first aspect, the antibody, anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment includes a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1.

[0180] In the 34th 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, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2.

[0181] In the 35th 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, the light chain variable region comprising the amino acid sequence of SEQ ID NO: 1.

[0182] In the 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 containing the amino acid sequence of SEQ ID NO: 2, and a light chain variable region, the light chain variable region containing the amino acid sequence of SEQ ID NO: 1.

[0183] In the 37th 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 the human immunoglobulins IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 heavy chain constant regions.

[0184] In the 38th 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 the 39th 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 the antibody containing the variable heavy chain region of SEQ ID NO: 2 and the variable light chain region of SEQ ID NO: 1.

[0186] In the 40th embodiment of the first aspect, the anti-CCR2 antibody includes the heavy chain region of SEQ ID NO: 3.

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

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

[0189] In a second aspect, the 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 the 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 cell death ligand 1 (PD-L1, CD274, or B7H1).

[0192] In a third aspect, the Disclosure provides a method for treating cancer in a subject requiring treatment for cancer, 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 the first embodiment of the third aspect, a method for 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 the second embodiment of the third aspect, the cancer treatment method involves administering to a subject a pharmaceutically acceptable amount The treatment comprises administering a compound of formula (I), or a pharmaceutically acceptable salt thereof, and an anti-PD-L1 antibody.

[0195] In the third embodiment of the third aspect, the compound of formula (I), or a pharmaceutically acceptable salt thereof, and an anti-PD-1 antibody are administered simultaneously.

[0196] In the fourth embodiment of the third aspect, the compound of formula (I), or a pharmaceutically acceptable salt thereof, and an anti-PD-1 antibody are administered sequentially.

[0197] In the fifth embodiment of the third aspect, the compound of formula (I), or a pharmaceutically acceptable salt thereof, and an anti-PD-L1 antibody are administered simultaneously.

[0198] In the sixth embodiment of the third aspect, the compound of formula (I), or a pharmaceutically acceptable salt thereof, and an anti-PD-L1 antibody are administered sequentially.

[0199] In the seventh embodiment of the third aspect, the method further includes administering radiation to a target. In the eighth embodiment of the third aspect, the radiation is particle radiation. In the ninth embodiment of the third aspect, the radiation is administered by external beam radiation.

[0200] In a fourth aspect, the Disclosure provides a method for stimulating an immune response in a subject requiring stimulation of an immune response, 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 drawing]

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

[0202] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains. All publications and patents provided herein are incorporated in their entirety by reference. .

[0203] The singular forms "a," "an," and "the" include plural nouns unless otherwise defined in the context.

[0204] As used herein, the term "or" means logical disjunction (i.e., and / or) and does not indicate exclusive disjunction unless explicitly indicated by the terms "either," "unless," "or," or other words having a similar effect.

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

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

[0207] [ka]

[0208] or provide a pharmaceutically acceptable salt thereof, in the formula,

[0209] a is an integer between 1 and 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, containing 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 bonded to Ab and also to the amino group on D.

[0213] STING Modulator Section This disclosure provides compounds comprising a STING activity modulator. 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 a guanine base, a guanine base derivative, an adenine base, or an adenine base derivative with an amino group. In some embodiments, the STING modulator is a cyclic dinucleotide or a cyclic dinucleotide-like compound (each a CDN).

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

[0215] [ka]

[0216] or a pharmaceutically acceptable salt thereof, in the formula,

[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 It is a C1-C4 alkyl group.

[0221] R 10 is hydrogen, fluoro, -OH, -NH2, -OR b , or -NHR b And,

[0222] R 20 is hydrogen or fluoro,

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

[0224] R 50 is hydrogen or fluoro,

[0225] R b These are C1-C6 alkyl, halo(C1-C6)alkyl, and 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, where ring A 10 It contains at least one N atom in the ring, and in the formula, Y b is ring A 10 It is bonded to the carbon atom,

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

[0228] However, ring A 10 or ring B 10 One of these is bonded to "L" in formula (I) via an -NH- group.

[0229] As described herein, Ring A 10 and ring B 10 It may contain one or more substituents and therefore may be optionally substituted. Preferred substituents on the unsaturated carbon atom of the heteroaryl group are -halo, -NO2, -CN, and -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 + R is independently a hydrogen atom, or an optionally substituted aliphatic, aryl, heteroaryl, alicyclic, or heterocyclyl group, or two independently existing R groups. + Together with the intervening atoms (multiple atoms are possible), they form optionally substituted 5-7 member aryl, heteroaryl, alicyclic, or heterocyclyl groups. Examples include, and are generally selected from these. In some embodiments, R + Hydrogen and C are independent of each other. 1-6 Aliphatic, or C 3-6 It is an alicyclic compound. Each R° is independently an arbitrarily substituted aliphatic, aryl, heteroaryl, alicyclic, or heterocyclyl group.

[0230] As detailed above, in some embodiments, two independent R +(or any other variable elements similarly defined herein and in the claims) together with their intercalating atoms(s) to form monocyclic or bicyclic rings selected from 3- to 13-membered alicyclic rings, 3- to 12-membered heterocyclines having 1- to 5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 6- to 10-membered aryl rings, or 5- to 10-membered heteroaryl rings having 1- to 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, where R 10 and R 40 Each of these is independently hydrogen, fluoro, -OH, or -OCH2CF3, and ring A 10 and B 10 This is defined for compounds of formula (Ii), provided that ring A 10 or ring B 10 One of them is bonded to "L" via an -NH- group.

[0234] In some embodiments, ring A 10 This is an optionally substituted six-membered monocyclic heteroaryl ring containing one, two, or three 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 Each of these can be 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 Each of these is independently hydrogen, halogen, -OH, -NH2, -CN, C1-C6 alkyl, halo(C1-C6)alkyl, or C3-C6 cycloalkyl.

[0242] In some embodiments, the STING modulator is

[0243] [ka]

[0244] or a pharmaceutically acceptable salt thereof, in the formula,

[0245] [ka]

[0246] This is the bond point to the "L" group of the parent molecule.

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

[0248] [ka]

[0249] or a pharmaceutically acceptable salt thereof, in the formula,

[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 Each is independently a hydrogen atom or a substituent, where R 105 and R 205 Each of them is independently bonded to the 2nd or 3rd position of the 5-membered ring to which they are joined.

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

[0256] [ka]

[0257] It is a group represented by R 13 , R 14 , R 15 , R 16 , and R 17 Each of these is independently a hydrogen atom or a substituent.

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

[0259] Y 11 , Y 12 , Y 13 , Y 14 , Y 15, and Y 16 Each of these can be independently N or CR 1a And,

[0260] Z 11 , Z 12 , Z 13 , Z 14 , Z 15 , and Z 16 Each is independently either N or C,

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

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

[0263] [ka]

[0264] It is a group represented by R 23 , R 24 , R 25 , R 26 , and R 27 Each of these is independently a hydrogen atom or a substituent.

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

[0266] Y 21 , Y 22 , Y 23 , Y 24 , Y 25 , and Y 26 Each of these can be independently N or CR 2a And,

[0267] Z 21 , Z 22 , Z 23 , Z 24 , Z 25 , and Z 26 Each is independently either N or C,

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

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

[0270] In this specification, the compounds of formula (III) and formula (IIIa) (described later) include 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 hydroxyl groups, optionally substituted sulfanyl (SH) groups, and optionally substituted silyl groups, wherein the optionally substituted groups have one or more substituents selected from the following substituent group A: "Substituent group A": (1) Halogen atom, (2) Nitro group, (3) Cyano group, (4) Oxo group, (5) Hydroxyl group, (6) C 1-6 Alkoxy group, (7)C 6-14 Aryloxy groups (e.g., phenoxy, naphthoxy), (8)C 7-16 Aralkyloxy group (e.g., benzyloxy), (9) 5-14 member aromatic heterocyclyloxy groups (e.g., pyridyloxy), (10) 3-14 member 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) 5-14 member aromatic heterocyclylcarbonyloxy group (e.g., nicotinoyloxy), (17) 3-14 member non-aromatic heterocyclylcarbonyloxy groups (e.g., morpholinylcarbonyloxy, piperidinylcarbonyloxy), (18) C 1-6 Alkyl sulfonyloxy groups (e.g., methyl sulfonyloxy, trifluoromethyl sulfonyloxy), (19)C 1-6 C arbitrarily substituted with alkyl groups 6-14 Aryl sulfonyloxy groups (e.g., phenylsulfonyloxy, toluenesulfonyloxy), (20) C 1-6 Alkylthio group, (21) Aromatic heterocyclic groups with 5 to 14 members, (22) 3-14 member non-aromatic heterocyclic groups, (23) Formyl group, (24) Carboxy group, (25) C 1-6 Alkyl-carbonyl group, (26)C 6-14 Aryl-carbonyl group, (27) Aromatic heterocyclylcarbonyl group with 5 to 14 members, (28) 3-14 member non-aromatic heterocyclylcarbonyl group, (29)C 1-6 Alkoxy-carbonyl group, (30)C 6-14 Aryloxycarbonyl groups (e.g., phenyloxycarbonyl, 1-naphthyloxycarbonyl, 2-naphthyloxycarbonyl), (31)C 7-16 Aralkyloxycarbonyl group (e.g., benzyloxycarbonyl, phenethyloxycarbonyl), (32) Carbamoyl group, (33) Thiocarbamoyl group, (34) Mono- or di-C 1-6 Alkyl-carbamoyl group, (35)C 6-14 Aryl-carbamoyl group (e.g., phenylcarbamoyl), (36) 5-14 member aromatic heterocyclylcarbamoyl groups (e.g., pyridylcarbamoyl, thienylcarbamoyl), (37) 3-14 member non-aromatic heterocyclylcarbamoyl groups (e.g., morpholinylcarbamoyl, piperidinylcarbamoyl), (38) C 1~6 Alkyl sulfonyl group, (39)C 6-14 Aryl sulfonyl group, (40) 5-14 member aromatic heterocyclylsulfonyl groups (e.g., pyridylsulfonyl, thienylsulfonyl), (41) C 1-6 Alkyl sulfinyl group, (42)C 6-14 Aryl sulfinyl groups (e.g., phenyl sulfinyl, 1-naphthyl sulfinyl, 2-naphthyl sulfinyl), (43) 5-14 member 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 group (e.g., phenylamino), (47) Aromatic heterocyclylamino groups with 5 to 14 members (e.g., pyridylamino), (48)C 7-16 Aralkylamino group (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 group (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 Aralkyloxycarbonylamino group (e.g., benzyloxycarbonylamino), (55)C 1-6 Alkylsulfonylamino group (e.g., methylsulfonylamino, ethylsulfonylamino), (56)C 1-6 C arbitrarily substituted with alkyl groups 6-14 Aryl sulfonylamino group (e.g., phenylsulfonylamino, toluenesulfonylamino), (57) C 1-6 alkyl group, (58)C 2-6 Alkenyl group, (59)C 2-6 Alkynyl group, (60)C 3-10 Cycloalkyl groups, (61)C 3-10 Cycloalkenyl group, and (62)C 6-14 Aryl group.

[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, in the formula, B 100 is, equation (B 1 -A) or formula (B 1 -B)

[0274] [ka]

[0275] It is a group represented by R 13 , R 14 , R 15 , R 16 , and R 17 Each of these is independently a hydrogen atom or a substituent. R 1000 is a hydrogen atom, or a bond to the carbonyl group of formula (I), Y 11 , Y 12 , Y 13 , Y 14 , Y 15 , and Y 16 Each of these can be 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 Each is independently either N or C, R 105 is a hydrogen atom or substituent, B 200 is, equation (B 2 -A) or formula (B 2 -B)

[0276] [ka]

[0277] It is a group represented by R 23 , R 24 , R 25 , R 26 , and R 27 Each of these is independently a hydrogen atom or a substituent. R 100’ is a bond to hydrogen or the carbonyl group of formula (I), Y 21 , Y 22 , Y 23 , Y 24 , Y 25 , and Y 26 Each of these can be 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 Each is independently either N or C, R 205 R is a hydrogen atom or substituent, where R is a hydrogen atom or substituent. 105 and R 205 Each of them is independently bonded to the 2nd or 3rd position of the 5-membered ring to which they are joined. 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 It is alkyl, R 19 It 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, in the formula, R 1 and R 2 Each of these is independently a hydroxyl group or a halogen atom. B 1 teeth,

[0283] [ka]

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

[0285] [ka]

[0286] and; Q 2 and Q 4 Each of these is independently either an oxygen atom or a sulfur atom.

[0287] In some embodiments, the cyclic dinucleotide is

[0288] [ka]

[0289] or a pharmaceutically acceptable salt thereof, in the formula,

[0290] [ka]

[0291] This is the dot of "L".

[0292] Linker section The group "L" is a linker. As used herein, the term "linker" means any chemical portion in the compounds of formulas (I) and (IV) to which an antibody, antibody fragment, or antigen-binding fragment (Ab) can be bound to the drug-containing portion. The linker may be branched and may be substituted with 1 to 20 drug-containing portions. In some embodiments, the linker may be substituted with 1 to 10 drug-containing portions. In some embodiments, the linker may be substituted with 1 to 5 drug-containing portions. In some embodiments, the linker may be substituted with 1 or 2 drug-containing portions. In some embodiments, the linker may be substituted with 1 drug-containing portion.

[0293] In some embodiments, linker "L" is a detachable linker. In the application, the linker is likely to undergo acid-inducible cleavage, photo-induced cleavage, enzymatic cleavage, etc., under conditions in which the drug and / or antibody can remain active. In some embodiments, the cleavable linker can be cleaved enzymatically. In some embodiments, the cleavable linker can be cleaved by proteases, peptidases, esterases, glycosidases, phosphodiesterases, phosphatases, or lipases. In some embodiments, the cleavable linker can be cleaved by proteases. Examples of proteases include, but are not limited to, cathepsin B and VAGP tetrapeptide.

[0294] In certain embodiments, the linkers may be any of those disclosed in PCT Publications WO2018 / 200812 and WO2018 / 100558, which are incorporated by reference in their entirety.

[0295] In a particular embodiment, "L" is the formula:

[0296] [ka]

[0297] It has, During the ceremony:

[0298] [ka]

[0299] This is a bonding point to the nitrogen atom,

[0300] [ka]

[0301] This is the connection point to Ab.

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

[0303] [ka]

[0304] It has, During the ceremony:

[0305] [ka]

[0306] This is a bonding point to the nitrogen atom,

[0307] [ka]

[0308] This is the binding site to the antibody.

[0309] The group "W" is either absent or a self-sacrificing group. In its use herein, the term "Self-sacrificing" refers to a group that undergoes an electron cascade, resulting in the release of a bonded group. In some embodiments, the self-sacrificing group includes one or more groups that can undergo 1,4-elimination, 1,6-elimination, 1,8-elimination, 1,6-cyclization-elimination, 1,5-cyclization-elimination, 1,3-cyclization-elimination, intramolecular 5-exo-trig cyclization, and / or 6-exo-trig cyclization. In certain embodiments, the self-sacrificing group may be any of those disclosed in PCT Publications WO2018 / 200812 and WO2018 / 100558, which are incorporated by reference in whole.

[0310] The group "Z" is either absent or a peptide of 2-5 amino acids. In certain embodiments, the peptide is a linker cleavage site, thereby facilitating drug release 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); phenylalanine Examples of peptides with three amino acids include, but are not limited to, n-lysine (FK or Phe-Lys), phenylalanine homolysine (Phe-Homolys), and N-methyl-valine-citrulline (Me-Val-Cit). Examples of peptides with three amino acids include, but are not limited to, glycine-valine-citrulline (Gly-Val-Cit) and glycine-glycine-glycine (Gly-Gly-Gly). The above combinations of amino acids can also exist in reverse order (i.e., Cit-Val).

[0311] The peptides of this disclosure may contain native and / or non-native amino acid residues. The term “native 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. “Non-native amino acids” (i.e., amino acids that do not exist in nature) include, without limitation, homoserine, homoarginine, citrulline, phenylglycine, taurine, iodotyrosine, selenocysteine, norleucine ("Nle"), norvaline ("Nva"), beta-alanine, L- or D-naphthalanine, ornithine ("Orn"). The peptides may 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 include both native and unnatural D-form amino acids. "D-" indicates an amino acid with a "D" (dextrorotatory) configuration, as opposed to the configuration in the native ("L-") amino acid. Native and unnatural amino acids may be commercially available (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

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

[0314] [ka]

[0315] These are some examples.

[0316] In the compounds of this disclosure, if "U" is present, U may be a branched 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 spacers may be any of those disclosed in PCT Publications WO2018 / 200812 and WO2018 / 100558, which are incorporated by reference in their entirety.

[0317] The group "Q" is a heterobifunctional group. In this disclosure, the term "heterobifunctional group" means a chemical portion that connects a linker, which is a part of it, to an antibody, antibody fragment, or antigen-binding fragment. See, for example, WO2017 / 191579. A heterobifunctional group is characterized by having different reactive groups at both ends of the chemical portion. A heterobifunctional group can be directly bound to "Ab," or alternatively, it can be connected via the linker "U." Binding to "Ab" can be achieved by chemical or enzymatic conjugation, or a combination of both. Chemical conjugation involves a controlled reaction of an accessible amino acid residue on the surface of an antibody having a reaction handle at "Q" or "U." Examples of chemical conjugation include, but are not limited to, lysine amide coupling, cysteine ​​coupling, and coupling via a non-natural amino acid incorporated by recombination; any non-natural amino acid residue having a desired reaction handle can be introduced to "Ab." In enzymatic conjugation, the enzyme mediates the coupling of a linker with an accessible amino residue on an antibody, antibody fragment, or antigen-binding fragment. Examples of enzymatic conjugation include, but are not limited to, peptide transfers using saltase, peptide transfers using bacterial transglutaminase, and N-glycan recombination. Chemical and enzymatic conjugations can also be used sequentially. For example, an enzymatic conjugation can be used to introduce a specific reaction handle on "Ab" that will be used in a later chemical conjugation. In certain embodiments, hete The roni functional groups may be any of those disclosed in PCT Publications WO2018 / 200812 and WO2018 / 100558, which are incorporated by reference in their entirety.

[0318] In some embodiments, "Q" is

[0319] [ka]

[0320] Selected from, During the ceremony,

[0321] [ka]

[0322] It is either a connection point to U, or, if U does not exist, a connection point to Z.

[0323] [ka]

[0324] It is either a connection point to U', or, if U' does not exist, a connection point to Ab.

[0325] In certain embodiments, this disclosure relates to compounds of formula (XX):

[0326] [ka]

[0327] or provides a pharmaceutically acceptable salt thereof, in which n, m, a, t, D-NH-, R 1 , R 2 , R 3 , R 3’ W, Z, and U are as described herein, where Q * Q is a reactive functional group that can be conjugated to an antibody, antibody fragment, or antigen-binding fragment. *Examples of groups include, but are not limited to, activated carboxylic acid groups such as acid chlorides-C(O)-Cl, and acid anhydrides, haloacetamides, maleimides, alkynes, cycloalkynes, such as cyclooctin, oxanorboradine, norbornene, azides, diaryltetrazine, monoaryltetrazine, aldehydes, ketones, hydroxylamines, vinyl sulfones, and aziridines. In certain embodiments, the reactive functional groups may be any of those disclosed in PCT publications WO2018 / 200812 and WO2018 / 100558, which are incorporated by reference in their entirety.

[0328] Anti-CCR2 antibody, antibody fragment, and antigen-binding fragment The group "Ab" is an anti-CCR2 antibody, an anti-CCR2 antibody fragment, or an anti-CCR2 antigen-binding fragment. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. Target antigens generally have numerous binding sites, also called epitopes, that are recognized by the CDRs of multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, one antigen may have two or more corresponding antibodies. The term "antibody" as used herein is used in its broadest sense and specifically includes monoclonal antibodies, single-domain antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, but only if they exhibit the desired biological activity. Antibodies may be mouse, 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) that bind to mammalian CC-chemokine receptor 2 (also known as CCR2, CKR-2, CD192, MCP-1RA, or MCP-1RB), or to a portion of the receptor. In one embodiment, the antibody or fragment is specific to 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 the function of the ligand to bind to the receptor (e.g., leukocyte trafficking). For example, as described herein, antibodies and fragments useful in this disclosure can bind to human or rhesus monkey CCR2, or a portion thereof, block the binding of chemokines (e.g., MCP-1, MCP-2, MCP-3, MCP-4) to the receptor and inhibit the function of the chemokine to bind to the receptor. In one embodiment, the antibody is a monoclonal antibody (mAb) LS132.1D9 (1D9), or an antibody that is competitive with 1D9 in terms of binding to human CCR2 or a portion of human CCR2. The functional fragment of the aforementioned antibody is also considered.

[0330] In some embodiments, a humanized immunoglobulin or its antigen-binding fragment having binding specificity to CCR2 is used, wherein the immunoglobulin comprises a non-human (e.g., rodent) antigen-binding region and at least a portion of a human immunoglobulin (e.g., a human framework region, a γ-type human constant region). In one embodiment, the humanized immunoglobulin or its fragment can compete with 1D9 for binding to CCR2. In one embodiment, the antigen-binding region of the humanized immunoglobulin is derived from a monoclonal antibody 1D9 (e.g., an immunoglobulin containing the variable light and heavy chain regions shown below).

[0331] For example, a humanized immunoglobulin or its antigen-binding fragment may include an antigen-binding region containing at least one complementarity-determining region (CDR) of non-human origin, and a framework region (FR) derived from a human framework region. In one embodiment, against CCR2 A humanized immunoglobulin with binding specificity comprises a light chain containing at least one CDR derived from a non-human antibody that binds to CCR2, and a FR derived from a human light chain (e.g., HF-21 / 28); and a heavy chain containing a CDR derived from a non-human antibody that binds to CCR2, and a FR derived from a human heavy chain (e.g., 4B4'CL). In another embodiment, the light chain comprises three CDRs derived from the light chain of a 1D9 antibody, and the heavy chain comprises three CDRs derived from the heavy chain of a 1D9 antibody.

[0332] In one embodiment, a humanized immunoglobulin having binding specificity to CCR2 comprises the light chains CDR1, CDR2, and CDR3 of the 1D9 antibody, as well as the human light chain FR, and the heavy chains CDR1, CDR2, and CDR3 of the 1D9 antibody, as well as the human heavy chain FR. In one embodiment, the humanized immunoglobulin comprises the humanized heavy and light chains described herein (e.g., a humanized light chain including the variable region of the light chain shown below, and a humanized heavy chain including the variable region of the heavy chain shown below). Humanized immunoglobulins comprising one or more humanized light and / or heavy chains also encompass the present invention.

[0333] The following shows the amino acid sequence of the κ light chain variable region (VL) of the humanized 1D9 antibody. The CDR is 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. CDR is 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 containing amino acids 24-39 of SEQ ID NO: 1; a light chain CDR2 containing amino acids 55-61 of SEQ ID NO: 1; a light chain CDR3 containing amino acids 94-102 of SEQ ID NO: 1; a heavy chain CDR1 containing amino acids 31-35 of SEQ ID NO: 2; a heavy chain CDR2 containing amino acids 50-68 of SEQ ID NO: 2; and a heavy chain CDR3 containing amino acids 101-106 of SEQ ID NO: 2.

[0338] In some embodiments, the anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment includes a heavy chain variable region containing 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 includes a light chain variable region containing 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 includes a heavy chain variable region and a light chain variable region, the heavy chain variable region being the A of SEQ ID NO: 2 Contains a mino 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, the light chain variable region comprising 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 containing the amino acid sequence of SEQ ID NO: 2, and a light chain variable region, the light chain variable region containing 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. In some embodiments, the heavy chain constant region is selected from the 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. In 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 the antibody containing 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 them. Alignments can be generated using programs known in the art. For the purposes of this specification, nucleotide sequence alignment can be performed using the blastn program set to default parameters, and amino acid sequence alignment can be performed using the blastp program set to default parameters (see ncbi.nlm.nih.gov on the World Wide Web, National Center for Biotechnology Information (NCBI)).

[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 residue 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, the antibody or antigen-binding fragment described herein is bound to human CCR2 and comprises six CDRs of the antibody described in SEQ ID NO: 1 and SEQ ID NO: 2, including a VH sequence that is at least 80% identical to the VH sequence of SEQ ID NO: 2, and a VL sequence that is at least 80% identical to the VL sequence of SEQ ID NO: 1. In certain embodiments, the antibody or antigen-binding fragment described herein is bound to human CCR2 and comprises six CDRs of the antibody described in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., three VH CDRs of the antibody described in SEQ ID NO: 2 and three VL CDRs of SEQ ID NO: 1), including a VH sequence that is at least 85% identical to the VH sequence of SEQ ID NO: 2, and a VL sequence that is at least 85% identical to the VL sequence of SEQ ID NO: 1.

[0349] In certain embodiments, the antibody or antigen-binding fragment described herein is used in human CCR. The antibody or its antigen-binding fragment is bound to human CCR2 and comprises six CDRs of the antibody described in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., three VH CDRs of SEQ ID NO: 2 and three VL CDRs of SEQ ID NO: 1), and includes a VH sequence that is at least 90% identical to the VH sequence of SEQ ID NO: 2, and a VL sequence that is at least 90% identical to the VL sequence of SEQ ID NO: 1. In certain embodiments, the antibody or its antigen-binding fragment is bound to human CCR2 and comprises six CDRs of the antibody described in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., three VH CDRs of the antibody and three VL CDRs of SEQ ID NO: 1), and includes a VH sequence that is at least 95% identical to the VH sequence of SEQ ID NO: 2, and a VL sequence that is at least 95% identical to the VL sequence of SEQ ID NO: 1.

[0350] In certain embodiments, the antibody or antigen-binding fragment described herein is bound to human CCR2 and comprises six CDRs of the antibody described in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., three VH CDRs of SEQ ID NO: 2 and three VL CDRs of SEQ ID NO: 1), including a VH sequence that is at least 96% identical to the VH sequence of SEQ ID NO: 2 and a VL sequence that is at least 96% identical to the VL sequence of SEQ ID NO: 1. In certain embodiments, the antibody or antigen-binding fragment described herein is bound to human CCR1 and comprises six CDRs of the antibody described in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., three VH CDRs of SEQ ID NO: 2 and three VL CDRs of SEQ ID NO: 1), including a VH sequence that is at least 97% identical to the VH sequence of SEQ ID NO: 2 and a VL sequence that is at least 97% identical to the VL sequence of SEQ ID NO: 1. In certain embodiments, the antibody or antigen-binding fragment described herein is bound to human CCR2 and comprises six CDRs of the antibody described in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., three VH CDRs of SEQ ID NO: 2 and three VL CDRs of SEQ ID NO: 1), including a VH sequence that is at least 98% identical to the VH sequence of SEQ ID NO: 2 and a VL sequence that is at least 98% identical to the VL sequence of SEQ ID NO: 1. In certain embodiments, the antibody or antigen-binding fragment described herein is bound to human CCR2 and comprises six CDRs of the antibody described in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., three VH CDRs of SEQ ID NO: 2 and three VL CDRs of SEQ ID NO: 1), including a VH sequence that is at least 99% identical to the VH sequence of SEQ ID NO: 2 and a VL sequence that is at least 99% identical to the VL sequence of SEQ ID NO: 1.

[0351] In certain embodiments, the antibody or antigen-binding fragment described herein binds to human CCR2 and comprises six CDRs of the antibody described in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., three VH CDRs of SEQ ID NO: 2 and three VL CDRs of SEQ ID NO: 1), comprising a VH sequence containing at least 80% identical to the VH sequence of SEQ ID NO: 2, and a VL sequence containing 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, the antibody or antigen-binding fragment described herein binds to CCR2 and comprises six CDRs of the antibody described in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., three VH CDRs of SEQ ID NO: 2 and three VL CDRs of SEQ ID NO: 1), comprising a VH sequence containing at least 85% identical to the VH sequence of SEQ ID NO: 2, and a VL sequence containing 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 antibody or antigen-binding fragment described herein binds to human CCR2 and comprises six CDRs of the antibody described in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., three VH CDRs of the antibody and three VL CDRs of SEQ ID NO: 1), including a VH sequence that is at least 90% identical to the VH sequence of SEQ ID NO: 2, and a VL sequence that is at least 90% 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 antibody or antigen-binding fragment described herein binds to human CCR2 and comprises six CDRs of the antibody described in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., three VH CDRs of SEQ ID NO: 2 and three VL CDRs of SEQ ID NO: 1), including a VH sequence that is at least 95% identical to the VH sequence of SEQ ID NO: 2, and a VL sequence that is at least 95% identical to the VL sequence of SEQ ID NO: 1, and binds to human, cynomolgus monkey, and rat , and / or binds to mouse CCR2.

[0353] In certain embodiments, the antibody or antigen-binding fragment described herein binds to human CCR2 and comprises six CDRs of the antibody described in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., three VH CDRs of SEQ ID NO: 2 and three VL CDRs of SEQ ID NO: 1), comprising a VH sequence containing at least 96% identical to the VH sequence of SEQ ID NO: 2 and a VL sequence containing 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, the antibody or antigen-binding fragment described herein binds to human CCR2 and comprises six CDRs of the antibody described in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., three VH CDRs of SEQ ID NO: 2 and three VL CDRs of SEQ ID NO: 1), comprising a VH sequence containing at least 97% identical to the VH sequence of SEQ ID NO: 2 and a VL sequence containing 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 antibody or antigen-binding fragment described herein binds to human CCR2 and comprises six CDRs of the antibody described in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., three VH CDRs of SEQ ID NO: 2 and three VL CDRs of SEQ ID NO: 1), comprising a VH sequence containing at least 98% identical to the VH sequence of SEQ ID NO: 2, and a VL sequence containing at least 98% 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 antibody or antigen-binding fragment described herein binds to human CCR2 and comprises six CDRs of the antibody described in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., three VH CDRs of SEQ ID NO: 2) It comprises a CDR, three VL CDRs of SEQ ID NO: 1, a VH containing a sequence at least 99% identical to the VH sequence of SEQ ID NO: 2, and a VL containing 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, the compound of formula (I) is combined with an antibody, an antibody fragment, or an antigen-binding fragment of an antibody that binds to PD-1, and / or an antibody, an antibody fragment, and / or an antigen-binding fragment of an antibody that binds to PD-L1. PD-1 is an immune checkpoint protein expressed in activated T cells, B cells, and monocytes that regulates the immune system by, for example, promoting apoptosis of antigen-specific T cells and reducing apoptosis of regulatory T cells when it binds to its ligand, PD-L1. PD-L1 is expressed by tumors and can help tumors evade detection and removal by the immune system. Antagonist inhibition of the PD-1 / PD-L1 interaction favorably increases T cell activation and improves the recognition and removal of tumor cells by the immune system. In certain embodiments, the anti-PD-1 antibody may be pembrolizumab, nivolumab, semiprimab, pimivalimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, 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, pemprimab, pukotenlimab, CX-188, zimvalerimab, and teboterimab, or human PD-1, or PD Regarding binding to part of -1, the antibody is selected from the group consisting of pembrolizumab, nivolumab, semiprimab, pimivalimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, 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, pemprimab, pcotenlimab, CX-188, zimvalerimab, or antibodies that can compete with teboterimab.

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

[0356] In certain embodiments, the anti-PD-L1 antibody may be atezolizumab, avelumab, durvalumab, kosiberimab, MSB-2311, ZKAB-001, FAZ-053, MDX-1105, CBT-502, IMC-001, RC-98, KL-A167, GR-1405, rodapolimab, sugemalimab, emvafolimab, opcolimab, and gariblimab, or human PD-L1 or PD -Regarding binding to a portion of L1, the antibody is selected from the group consisting of atezolizumab, avelumab, durvalumab, kosiberimab, MSB-2311, ZKAB-001, FAZ-053, MDX-1105, CBT-502, IMC-001, RC-98, KL-A167, GR-1405, rodapolimab, sugemalimab, emvafolimab, opcolimab, or antibodies that can compete with gariblimab.

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

[0358] Further anti-PD-1 antibodies useful in combination with the compound 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 This antibody is one of the following: BioSite; PDCD1 (BosterBio); 10B3 (ProSci Inc.); 4C7 (ProSci Inc.); mhT28 blocking (Sino Biological Inc.); HF06 neutralization (Sino Biological Inc.); or TK12-02 (Creative Diagnostics), or an antibody that can compete with any one of the aforementioned antibodies for binding to PD-1 or a portion of PD-1.

[0359] Further anti-PD-L1 antibodies useful in combination with the compound 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 aforementioned antibodies for binding to PD-L1 or a portion of PD-L1.

[0360] As used herein, the term “antibody” also means a full-length immunoglobulin molecule, or a molecule containing an immunologically active portion of a full-length immunoglobulin molecule, i.e., an antigen-binding site that immunely binds to a target antigen or a portion thereof, such targets include, but are not limited to, cancer cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins may originate from any species; however, in one embodiment, the immunoglobulins are of human, mouse, or rabbit origin.

[0361] Also known as nanobodies, the term "single-domain antibody" refers to an antibody fragment consisting of a single monomer variable antibody domain with a molecular weight of approximately 12 kDa to 15 kDa. Single-body antibodies can be based on a heavy chain variable region or a light chain. An example of a single-domain antibody is V H H fragment, and V NAR Some examples are given, but are not limited to, Harmsen MM et al. Applied Microbiology and Biotechnology 77(1):13-22.

[0362] An "antibody fragment" is a part of an intact antibody, generally its antigen-binding region or other part. This includes variant regions. Examples of antibody fragments include Fab, Fab', F(ab').sub2, and Fv fragments; diabodies; linear antibodies; fragments produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies, CDRs (complementarity-determining regions), and any of the above-mentioned epitope-binding fragments, single-chain antibody molecules that bind immunospecifically to cancer cell antigens, viral antigens, or bacterial antigens; as well as multispecific antibodies formed from antibody fragments.

[0363] An "intact antibody" is an antibody that contains 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 the constant domains of the natural sequence (e.g., the constant domains of the human natural sequence) or amino acid sequence variants thereof.

[0364] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting that population are identical except for the possibility of naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which contain different antibodies directed against different determinants (epitopes), each monoclonal antibody targets a single determinant on an antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized without contamination by other antibodies. The modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with this disclosure may be produced by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or by the recombinant DNA method (see, e.g., US 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J.Mol.Biol., 222:581-597.

[0365] The term "monoclonal antibody" as used herein specifically includes "chimeric" antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody of a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody of a different species or belonging to a different antibody class or subclass, as well as fragments of such antibodies, insofar as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). The chimeric antibodies used herein include "primatized" antibodies that include a variable domain antigen-binding sequence derived from a non-human primate (e.g., Old World monkeys, apes, etc.) and a human constant region sequence.

[0366] Various methods have been used to produce monoclonal antibodies (MAbs). "Hybridoma technology," which refers to cloned cell lines that produce antibodies of a single species, uses cells from various species, including mice, hamsters, rats, and humans. Another method for preparing MAbs involves genetic recombination, including recombinant DNA technology. Among the monoclonal antibodies produced using these technologies are chimeric antibodies and humanized antibodies. Chimeric antibodies combine DNA encoding regions from two or more species. For example, a chimeric antibody may be derived from the variable region of a mouse and the constant region of a human. Humanized antibodies are primarily derived from humans but also contain non-human portions. Similar to chimeric antibodies, humanized antibodies may also contain the complete human constant region. However, Unlike chimeric antibodies, the variable region can be partially derived from humans. The non-human synthetic portion of humanized antibodies is often derived from the CDR of mouse antibodies. In any case, these regions are crucial for enabling the antibody to recognize and bind to a specific antigen. While useful for diagnosis and short-term therapy, mouse antibodies cannot be administered to humans for extended periods without increasing the risk of a harmful immunogenic response. This response, called human anti-mouse antibody (HAMA), occurs when the human immune system recognizes the mouse antibody as foreign and attacks it. 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 may have additional benefits, such as activating secondary human immune responses, including antibody-dependent cell-mediated cytotoxicity.

[0368] An intact antibody may possess one or more "effector functions," which refer to biological activity that may be attributed to the antibody's Fc region (either the Fc region of the natural sequence or the Fc region of an amino acid sequence variant). Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and downregulation of cell surface receptors (e.g., B cell receptors; BCRs).

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

[0370] Useful non-immunoreactive proteins, polypeptides, 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 factor ("TGF") (e.g., TGF-α and TGF-β), varicella growth factor ("VGF"), insulin and insulin-like growth factors I and II, lectins, and apolipoproteins derived from low-density lipoproteins.

[0371] Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the serum of immunized animals. Various procedures known in the art can be used to produce polyclonal antibodies against a target antigen. For example, various host animals, including but not limited to rabbits, mice, rats, and guinea pigs, can be immunized by injection with the target antigen or a derivative thereof for the production of polyclonal antibodies. Various adjuvants can be used to enhance the immune response depending on the host species, and adjuvants include, but are not limited to, Freund's (complete and incomplete) adjuvants, 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 BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.

[0372] Useful monoclonal antibodies are those that target specific antigenic determinants (e.g., cancer cell antigens, viral antigens, bacterial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids, or fragments thereof). This is a homogeneous population of antibodies against the target antigen. Monoclonal antibodies (mAbs) against the target antigen can be prepared by using any technique known in the art that results in the production of antibody molecules by a continuous cell line in a culture medium. 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 mAb-producing hybridomas used in this disclosure can be cultured 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 are produced 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). It can be prepared by any of the following methods (et al., 1982, Meth. Enzymol. 92, 3-16).

[0374] Antibodies can also be bispecific antibodies. Methods for producing bispecific antibodies are known in the art. Conventional production of full-length bispecific antibodies is based on the simultaneous expression of two immunoglobulin heavy-light chain pairs, each having two different specificities (Milstein et al., 1983, Nature 305:537-539). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) can produce a mixture of 10 different antibody molecules, of which only one 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 WO93 / 08829 and Traunecker et al., EMBO J.10:3655-3659 (1991).

[0375] According to different approaches, an antibody variable domain (antibody-antigen binding site) with desired binding specificity is fused to an immunoglobulin constant domain sequence. The fusion may be with an immunoglobulin heavy chain constant domain containing at least a portion of the hinge, C.sub.H2, and C.sub.H3 regions. The first heavy chain constant region (C.sub.H1) may contain a site necessary for light chain binding, which is present in at least one of the fusions. The immunoglobulin heavy chain fusions, and, if desired, nucleic acids containing sequences encoding the immunoglobulin light chain, are inserted into separate expression vectors and simultaneously transfected into suitable host organisms. This provides excellent flexibility in adjusting the relative proportions of the three polypeptide fragments, in embodiments where an unequal ratio of the three polypeptide chains used in construction yields optimal yield. However, if high yield is obtained by expressing at least two polypeptide chains in equal ratios, or if their ratios are not particularly important, it is possible to insert the coding sequences of two or all three polypeptide chains into a single expression vector.

[0376] A bispecific antibody is a hybrid immunoglobulin heavy chain that has a first binding specificity in one arm and a hybrid that has a second binding specificity in the other arm. It may possess immunoglobulin heavy-light chain pairs. The presence of immunoglobulin light chains on only half of the bispecific molecule provides an easy separation method (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). Therefore, this asymmetric structure facilitates the separation of desired bispecific compounds from unnecessary immunoglobulin chain combinations. By using such techniques, bispecific antibodies for conjugation can be prepared 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, in particular, using crosslinking agents or disulfide crosslinking reagents, and may include whole antibodies or fragments thereof (EP105360; WO 83 / 03679; EP217577).

[0378] The antibody may be an antibody that immune-specifically binds to cancer cell antigens, viral antigens, or bacterial antigens, or a functionally active fragment, derivative, or analog of another antibody that binds to tumor cells or the matrix. In this regard, “functionally active” means that the fragment, derivative, or analog can induce an anti-anti-idiotype antibody that recognizes the same antigen recognized by the antibody from which the fragment, derivative, or analog is derived. Specifically, in exemplary embodiments, the antigenicity of an idiotype of an immunoglobulin molecule can be enhanced by deleting a framework and CDR sequence located at the C-terminus relative to a CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind to the antigen, synthetic peptides containing CDR sequences can be used in a binding assay with the antigen using any binding assay method known in the art (e.g., BIA core assay) (see, for example, 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 a variable region, a light chain constant region, and a heavy chain CH1 domain, which can be produced by pepsin degradation of the antibody molecule, and Fab fragments, which can be produced by reducing the disulfide crosslinks of the F(ab')2 fragment. Other useful antibodies include antibodies such as FV or single-chain antibodies (SCA), or heavy and light chain dimers of any smallest fragment thereof (e.g., those described in U.S. Patent No. 4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., (1989) Nature 334:544-54), or any other molecule having the same specificity as the antibody.

[0380] In addition, recombinant antibodies such as chimeric and humanized monoclonal antibodies, which contain both human and non-human portions and can be produced using standard recombinant DNA technology, are useful antibodies. Chimeric antibodies are molecules in which different parts originate from different animal species, such as those having variable regions derived from mouse monoclonal and human immunoglobulin constant regions. (e.g., Cabilly et al., U.S. Patent No. 4,816,567; and Boss et al.) See U.S. Patent No. 4,816,397.) A humanized antibody is a non-human antibody molecule having one or more complementarity-determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule. (For example, see Queen, U.S. Patent No. 5,585,089.) Such chimeric and humanized monoclonal antibodies are, for example, WO87 / 02671;EP184,187;EP171496;EP173494;WO86 / 01533;U.S. Patent No. 4,816,567;EP12023;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 using recombinant DNA techniques known in the art, using the method described in al., 1988, J.Immunol. 141:4053-4060.

[0381] Fully human antibodies can be produced using transgenic mice that cannot express endogenous immunoglobulin heavy and light chain genes but can express human heavy and light chain genes. The transgenic mice are immunized in the usual manner with a selected antigen, for example, all or part of the polypeptides disclosed herein. Monoclonal antibodies targeting the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes possessed by the transgenic mice can be rearranged during B cell differentiation and subsequently undergo class switching and somatic mutation. Therefore, therapeutically useful IgG, IgA, IgM, and IgE antibodies can be produced using such technology. 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, for example, U.S. Patents 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806. Other human antibodies are commercially available, for example, from Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).

[0382] Fully human antibodies that recognize selected epitopes can be generated using a technique called "inducible selection." In this approach, selected non-human monoclonal antibodies, such as mouse antibodies, can be used to guide the selection of fully human antibodies that recognize the same epitope (Jespers et al. (1994) Biotechnology 12:899-903). Human antibodies can also be produced using various 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, an antibody fusion protein or a functionally active fragment thereof, formed by the antibody covalently fusing (e.g., via a peptide bond) to the N-terminus or C-terminus of the amino acid sequence of another protein (or a portion thereof, e.g., a portion of at least 10, 20, or 50 amino acids of a protein) that is not an antibody. The antibody or its fragment can covalently bond to other proteins at the N-terminus of its constant domain.

[0384] Antibodies include analogs and derivatives in which any modification (i.e., by covalent bonding of any type of molecule) is performed, to the extent that such covalent bonding allows the antibody to retain its antigen-binding immunospecificity. For example, but are not limited to, antibody derivatives and analogs that are further modified by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or binding to cellular antibody units or other proteins. Any of the numerous chemical modifications can be carried out by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, or metabolic synthesis in the presence of tunicamycin. In addition, analogs or derivatives may contain one or more non-natural amino acids.

[0385] Antibodies in antibody-drug conjugates include those having modifications (e.g., substitutions, deletions, or additions) to amino acid residues that interact with the Fc receptor. Specifically, antibodies include those having modifications to amino acid residues identified as being involved in the interaction between the anti-Fc domain and the FcRn receptor (see, for example, WO97 / 34631). Antibodies immune-specific to cancer cell antigens can be commercially available, for example, from Genentech (San Francisco, Calif.), or can be produced by any method well known to those skilled in the art, such as chemical synthesis or recombinant expression techniques. Nucleotide sequences encoding antibodies immune-specific to cancer cell antigens can be obtained, for example, from the GenBank database or similar databases, literature publications, or by routine cloning and sequencing.

[0386] The antibody in the ADC can be a monoclonal antibody, such as a mouse monoclonal antibody, a chimeric antibody, or a humanized antibody. The antibody can also be an antibody fragment, such as a Fab fragment.

[0387] Known anti-CCR2 antibodies for the treatment or prevention of cancer can be conjugated as ADCs. Immunospecific antibodies against cancer cell antigens are commercially available or can be produced by any method known to those skilled in the art, such as recombinant expression technology. Nucleotide sequences encoding immunospecific antibodies against 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 available for cancer treatment 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 incorporated herein by reference), 4.40A68G (Pfizer / Amgen; described in U.S. Patent No. 8710191 incorporated herein by reference), UniTI-101 (CSF-1R × CCR2 bispecific antibody, Elstar Therapeutics), and those described in WO97 / 31949 incorporated herein by reference.

[0388] The term "amino acid sequence variant" refers to a polypeptide that differs to some extent from its natural sequence. This refers to polypeptides having a specific amino acid sequence. Typically, amino acid sequence variants have at least about 70% sequence identity with at least one receptor-binding domain of a native antibody or at least one ligand-binding domain of a native receptor, and typically the sequence will be at least about 80%, more typically about 90%, homologous to such receptor or ligand-binding domains. Amino acid sequence variants have substitutions, deletions, and / or insertions at specific positions within the amino acid sequence of the native amino acid sequence. Amino acids are indicated by their conventional names, one-letter, and three-letter codes.

[0389] "Sequence identity" is defined as the percentage of amino acid sequence variant residues that are identical after aligning sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage. 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, along with its user documentation, is licensed under the United States Copyright Office. It was filed with Office Washington, DC20559 on December 10, 1991.

[0390] The terms "Fc receptor" or "FcR" are used to describe receptors that bind to the Fc region of an antibody. Exemplary FcR is the naturally occurring human FcR. Furthermore, FcR may bind to an IgG antibody (γ receptor) and include the FcγRI, FcγRII, and FcγRIII subclass receptors, the RIII subclass of which includes allelic variants and alternative spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine system inhibitory motif (ITIM) in its cytoplasmic domain. (See the overview in M. in Daeron, Annu. Rev. Immunol., 15:203-234 (1997).) For FcR, see Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991); Capel et al. This is outlined in al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs, including FcRs identified in the future, are included in the term “FcR” herein. This term also includes 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., an antibody) conjugated with an alloantigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), may be performed.

[0392] "Natural antibodies" are typically heterotetrameric glycoproteins with approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is attached to the heavy chain by one covalent disulfide bond, while the number of disulfide bonds differs between 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 at the other end... It possesses a constant domain. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is thought that specific amino acid residues 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 parts of the variable domain have significantly different sequences within an antibody, and these differences are used to determine the binding and specificity of each particular antibody to a specific antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called hypervariable regions in both the light chain and heavy chain variable domains. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of the native heavy and light chains each contain four FRs that primarily take a beta-sheet configuration, linked by three hypervariable regions that bind a portion of the beta-sheet structure and, in some cases, form loops that make up that portion. The hypervariable regions in each chain are held in close proximity to the hypervariable region of the other chain by the FRs, contributing to the formation of the antibody's antigen-binding site (see Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The constant domain is not directly involved in antibody binding to antigens, but it exhibits various effector functions, such as involvement in antibody-dependent cytotoxicity (ADCC).

[0394] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody responsible for antigen binding. The hypervariable region generally includes amino acid residues from the "complementarity-determining region" or "CDR" (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 (see above)) and / or residues from the "hypervariable loop" (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). A "framework region" or "FR" residue is a variable domain residue other than the hypervariable region residues as defined herein.

[0395] Papain digestion of the antibody produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and the remaining "Fc" fragment, whose name reflects its ability to readily crystallize. Pepsin treatment yields the F(ab')2 fragment, which still has two antigen-binding sites and can crosslink to the antigen.

[0396] "Fv" is the smallest antibody fragment containing a complete antigen recognition site and an antigen binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in a tight non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define the 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 Fv containing only three antigen-specific hypervariable regions) has the ability to recognize and bind to the antigen, but with lower affinity than the full binding site.

[0397] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines derived from the antibody hinge region. In this specification, Fab'-SH is defined as having one or more cysteine ​​residues in the constant domain. This is a notation for Fab' having at least one free thiol group. The F(ab')2 antibody fragment was originally produced as a pair with a Fab' fragment having a hinged cysteine ​​in between. 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 distinct types, called κ and λ, based on the amino acid sequence of their constant domains.

[0399] "Single-chain Fv" or "scFv" antibody fragments contain the VH and VL domains of the antibody, which are located within a single polypeptide chain. The Fv polypeptide may further contain a polypeptide linker between the VH and VL domains, which allows the scFv to form a structure desirable for antigen binding. For an overview of scFv, see, for example, 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. WO93 / 16185, 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, on the same polypeptide chain (VH-VL), contains a variable heavy domain (VH) attached to a variable light domain (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 a complementary domain on another chain, thereby generating two antigen-binding sites. Diabodies are fully described, for example, in EP404,097;WO93 / 11161; and Hollinger et al (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0401] The "humanization" of non-human (e.g., rodent) antibodies is a chimeric antibody containing a minimal amount of sequences derived from non-human immunoglobulins. Humanization is a method for transferring mouse antigen-binding information into non-immunogenic human antibody acceptors, resulting in numerous therapeutically useful drugs. The humanization method 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 grafted with these CDRs generally do not retain their original affinity for antigen binding, and in fact, affinity is often severely impaired. In addition to the CDRs, selected non-human antibody framework residues also need to be incorporated to maintain the appropriate CDR configuration (Chothia et al, (1989) Nature 342:877). To support the structural arrangement of grafted CDRs, it has been shown that antigen binding and affinity can be restored by transferring key mouse framework residues to human acceptors (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). In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, that possess the desired specificity, affinity, and capabilities. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced by corresponding These are replaced with non-human residues. Furthermore, humanized antibodies may contain residues not found in either the recipient or donor antibody. These modifications are made to further improve antibody performance. Generally, humanized antibodies contain substantially all of at least one, typically two, variable domains, all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. Humanized antibodies also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the 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 containing an amino acid sequence derived from a sequence in which one or more amino acid residues are replaced by one or more cysteine ​​residues. Parent antibodies may contain natural or wild-type sequences. Parent antibodies may have existing amino acid sequence modifications (such as additions, deletions, and / or substitutions) compared to other natural, wild-type, or modified forms of the antibody. Parent antibodies are targeted against the target antigen. Antibodies targeting non-polypeptide antigens (such as tumor-associated glycolipid antigens; see U.S. Patent No. 5,091,178) are also considered.

[0403] An “isolated” antibody is one that has been identified and separated from components of its natural environment and / or recovered. These contaminants from the natural environment are materials that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody is purified to (1) more than 95% by weight or more than 99% by weight as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a gas-phase protein sequencer, or (3) to be homogeneous by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Because isolated antibodies lack at least one component of the antibody’s natural environment, they contain antibodies in situ within recombinant cells. However, isolated antibodies are typically prepared by at least one purification step.

[0404] An antibody that "binds" to a target molecular or antigen is one that can bind to that antigen with sufficient affinity to be useful in targeting cells that express the antigen.

[0405] The terms “to treat” or “treatment” refer to both therapeutic and preventive or protective measures aimed at preventing or slowing (mitigating) undesirable physiological changes or impairments (such as the onset or transmission of cancer). For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, symptom reduction, attenuation of disease severity, a stable (i.e., non-worsening) disease state, delayed or slowed disease progression, relief or remission of the disease state, and remission (whether partial or complete), whether detectable or undetectable. “Treatment” may also mean extending survival compared to the survival expected without treatment. Persons requiring treatment include those who already have a disease or impairment, those prone to developing a disease or impairment, or those who need to prevent a disease or impairment.

[0406] "Phage display" is a technique in which variant polypeptides are presented as fusion proteins with the coating proteins on the surface of phages, such as filamentous phage particles. One of the advantages of phage display is that it allows for the rapid and efficient classification of large libraries of random protein variants based on sequences that bind to target molecules with high affinity. The key is the fact that this is possible. Displaying peptide and protein libraries on phages has been used to screen millions of polypeptides for those with specific binding properties. Multivalent phage display methods have been used to display small random peptides and small proteins, typically through fusion to either PIII or PVIII of filamentous phages. Wells and Lowman, Curr. Opin. Struct. Biol., 3:355-362 (1992), and the references cited herein. In monovalent phage display, a protein or peptide library is fused to the phage coat protein or a portion thereof and expressed at low levels in the presence of wild-type protein. The binding force effect is reduced for multivalent phages so that selection is based on endogenous ligand affinity, and phagemid vectors are used, which simplifies DNA manipulation. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991). Phage display includes techniques for producing antibody-like molecules (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., Garland Publishing, New York, pp. 627-628).

[0407] A "phagemide" is a plasmid vector containing a bacterial replication origin, e.g., Co1E1, and a copy of the intergenetic region of a bacteriophage. Phagemids can be used with any known bacteriophage, including filamentous and lambdoid bacteriophages. Plasmids will also commonly contain selective markers for antibiotic resistance. Segments of DNA cloned into these vectors can be grown as plasmids. When cells containing these vectors are provided with all the genes necessary for the production of phage particles, the plasmid replication mode changes 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 a fragment thereof, conjugated as a gene fusion to a heterologous polypeptide gene so that the heterologous polypeptide is displayed on the surface of the phage particle. The compounds described herein may be in the form of pharmaceutically acceptable salts. In some embodiments, such salts are derived from inorganic or organic acids or bases. For a review of suitable salts, see, for example, 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 this disclosure, a group "Ab" (i.e., an antibody, an antibody fragment, and / or an antigen fragment) can be conjugated to two or more drug-containing moieties. In some embodiments, "Ab" can be conjugated to 1 to 20 drug-containing moieties. In some embodiments, "Ab" can be conjugated to 1 to 10 drug-containing moieties. In some embodiments, "Ab" can be conjugated to 1 to 5 drug-containing moieties. In some embodiments, "Ab" can be conjugated to 1 or 2 drug-containing moieties. In some embodiments, "Ab" can be conjugated to 1 drug-containing moiety.

[0409] In some aspects of this 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 pharmaceutically acceptable salts or pharmaceutically acceptable salts. This is possible. In some embodiments, such salts are derived from inorganic or organic acids or bases. For a review 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] Suitable examples of acid addition salts include acetate, adipine, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivaphosphate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate.

[0412] Suitable examples of base addition salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts containing organic bases such as dicyclohexylamine salts and N-methyl-D-glucamine, and salts containing amino acids such as arginine, lysine, and the like.

[0413] For example, Berge lists the following commercially available salts approved by the FDA: anionic acetates, besilates (benzenesulfonates), benzoates, bicarbonates, tartarates, bromides, calcium edetate (ethylenediaminetetraacetate), cansilates (camphor sulfonates), carbonates, chlorides, citrates, dihydrochlorides, edetates (ethylenediaminetetraacetate), edisylates (1,2-ethanedisulfonates), and edetates. Straight (lauryl sulfate), esylate (ethanesulfonate), fumarate, gluceptate (glucoheptonate), gluconate, glutamate, glycolylarsanilate (glycolamide phenylarsonate), hexylresorcinate, hydravamin (N,N'-di(dehydroabiethyl)ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, i Cetionates (2-hydroxyethanesulfonates), lactates, lactobionates, malates, maleates, mandelates, mesylates (methanesulfonates), methyl bromide, methyl nitrate, methyl sulfate, mucate, napsylates (2-naphthalenesulfonates), nitrates, pamoates (embolates), pantothenates, phosphates / diphosphates, polygalactuloses, salicylates, stearates, basic acetates, succinates, sulfates, tannates, tartrates, theoclates (8-chlorotheophylline), and triethiozides; organic cations such as benzathine (N,N'-dibenzylethylenediamine), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine; and metal cations such as aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.

[0414] Berge further lists the following salts that are not FDA approved and are commercially available (outside the U.S.): anions adipine, alginate, aminosalicylate, anhydromethylene citrate, arecoline, aspartate, bisulfate, butyl bromide, camphorate, digluconate, hydrobromide, disuccinate, glycerophosphate, hemisulfate, hydrofluoric acid, hydroiodide, methylenebis(salicylate), napadisylate (1,5-naphthalenedisulfonate), oxalate, pectinate, persulfate, and flu. Phenylethyl barbiturates, picrates, propions, thiocyans, tosylates, and undecanoates; the organic cations benethamine (N-benzylphenethylamine), cremisole (1-p-chlorobenzyl-2-pyrrolidine-1'-ylmethylbenzimidazole), diethylamine, piperazine, and tromethamine (tris(hydroxymethyl)aminomethane); and the metal cations barium and bismuth.

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

[0416] In some embodiments, the pharmaceutical composition can be formulated as a suitable parenteral dosage form. Suitable formulations can be prepared by various methods known in the art. The pharmaceutical composition can be administered directly into the bloodstream, muscle, or organs. Suitable means of parenteral administration include intravenous, intraarterial, intraperitoneal, subarachnoid, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous. Suitable devices for parenteral administration include needled syringes, needleless syringes, and injection techniques.

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

[0418] For example, the preparation of parenteral compositions under sterile conditions by freeze-drying can be quickly achieved 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. Accordingly, compositions can be formulated as solids, semi-solids, or thixotropic liquids for administration as implantable depots resulting in modified release of the active compound.

[0420] Parenteral formulations may be mixed with other suitable pharmaceutically acceptable excipients used in parenteral dosage forms, such as preservatives, but are not limited to these.

[0421] In another embodiment, the pharmaceutical composition can be formulated as a suitable oral dosage form, such as tablets, capsules, powders, pellets, suspensions, solutions, or emulsions. Other suitable carriers may be present, such as disintegrants, diluents, chelating agents, binders, lubricants, fillers, and anti-adhesives.

[0422] Oral formulations may also contain other suitable pharmaceutical excipients, such as sweeteners, vehicle / humectants, colorants, flavoring additives, preservatives, and viscosity enhancers / thickeners.

[0423] The dosage of the pharmaceutical composition disclosed herein can be determined for individual patients.

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

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

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

[0427] Methods of using compounds and compositions The specific compounds described herein are STING agonists and are therefore useful for stimulating an immune response in their target. The compositions can be used in the treatment of cancer.

[0428] The compounds of this disclosure exhibit STING-modulating / agonist activity. Certain compounds of this 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, enzyme stability).

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

[0430] The compounds disclosed herein are used to treat diseases that may be affected by STING (hereinafter referred to as "STING-related diseases"), such as cancer, such as colorectal cancer (e.g., colorectal cancer, rectal cancer, anal cancer, familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumors), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma), mesothelioma, pancreatic cancer (e.g., ductal carcinoma, pancreatic endocrine tumors), pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma), duodenal cancer, small intestine cancer, and 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, low-grade ovarian 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 cholangiocarcinoma), 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 body cancer, uterine sarcoma), choriocarcinoma of pregnancy, brain tumor (e.g., medulloblastoma, glioma, pineal It can be used as a pharmaceutical product for the prevention or treatment of cancers of unknown primary origin, such as a cancer growth inhibitor, a cancer metastasis inhibitor, an apoptosis promoter, and a drug for the treatment of precancerous lesions (e.g., myelodysplastic syndromes).

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

[0432] In certain embodiments, the compounds of this disclosure can be used concurrently with antibody therapy. In some embodiments, the antibody therapy includes an anti-PD-1 antibody. In some embodiments, the antibody therapy includes an anti-PD-L1 antibody.

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

[0434] Furthermore, the compounds of this disclosure can be used in conjunction with non-pharmacological therapies. More precisely, the compounds of this disclosure or the combination agents of this disclosure can be used in combination with non-pharmacological therapies, such as (1) surgery, (2) antihypertensive chemotherapy using angiotensin II, etc., (3) gene therapy, (4) hyperthermia, (5) cryotherapy, (6) laser ablation, and (7) radiotherapy.

[0435] For example, by using the compounds of this disclosure before or after the above-mentioned surgery, or before or after combination therapies of two or three of these, effects such as prevention of resistance development, extension of disease-free survival, suppression of cancer metastasis or recurrence, and life extension may be obtained.

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

[0437] In some embodiments, the present disclosure relates to a method for treating cancer in a patient, comprising administering to a patient in need of the above treatment a combination of a compound of formula (I) or a pharmaceutically acceptable salt thereof, one or more checkpoint inhibitors, and radiation. In some embodiments, the one or more checkpoint inhibitors include antibodies. In some embodiments, the one or more checkpoint inhibitors include anti-PD-1 antibodies. In some embodiments, the one or more checkpoint inhibitors include anti-PD-L1 antibodies.

[0438] In some embodiments, the present disclosure relates to the use of compounds of formula (I) or pharmaceutically acceptable salts thereof in combination with checkpoint inhibitors and radiation for the treatment of cancer in patients.

[0439] In some embodiments, the disclosure relates to compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of cancer in a patient, wherein the patient is also being treated with one or more checkpoint inhibitors and radiation. In some embodiments, the disclosure relates to compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of 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 checkpoint inhibitors, radiation, and / or combinations thereof. It can be administered simultaneously or consecutively. In some embodiments, the present disclosure relates to a method for 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 may be administered at least 5 hours before administration of the checkpoint inhibitor and / or the compound of formula (I). In some embodiments, radiation may be administered at least 10 hours before administration of the checkpoint inhibitor and / or the compound of formula (I). In some embodiments, radiation may be administered at least 20 hours before administration of the checkpoint inhibitor and / or the compound of formula (I). In some embodiments, radiation may be administered at least 40 hours before administration of the checkpoint inhibitor and / or the compound of formula (I). In some embodiments, radiation may be administered at least 80 hours before administration of the checkpoint inhibitor and / or the compound of formula (I).

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

[0442] In some embodiments, radiation can be administered on any two days between the first and fifth days of each week and repeated for 5 to 8 weeks. In some embodiments, radiation can be administered on any two days between the first and fifth days of each week and repeated for 6 to 8 weeks. In some embodiments, radiation can be administered on any two days between the first and fifth days of each week and repeated for 5 weeks. In some embodiments, radiation can be administered on any two days between the first and fifth days of each week and repeated for 6 weeks. In some embodiments, radiation can be administered on any two days between the first and fifth days of each week and repeated for 7 weeks. In some embodiments, radiation can be administered on any two days between the first and fifth days of each week and repeated for 8 weeks.

[0443] In some embodiments, checkpoint inhibitors may 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, checkpoint inhibitors may be administered once every 2 weeks. In some embodiments, checkpoint inhibitors may be administered once every 3 weeks. In some embodiments, checkpoint inhibitors may be administered once every 4 weeks. In some embodiments, checkpoint inhibitors may be administered once every 12 weeks.

[0444] In certain embodiments, radiation is administered at least 40 hours before administration of the checkpoint inhibitor and / or compound of formula (I). In certain embodiments, radiation is administered at least 30 hours before administration of the checkpoint inhibitor and / or compound of formula (I). In certain embodiments, radiation is administered at least 20 hours before administration of the checkpoint inhibitor and / or compound of formula (I). In certain embodiments, radiation is administered at a certain time. The radiation is administered at least 10 hours before the administration of the checkpoint inhibitor and / or the compound of formula (I). In certain embodiments, the radiation is administered at least 5 hours before the administration of the checkpoint inhibitor and / or the compound of formula (I). In certain embodiments, the 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 the checkpoint inhibitor can be administered to the patient 1 to 3 months after the patient receives radiation therapy. In some embodiments, the compound of formula (I) and / or the checkpoint inhibitor can be administered to the patient 1 to 2 months after the patient receives radiation therapy. In some embodiments, the compound of formula (I) and / or the checkpoint inhibitor can be administered to the patient 1 to 1 month after the patient receives radiation therapy. In some embodiments, the compound of formula (I) and / or the checkpoint inhibitor can be administered to the patient 1 to 15 days after the patient receives radiation therapy. In some embodiments, the compound of formula (I) and / or the checkpoint inhibitor can be administered to the patient 1 to 7 days after the patient receives radiation therapy.

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

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

[0448] In some embodiments, radiation can be administered in divided doses. In some embodiments, radiation can be administered in 1 to 10 divided doses. In some embodiments, radiation can be administered in 1 to 5 divided doses. In some embodiments, radiation can be administered in one dose, or divided into two, three, four, or five divided doses. In some embodiments, radiation can be administered in one dose or divided into three doses.

[0449] In some embodiments, radiation can be administered in 1 to 3 fractions, with fractional doses of approximately 1 to 5 Gy. In some embodiments, radiation can be administered in 1 to 3 fractions, with fractional doses of approximately 5 to 10 Gy. In some embodiments, radiation can be administered in 1 to 3 fractions, with fractional doses of approximately 10 to 15 Gy. In some embodiments, radiation can be administered in 1 to 3 fractions, with fractional doses of approximately 15 to 20 Gy. In some embodiments, radiation can be administered in 1 to 3 fractions, with fractional doses of approximately 5 to 10 Gy, or in 1 to 3 fractions, with fractional doses of approximately 15 to 20 Gy. In some embodiments, radiation can be administered in a single dose, with fractional doses of approximately 8 Gy. In some embodiments, radiation can be administered in 3 fractions, with fractional doses of approximately 8 Gy. In some embodiments, radiation can be administered in a single dose, with fractional doses of approximately 16 Gy. In some embodiments, radiation can be administered in a single fractional dose of approximately 8 Gy, or divided into three fractional doses of approximately 8 Gy, or in a single fractional dose of approximately 16 Gy.

[0450] Furthermore, treatment with the compounds of this disclosure or the combination agents of this disclosure can be combined with supportive care, such as (i) administration of antibiotics (e.g., β-lactam antibiotics such as Pansporin, macrolide antibiotics such as clarithromycin) for complications of various infections, (ii) administration of high-calorie intravenous fluids, amino acid preparations, or multivitamins to improve malnutrition, (iii) administration of morphine for pain relief, (iv) administration of drugs to improve side effects such as nausea, vomiting, loss of appetite, diarrhea, leukopenia, thrombocytopenia, decreased hemoglobin concentration, alopecia, liver damage, kidney damage, DIC, fever, etc., and (v) administration of drugs to suppress multidrug resistance in cancer. [Examples]

[0451] definition Ab antibody ACN Acetonitrile ADA anti-drug antibody ADC Antibody-drug conjugate BLQ lower limit of quantification Celsius CCR2 CC motif chemokine receptor 2 CR (Complete Remission) CD Differential Cluster DAR drug antibody ratio DMA N,N-dimethylacetamide DMSO (Dimethyl Sulfoxide) DTT (Dithiothreitol) ε absorption coefficient Absorption coefficient of E0.1% 0.1% solution EC 50 Half-effective concentration EDTA (Ethylenediaminetetraacetic acid) h time HIC Hydrophobic Interaction Matriculation hIgG Human Immunoglobulin G HPLC (High-Pressure Liquid Chromatography) IACUC Animal Experimentation Committee IFN (Interferon) IgG (Immunoglobulin G) IgM (Immunoglobulin M) IL Interleukin IP Interferon-γ-inducible protein LC (Reset Chromatography) LCMS (Liquid Chromatography Mass Spectrometry) μM micromoles MCP (Monocyte Chemotropic Protein) MDSC (Myelosuppressive 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 interferon gene stimulator TCEP (Tris(2-carboxyethyl)phosphine) TNF (tumor necrosis factor) TPPTS 3,3',3''-Phosphantriyltris(benzenesulfonic acid) trisodium salt Tris (Hydroxymethyl)aminomethane UFLC Ultra-High-Speed ​​Liquid Chromatography 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 with a diode array detector, using an SEC column (typically Tosoh Biosep TSK Gel, G3000SWxl; P / N 8541; 250A; 5um; 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 performed at constant composition 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 Tosoh Butyl-NPR, 4.6 × 35 mm, 2.5 μm, 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-minute run, a 12-minute linear gradient of 95% / 5% A / B to 100% B is used between the initial and final intervals of constant-composition flow.

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

[0455] [Table 1]

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

[0457] LC / MS / MS conditions: LC / MS / MS analysis was performed using a Shimadzu UFLC LC-20AD XR2 pump, a SIL-30AC MP autosampler system, and an AB SCIEX Triple Quad 4500 ESI mass spectrometer.

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

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

[0460] ADC concentration: The ADC concentration was calculated from the UV absorbance at 280 nm, measured using the NanoDrop (2000c; Fisher Scientific) coefficient, after subtracting the UV absorbance from the corresponding linker-payload construct.

[0461] Table 2 lists the linker-payload constructs used in the preparation of the ADC. The compound contains either compound number 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 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 was generated as described in US7,473,421B2, consisting of the humanized variable domains of the heavy and light chains of the 1D9 mouse monoclonal antibody, and the constant domains of the human IgG1 heavy chain and human κ light chain (also known as humanized 1D9, TAK-202, and possibly also referred to as the hIgG1 isotype below). The hIgG4 isotype of humanized 1D9 was prepared in the same manner as described in Anticancer Research March-April 2006 vol.26 no.2A 1057-1063. Humanized 1D9 sequence 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 (Sequence No. 4) Humanized 1D9 hIgG4 isotype sequence Heavy chain: EVQLVESGGGLVKPGGSLRLSCAASGFTFSAYAMNWVRQAPGKGLEWVGRIRTKNNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTFYGNGVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (Sequence ID 5) Light chain: DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTFLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 6)

[0468] Example 1 Procedure for preparing 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. Next, the desired linker-payload construct (5 mM DMA solution, 6-9 equivalents) was slowly added to the mixture. The reaction mixture was purged with argon and incubated at room temperature or °C for a further 1-2 hours with gentle shaking. The reaction mixture was purified according to the preparative SEC method described herein to obtain ADC. The ADC concentration, aggregation rate, and DAR were measured by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical method.

[0470] A schematic diagram of this procedure is shown in Figure 1.

[0471] Other antibody conjugates were prepared using a procedure similar to that described above.

[0472] Example 2 Preparation of further Ab-STING agonist conjugates by stochastic cysteine ​​conjugation. Using the linker-payload construct and the antibodies shown as starting materials, antibody-drug conjugates listed in Table 3 were prepared as described in Example 1.

[0473] [Table 3]

[0474] Example 3 Procedure for preparing Ab-STING agonist conjugates by transglutaminase conjugation Deglycosylation: A solution of anti-CCR2 antibody (humanized 1D9, produced as described in US7,473,421B2, 60 mg / mL) in 50 mM histidine, 125 mM arginine, and pH 6.1 buffer was diluted with an equal volume of pH 7.2 PBS. N-glycosylase F (New England Biolabs, P0704S, 500,000 units / mL, 300 units per 1 mg of antibody) was added to the solution, and the reaction mixture was heated gently overnight with stirring to 37°C. The resulting deglycosylated humanized 1D9 was buffer-replaced with PBS (pH 7.2).

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

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

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

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

[0479] [Table 4]

[0480] Example 5 Procedure for preparing Ab-STING agonist conjugates by transglutaminase conjugation Transglutaminase conjugation (after modification, follow the procedure described in Tumy, LNet al. Mol. Pharmaceuticals 2019, 16, 6, 2795-2807): Transglutaminase (ACTIVA®, Ajinomoto, 50 mg per 1 mg of antibody) was added to a pH 6.1 phosphate buffer solution, followed by a PBS solution of deglycosylated humanized 1D9 (10-20 mg / mL, prepared according to the deglycosylation procedure described in Example 3), and then a pH 6.1 phosphate buffer solution of 30 mM cystamine·2HCl (50 equivalents). The reaction mixture was heated gently overnight with stirring to 37°C. 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 citrate (pH 4.0). Further purification was performed using the preparative SEC method described herein to obtain humanized 1D9-NH-(CH2)2-SS-(CH2)2-NH2.

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

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

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

[0484] [Table 5]

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

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

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

[0488] [Table 6]

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

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

[0491] Example 10 Further mouse Ab-STING jaw development through probabilistic cysteine ​​conjugation Preparation of stent conjugates Using the linker-payload construct and the antibodies shown as starting materials, antibody-drug conjugates listed in Table 7 were prepared as described in Example 9.

[0492] [Table 7]

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

[0494] Using a gradient consisting of a 0.1% aqueous solution of formic acid (solvent A) and a 0.1% acetonitrile solution of formic acid (solvent B), at 40°C, Synergi 2.5μ Polar-RP Each sample was separated by reverse-phase HPLC using a 100A C18 column (2.0 mm × 30 mm) (Phenomenex®). Samples were analyzed by positive ion spray in multi-reaction monitoring (MRM) mode using a SCIEX API 4500 QTRAP instrument. Payload loss rates in human, primate, and mouse plasma at various time points are 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 #thpd-r232) was induced from human THP-1 monocyte cell lines by 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 the inducible secretory Lucia luciferase reporter gene under the control of the ISG54 (interferon-stimulating gene) minimal promoter in combination with five IFN-stimulated response elements (ISREs). Reporter gene expression allows for the study of the IFN regulatory factor (IRF) pathway by evaluating the activity of Lucia luciferase. In addition to human STING and luciferase, these cells can be recombined to stably express human CCR2 and study the activation of target-mediated IRF pathways. THP-1 cells express endogenous human CCR2 at a much lower density compared to endogenous human CCR2 in cells recombined 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 placed in growth medium (RPMI1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 100 μg / mL Normocin (trademark), 100 U / mL~100 μg / mL Pen-Strep, 10 μg / mL blastosidine, 100 μg / mL zeosin, and 1 μg / mL puromycin) at a density of 15,000 cells / 25 μL in a white 384-well plate. Cells were plated on Corning 356661. 5 μL of hCCR2-targeted-ADC sample or compound sample was added to the cell plate, followed by incubation at 37°C for 20 hours. At the end of incubation, 10 μL / well of QUANTI-Luc™ (InvivoGen number rep-qlc1) was added, and luminescence was immediately measured using LeadSeeker.

[0498] For the assay methods described above, the luminescence signal induction percentages for each test ADC or test compound at various concentrations were calculated compared to untreated and control samples. The compound concentration vs. signal induction percentage curves were fitted to the EC2. 50 A value was generated. A person skilled in the art would know the EC 50 You will understand that the values ​​generated are affected by experimental variations. 50 The Emax values ​​are reported in Table 9. The data in Table 9 clearly show that conjugation of either compound number 14 or compound I-5c to humanized 1D9 or its IgG4 isotype dramatically increases the in vitro potential in THP1 cell lines overexpressing hCCR2.

[0499] [Table 9]

[0500] Example 13 Pharmacokinetic evaluation in mice For in vivo evaluation of ADC in naive Balb / C mice, 6-8 week old mice Female Balb / c mice (purchased from Jackson Laboratory) were used. The mice were fed a normal diet and followed the guidelines for the care and use of laboratory animals. In accordance with the regulations of the Board on Care and Use of Laboratory Animals (SPF), the animals were housed in an SPF animal facility. The animals were maintained in a 12-hour intermittent light-dark cycle with a temperature of 18–26°C, a relative humidity of 50±20%, and free access to food and water.

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

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

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

[0504] The PK profiles of ADC-B14, ADC-B15, ADC-B16, ADC-B17, and ADC-B18 are summarized in Table 10. Graphs of plasma PK are shown in Figures 4-8.

[0505] [Table 10]

[0506] Example 14 Tolerability assessment in mice The tolerability of ADC was evaluated in naive C57BL / 6 mice. On day 0 of the study, the animals were weighed, and then the indicated dose of ADC (by payload concentration) was administered intravenously. Subsequently, the animals were regularly weighed for at least 14 days after administration (with no more than 3 days between each measurement), and after each measurement, weight loss was calculated based on the baseline weight before administration. All animals that experienced a weight loss of more than 20%, or that were mortally injured or otherwise exhibited suffering exceeding the humane endpoint of the study, were removed from the study and euthanized according to the guidelines of the IACUC protocol. The maximum tolerated dose (MTD) was calculated as the maximum dose (by payload concentration) at which any animal did not need to be removed from the study due to not being found dead, having experienced a weight loss of more than 20%, or otherwise exceeding the humane endpoint. The MTD for ADC-B17 was 200 μg / kg (depending on payload concentration, Figure 9), and the MTD for ADC-B20 was 250 μg / kg (depending on payload concentration, Figure 10).

[0507] Example 15 Evaluation of antitumor activity in mice The efficacy of ADC-B21 compared to compound number 14 was evaluated in a C57BL / 6 mouse model with MC38 (mouse colon adenocarcinoma) tumors. For tumor transplantation, 1 × 10⁶ 6 Individual MC38 cells were subcutaneously injected into C57BL / 6 mice, and tumor growth in the mice was then monitored. The tumor volume was approximately 100 mm². 3 When the mean was reached, animals were randomized by tumor volume and administered intravenously either 100 μL of vehicle, compound number 14 (2000 μg / kg), or ADC-B21 (50 μg / kg). The first day of administration was considered day 0 of the study. Compound number 14 and vehicle were administered again on days 3 and 6 of the study, while ADC-B21 was administered as a single dose on day 0 of the study. Tumor volume and body weight measurements were performed at least twice a week until the end of the study, when body weight decreased by more than 20% from the baseline, or when body weight exceeded 2000 mm. 3 Animals with tumor volume exceeding a certain amount were removed. By day 63 of the study, animals treated with compound number 14 had achieved complete remission in a total of 4 out of 6 trials of ADC-B21 treatment. Compared to the other group, the patient achieved complete remission once out of a total of six cycles.

[0508] A graph of the observed antitumor activity is shown in Figure 11, which demonstrates the significantly improved efficacy of anti-CCR2 ADCs at much lower dose levels compared to the payload alone.

[0509] Example 16 Toxicity / pharmacodynamic evaluation in non-human primates Two ADC variants were evaluated in toxicity tests in cynomolgus monkeys.

[0510] Single-dose studies were conducted using intravenous administration of ADC-B2 at doses of 0.15, 0.5, 1.5, or 5 mg / kg (2 monkeys / sex / group) (protein dose). Administration of 5 mg / kg of ADC-B2 was associated with early death in two animals on day 2, attributable to pulmonary toxicity similar to that observed in previous studies using a non-conjugated payload (compound number 14) (clinical signs including macroscopic red discoloration, increased alveolar edema and fibrin, increased alveolar macrophage and neutrophil infiltration, decreased mucosal pallor and decreased heart sounds, correlated with pleural and pericardial effusion in some animals, and histological findings of mild pulmonary vascular congestion and acute alveolar hemorrhage). Other findings specific to these early-death animals were found in the bone marrow (decreased hematopoietic cell solidity, single-cell necrosis, and increased histiocytes), liver (multifocal random foci of necrosis), and lymphoid tissue (decreased germinal center cell solidity and / or necrosis in the spleen and tonsils, and single-cell necrosis in the thymus). Clinicopathological and cytokine analysis of one of the early-death animals from which samples were available clearly showed pro-inflammatory / acute phase responses and elevations in IP-10, IL-6, MCP1, and TNF-α cytokine levels, similar to those of animals that survived to terminal euthanasia. Histological findings in animals that survived to terminal euthanasia were limited to increased lymph node cell solidity (due to increases in lymphocytes and histiocytes) at ≥1.5 mg / kg, and, in one animal, germinal center necrosis in lymph nodes at 5 mg / kg. The pharmacological endpoints included in the study consisted of flow cytometry to assess the monocyte population, identifying myeloid-derived immunosuppressive cells (MDSCs) at 6 and 24 hours post-administration on day 1, with dose-dependent decreases in the relative proportions of classical, intermediate, and non-classical monocytes, as well as partial recovery up to 48 hours post-administration on day 1.

[0511] The repeated dose study involved intravenous administration of ADC-B17 at a total of three doses, scheduled every two weeks, at doses of 0.3, 1, or 3 mg / kg (protein dose) (two monkeys / sex / group). However, two animals in the 3 mg / kg dose group died prematurely after the second dose on day 15, so the remaining two animals in group 4 received a dose reduction of 2 mg / kg on day 29 (third / final dose). Repeated administration of ADC-B17 at ≥0.3 mg / kg was associated with an increase in anti-drug antibodies (ADAs) in 10 out of 12 animals at one or more time points after day 15 (signal / noise ratio increased by 1–3 grades), most of which were directed towards the immune activation payload of ADC, with some ADAs observed directed towards the antibody components of ADC at the end of the time course. These ADAs were associated with a decrease in exposure (Cmax) after the third dose in most ADA-positive animals. Early deaths associated with ADC-B17 were observed at doses ≥1 mg / kg. One animal at 1 mg / kg was euthanized under mortal conditions on day 29, approximately 7 hours after administration. At 3 mg / kg, death was detected in one animal approximately 6 hours after administration on day 15, and one animal was euthanized under mortal conditions approximately 7 hours after administration on day 15. Clinical signs associated with ADC-B17 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 rate and / or respiratory rate. The cause of death was likely an immune-related immunogenic / hypersensitivity reaction. While the effects were attributed to the overall condition, the direct impact of ADC-B17 could not be ignored. Serological chemistry findings from all three animals that died early were generally similar to those of animals that survived to terminal euthanasia, consistent with a systemic pro-inflammatory response and muscle and / or hepatocyte damage. Hematological and coagulation parameters were assessed in animals euthanized at 29 days, rather than in animals at 15 days. Stress-induced lymphocyte and eosinophil reductions were minimal, and there were no changes in coagulation parameters. The observed immunophenotypic changes were similar to those of surviving animals, as will be discussed later. On day 15, most microscopic findings in animals that died early were similar to those in animals that survived to terminal euthanasia, but more severe and consistent with immune-mediated effects (immune cell infiltrations in the liver sinusoids, adrenal glands, pulmonary interstitium, and spleen; thrombosis in the pulmonary capillaries; necrosis / fibrin deposition in the spleen; myocardial degeneration; and microhemorrhages in the adrenal glands and epicardial fat), consisting of minimal hepatocyte necrosis and physical findings. Further findings specific to animals that died early were considered secondary to stress or the mortal state (decreased thymic weight and decreased thymic cell solidity, correlated with pancreatic acinar cell degradation). In animals euthanized at mortal age on day 29, the only finding was minimal adrenal hemorrhage.

[0512] In animals that survived to terminal euthanasia, clinicopathological findings consisted of mild to moderate increases in one or more of the following: aspartate aminotransferase, alanine aminotransferase, glutamate dehydrogenase, and creatine kinase, observed at ≥0.3 mg / kg on day 3. These findings were consistent with those originating from muscle and / or hepatocytes and lacked clear histological correlation. Other findings on day 3 were consistent with inflammatory cell infiltration in multiple tissues or dehydration without histological correlation (mild increases in urea, creatinine, and phosphorus) and a histologically correlated systemic pro-inflammatory / acute response (minimal to mild increases in globulin and c-reactive protein, as well as minimal to mild decreases in overall protein, albumin, and albumin / globulin ratio). Each of these changes recovered partially to completely by day 30. In males, further serological changes at days 14 and / or 30 consisted only of a mild increase in globulin and a mild rise in total bilirubin, both of which were consistent with an ongoing acute inflammatory response. Hematological and coagulation findings in individual animals at terminal euthanasia on day 30, at ≥0.3 mg / kg, consisted of a mild increase in leukocyte count, neutrophil count, fibrinogen, and activated partial thromboplastin time, as well as a mild decrease in erythrocyte count, hemoglobin, and hematocrit. These findings were consistent with a systemic pro-inflammatory / acute response.

[0513] Changes in monocytes and MDSCs in plasma samples were assessed using a flow cytometry panel designed to evaluate monocyte and MDSC counts, as well as CCR2, CD80, and CD86 expression in monocytes. Findings from this assessment were consistent with the expected pharmacology of ADC-B17 at ≥0.3 mg / kg and consisted of dose-responsive mild to severe decreases in the absolute numbers of classical monocytes, non-classical monocytes, and myeloid-derived immunosuppressive cells (MDSCs), measured by flow cytometry, with recovery towards or beyond baseline before each subsequent dose. CCR2 expression in classical monocytes decreased after administration and recovered to near baseline for all doses before subsequent doses (Figure 12, top). Furthermore, CD80 expression in 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] Cytokine changes were also assessed in plasma samples, showing ADC-B17 at ≥0.3 mg / kg, consisting of large dose-independent increases in serum IP-10 and MCP-1 concentrations, a potential biomarker of pharmacology. This peaked 6 hours after administration and returned to or tended to return to baseline values ​​24 hours after administration. Further increases were observed in IL-1RA, IL-6, TNF-α, and IFN-γ, peaking 6 hours after administration and returning to or tending to return to baseline levels 24 hours after administration or before subsequent administrations (Figure 13).

[0515] Histological findings in animals undergoing terminal euthanasia consisted of multifocal hepatocyte necrosis without clinicopathological correlation at ≥0.3 mg / kg. At ≥1 mg / kg, there was minimal to mild decrease in the cellular solidity of both erythrocytes and myeloid progenitor cells in the bone marrow (corresponding to mildly decreased erythrocytes in hematological findings and markedly decreased lymphocytes in one animal), mixed cellular infiltrations sporadically observed in the adrenal glands and hepatic synusoids, increased cellular solidity of splenic red medulla (mixed cells) corresponding to mildly increased splenic weight, and minimal local hemorrhage in the duodenum or heart. These organs with inflammatory cell infiltration / hemorrhage were considered likely to be part of a systemic pro-inflammatory response and were not considered direct target organ toxicity. Immunohistochemistry against human IgG, monkey IgG and IgM, C3, and / or C9 was performed to measure whether immune complex formation and tissue adhesion were present in areas of immune cell infiltration and / or tissue damage. No granule adhesions indicating immune complex formation were detected.

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

[0517] The circulating free payload was also measured by LC / MS after plasma protein precipitation. Briefly, monkey plasma was first spiked with stable isotope-labeled compound number 14, then proteins were precipitated using methanol, and the mixture was evaporated to dryness under a gentle nitrogen stream. Finally, the residue was reconstituted with ammonium acetate solution and then analyzed by LC / MS.

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

[0519] [Table 11]

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

[0521] Table 12 shows the available ADC and anti-PD-1 / anti-PD-L1 combinations.

[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. Since the anti-PD-1 antibody pembrolizumab does not cross-react with rodent PD-1, mice 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 day 0 of the study, after weighing the animals, the indicated dose of ADC can be administered intravenously in combination with the indicated dose of anti-PD-1 and / or anti-PD-L1 antibody. Next, for at least 14 days after administration, the animals may be weighed regularly (no more than 3 days between each weighing), and after each weighing, weight loss can be calculated based on the pre-administration baseline weight. Any animals with a weight loss exceeding 20%, or that appear mortally wounded, or otherwise exhibiting suffering exceeding the humane endpoint of the study, may be removed from the study and euthanized in accordance with the guidelines of the IACUC protocol. The maximum tolerated dose (MTD) can be calculated as the maximum dose (payload concentration + PD-1 / PD-L1 antibody concentration) at which any animal did not need to be removed from the study due to death not being detectable, weight loss exceeding 20%, or otherwise exceeding the humane endpoint. If satisfactory tolerability is achieved with ADC and either anti-PD-1 or anti-PD-L1 antibody, then ADC and anti-PD-1 and anti Combination therapy with PD-L1 antibodies can be performed using the same method.

[0525] Efficacy study of combination therapy in mice The efficacy of the ADCs shown in Table 12, combined with the anti-PD-1 antibody J43 or the anti-PD-L1 antibody MIH5, can be tested in a C57BL / 6 mouse model with MC38 (mouse colon adenocarcinoma) tumors. For tumor transplantation, 1 × 10⁶ 6 Individual MC38 cells can be subcutaneously injected into C57BL / 6 mice, and tumor growth in the mice can then be monitored. Tumor volume is approximately 100 mm². 3 When the mean is reached, animals may be randomized by tumor volume and administered intravenously with 100 μL of vehicle, the corresponding ADC (50 μg / kg) from Table 12, and either J43 (0.5, 5, or 50 mg / kg) or MIH5 (0.5, 5, or 50 mg / kg). The first day of administration may be considered day 0 of the study. Tumor volume and body weight measurements may be performed at least twice a week until the end of the study, when there is a weight loss of more than 20% from the starting body weight, or 2000 mm 3 Animals with tumor volume exceeding a certain threshold can be removed from the study. Complete and partial remission in animals can be assessed on day 63 of the study. If satisfactory reduction in tumor volume is not achieved with the combination of ADC and either an anti-PD-1 or anti-PD-L1 antibody, combination therapy with ADC and both anti-PD-1 and anti-PD-L1 antibodies can be administered in a similar manner.

[0526] Research on the effectiveness of combination therapy in non-human primates ADCs can be administered intravenously to cynomolgus monkeys every two weeks (days 1-29) in a total of three doses at 0.3, 0.5, or 1 mg / kg (protein dose) (2 monkeys / sex / group) in combination with the anti-PD-1 antibody pembrolizumab or the anti-PD-L1 antibody atezolizumab. Pembrolizumab can be administered at 0.5 or 15 mg / kg, and atezolizumab at 0.5 or 15 mg / kg. Hematological and coagulation parameters and overall serological chemistry of the animals can be evaluated, and histological evaluation can be performed at the end of the study.

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

[0528] Example 19 (Expected) Combination therapy with radiation Tolerability research The tolerability of anti-PD-1 and / or anti-PD-L1 antibodies, as well as ADCs in combination with radiation, can be evaluated in naive C57BL / 6 mice.

[0529] Table 13 shows the available combinations of ADCs, anti-PD-1 and / or anti-PD-L1 antibodies, and radiation.

[0530] [Table 13]

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

[0532] On day 0 of the study, it was possible to measure the weight of the animals and the amount of radiation, specifically anti-PD-1. Combined with J43 and / or anti-PD-L1 MIH5 antibody, the indicated amount of ADC It can be administered approximately 5 hours before intravenous administration. Next, for at least 14 days after administration, animals should be regularly measured (no more than 3 days between measurements), and after each measurement, weight loss can be calculated based on the baseline weight before administration. Any animals with a weight loss of more than 20%, or that appear to be mortally wounded, or otherwise exhibiting suffering exceeding the humane endpoint of the study, may be removed from the study and euthanized in accordance with the guidelines of the IACUC protocol. The maximum tolerated dose (MTD) can be calculated as the maximum dose of radiation in the combination therapy regimen for any animal that did not need to be removed from the study because death was not detectable, or because of a weight loss of more than 20%, or otherwise exceeding the humane endpoint. If satisfactory tolerability is achieved with ADC, anti-PD-1 or anti-PD-L1 antibody, and radiation, combination therapy with ADC, anti-PD-1 and anti-PD-L1 antibodies, and radiation can be carried out in a similar manner.

[0533] Efficacy study of combining radiotherapy with other therapies in mice. The efficacy of ADCs in combination with anti-PD-1 and / or anti-PD-L1 antibodies, as shown in Table 13, can be tested in a C57BL / 6 mouse model with MC38 (mouse colon adenocarcinoma) tumors. For tumor transplantation, 1 × 10⁶ 6 Individual MC38 cells can be subcutaneously injected into C57BL / 6 mice, and tumor growth in the mice can then be monitored. Tumor volume is approximately 100 mm². 3When the mean is reached, animals may be randomized by tumor volume and irradiated with either 0.5 Gy or 1 Gy of radiation, and intravenously administered either 100 μL of vehicle, the corresponding ADC (50 μg / kg) from Table 13, and either anti-PD1 antibody J43 (0.5, 5, or 50 mg / kg) or anti-PD-L1 antibody MIH5 (0.5, 5, or 50 mg / kg). The first day of administration may be considered day 0 of the study. Tumor volume and body weight measurements may be performed at least twice a week until the end of the study, when there is a weight loss of more than 20% from the starting body weight, or 2000 mm. 3 Animals with tumor volume exceeding a certain threshold can be removed from the study. Complete and partial remission in animals can be assessed on day 63 of the study. If satisfactory reduction in tumor volume is not achieved with a combination of ADC, radiation, and either an anti-PD-1 or anti-PD-L1 antibody, combination therapy with ADC, radiation, and anti-PD-1 and anti-PD-L1 antibodies can be administered in a similar manner.

[0534] Efficacy study of combination therapy with radiotherapy in non-human primates Before administering 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 radiotherapy, ADC can be administered intravenously to cynomolgus monkeys every two weeks (days 1-29) in a total of three doses at 0.3, 0.5, or 1 mg / kg (protein dose) (2 monkeys / sex / group) at anti-PD-1 antibody pembrolizumab or anti-PD-L1 antibody atezolizumab. Pembrolizumab can be administered at 0.5 or 15 mg / kg, and atezolizumab at 0.5 or 15 mg / kg. Hematological and coagulation parameters and overall serological chemistry of the animals can be evaluated and histologically assessed at the end of the study.

[0535] Blood samples can be collected from non-human primates at various time points, and the levels of total antibodies and conjugated payloads in monkey plasma can be measured as described above by a 2-in-1 immunocapture-based LC / MS assay using a Shimadzu UHPLC system interfaced to a Sciex 6500+ QTRAP mass spectrometer. Circulating free payloads can also be measured by LC / MS after plasma protein precipitation, as described above. Post-radiation hematological recovery and extramyelotoxicity can be evaluated in animals.

[0536] It should be understood that the section describing embodiments for carrying out the invention, rather than the section describing the summary and abstract of the invention, is intended to be used to interpret the claims. The sections describing the summary and abstract may, as intended by the inventor(s), describe one or more but not all embodiments of the present disclosure and shall not in any way limit the scope of the present disclosure and the accompanying claims.

[0537] This disclosure is described above using functional building blocks that illustrate the implementation and relationships of specific functions. The boundaries of these functional building blocks are arbitrarily defined herein for descriptive convenience. Different boundaries may be defined insofar as the specific functions and relationships are adequately performed.

[0538] The above descriptions of specific embodiments fully disclose the general features of this disclosure, that by applying knowledge within the scope of the art, others may readily modify and / or alter such specific embodiments for various applications without excessive experimentation and without deviating from the general concepts of this disclosure. Therefore, such modifications and alterations are intended to fall within the meaning and scope of the equivalents of the disclosed embodiments, based on the teachings and guidance presented herein. The language or terminology used herein is for illustrative purposes only and not intended to limit, so that it should be understood by those skilled in the art in light of the teachings and guidance.

[0539] The scope and breadth of this disclosure should not be limited by any of the exemplary embodiments described above, but should be defined solely in accordance with the following claims or their equivalents.

Claims

1. Compound of formula (I): 【Chemistry 1】 or a pharmaceutically acceptable salt thereof. [In the formula, a is an integer between 1 and 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, comprising a guanine base, a guanine base derivative, an adenine base, or an adenine base derivative with an amino group. L is a linker that is covalently bonded to Ab and also to the aforementioned amino group on D.

2. The compound according to claim 1, wherein D-L is represented by formula (Ia), or a pharmaceutically acceptable salt thereof. 【Chemistry 2】 [In the formula: 【Transformation 3】 This indicates the connection point to Ab, b is an integer between 1 and 20. m is 0, 1, 2, 3, or 4. n is either 0 or 1, Each R １ C １ -C ４ Alkyl, O-C １ -C ４ Selected independently from alkyl and halogen, R ２ is selected from C １ -C ４ alkyl and -(CH ２ CH ２ O) ｓ -CH ３ [where s is an integer from 1 to 10]. R ３ and R ３’ These are hydrogen and C, respectively. １ -C ３ Selected independently of alkyl, L １ This is a linker fragment that can be cut.

3. a is an integer from 1 to 8, b is an integer between 1 and 10, The compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein m is 0.

4. m is 0, n is 0, R ３ and R ３’ The compound according to claim 2 or 3, or a pharmaceutically acceptable salt thereof, wherein each of the atoms is hydrogen.

5. L １ but 【Chemistry 4】 The compound according to any one of claims 2 to 4, or a pharmaceutically acceptable salt thereof. [In the formula: 【Transformation 5】 This is the bond point to the nitrogen atom in equation (Ia), 【Transformation 6】 is a connection point to Ab, t is an integer between 1 and 10. W either does not exist or is a self-sacrificing element. Z is either absent or a peptide of 2 to 5 amino acids. U and U' are either non-existent or spacers, independently. Q is a heterodifunctional group. However, it is not possible for both W and Z to be absent.

6. W is 【Transformation 7】 The compound according to claim 5, or a pharmaceutically acceptable salt thereof, wherein the self-sacrificing group is selected from the above. [In the formula: 【Transformation 8】 This is a bonding point to the carbonyl group, 【Chemistry 9】 This is a connection point to Z.

7. W 【Chemistry 10】 The compound according to claim 5 or 6, or a pharmaceutically acceptable salt thereof.

8. W 【Chemistry 11】 The compound according to any one of claims 5 to 7, or a pharmaceutically acceptable salt thereof.

9. A compound according to any one of claims 5 to 8, wherein Z is a peptide that can be cleaved by an enzyme, or a pharmaceutically acceptable salt thereof.

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

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

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

13. U' does not exist, and U is 【Chemistry 12】 A compound according to any one of claims 5 to 12, selected from, or a pharmaceutically acceptable salt thereof. [In the formula: 【Chemistry 13】 is a connection point to Z, 【Chemistry 14】 is a connection point to Q, p is an integer from 1 to 6, q is an integer between 1 and 20. X is O, or -CH ２ - and Each r is independently either 0 or 1.

14. U' does not exist, and U is 【Chemistry 15】 The compound according to any one of claims 5 to 13, or a pharmaceutically acceptable salt thereof.

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

16. Q is 【Chemistry 16】 A compound according to any one of claims 5 to 15, selected from, or a pharmaceutically acceptable salt thereof. [During the ceremony 【Chemistry 17】 It is either a connection point to U, or, if U does not exist, a connection point to Z. [Chemistry 18] It is either a connection point to U', or, if U' does not exist, a connection point to Ab.

17. Q is 【Chemistry 19】 The compound according to any one of claims 5 to 16, or a pharmaceutically acceptable salt thereof.

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

19. R ２ ga-CH ３ And R ３ and R ３’ A compound according to any one of claims 2 to 18, or a pharmaceutically acceptable salt thereof, wherein each of the atoms is hydrogen.

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

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

22. The amino-substituted compound that controls STING activity is the compound of formula (II): 【Chemistry 20】 The compound according to any one of claims 1 to 21, or a pharmaceutically acceptable salt thereof. [In the 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, R ａ is C １ -C ４ It is alkyl. R １０ is hydrogen, fluoro, OH, NH ２ , OR ｂ , or NHR ｂ And, R ２０ is hydrogen or fluoro, R ３０ is hydrogen, R ４０ is hydrogen, fluoro, OH, NH ２ , OR ｂ , or NHR ｂ is 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 It is a cycloalkyl, Ring A １０ It contains 1 to 4 heteroatoms selected from N, O, or S. A optionally substituted 5 or 6-membered monocyclic heteroaryl ring, 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 It contains at least one N atom in the ring, and in the formula, Y ｂ is ring A １０ It is bonded to the carbon atom, Ring B １０ is an optionally substituted 9 or 10-membered bicyclic heteroaryl ring containing 2 to 5 heteroatoms selected from N, O, or S, wherein ring B １０ The ring contains at least two N atoms. However, ring A １０ or Ring B １０ One of these is bonded to "L" in formula (I) via an amino group.

23. The amino-substituted compound that controls STING activity is 【Chemistry 21】 The compound according to any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof. [In the formula, 【Chemistry 22】 This is the connection point to "L" in equation (I).

24. The amino-substituted compound that controls STING activity is the compound of formula (III): 【Chemistry 23】 The compound according to any one of claims 1 to 21, or a pharmaceutically acceptable salt thereof. [In the formula] 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 equation (B １ -A) or formula (B １ -B) 【Chemistry 24】 It is a group represented by R １３ , R １４ , R １５ , R １６ , and R １７ Each of these is independently a hydrogen atom or a substituent. R １０００ is a hydrogen atom, or a bond to the carbonyl group of formula (I), Y １１ , Y １２ , Y １３ , Y １４ , Y １５ , and Y １６ Each is independently N or CR １ａ [In the formula, R １ａ is hydrogen or a substituent. 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 substituent, B ２００ is equation (B ２ -A) or formula (B ２ -B) 【Chemistry 25】 is a group represented by, R ２３ , R ２４ , R ２５ , R ２６ , and R ２７ are each independently a hydrogen atom or a substituent, R １００’ is a bond to hydrogen or the carbonyl group of formula (I), Y ２１ , Y ２２ , Y ２３ , Y ２４ , Y ２５ , and Y ２６ Each is 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 Each of them is independently N or C, R ２０５ R is a hydrogen atom or substituent, where R is a hydrogen atom or substituent. １０５ and R ２０５ Each of them is independently bonded to the 2nd or 3rd position of the 5-membered ring to which they are joined. however, B １００ or B ２００ One of them is bonded to "L" in formula (I) via an amino group.

25. The amino-substituted compound that controls STING activity is the compound of formula (IIIa): 【Chemistry 26】 The compound according to any one of claims 1 to 21 and 24, or a pharmaceutically acceptable salt thereof. [In the formula] B １００ is equation (B １ -A) or formula (B １ -B) 【Chemistry 27】 It is a group represented by R １３ , R １４ , R １５ , R １６ , and R １７ Each of these is independently a hydrogen atom or a substituent. R １０００ is a hydrogen atom, or a bond to the carbonyl group of formula (I), Y １１ , Y １２ , Y １３ , Y １４ , Y １５ , and Y １６ Each is independently N or CR １ａ [In the formula, R １ａ is hydrogen or a substituent. Z １１ Z １２ Z １３ Z １４ Z １５ , and Z １６ Each of them is independently N or C, R １０５ is a hydrogen atom or substituent, B ２００ is equation (B ２ -A) or formula (B ２ -B) 【Chemistry 28】 It is a group represented by R ２３ , R ２４ , R ２５ , R ２６ , and R ２７ Each is independent and are hydrogen atoms or substituents, R １００’ is a bond to hydrogen or the carbonyl group of formula (I), Y ２１ , Y ２２ , Y ２３ , Y ２４ , Y ２５ , and Y ２６ Each is 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 Each of them is independently N or C, R ２０５ R is a hydrogen atom or substituent, where R is a hydrogen atom or substituent. １０５ and R ２０５ Each of them is independently bonded to the 2nd or 3rd position of the 5-membered ring to which they are joined. however, B １００ or B ２００ One of them is, 【Chemistry 29】 And, During the ceremony: R １８ is hydrogen, or C １－６ It is alkyl, R １９ It is a halogen atom, Furthermore, the other is bonded to the "L" group in formula (I) via a -NH- group.

26. The amino-substituted compound that controls STING activity is a compound of formula (IV): 【Transformation 30】 The compound according to any one of claims 1 to 21 and 24, or a pharmaceutically acceptable salt thereof. [During the ceremony R １ and R ２ Each of these is independently a hydroxyl group or a halogen atom. B １ teeth, 【Chemistry 31】 And, R １８ is hydrogen, or C １－６ It is alkyl, R １９ It is a halogen atom, B ２ teeth, 【Chemistry 32】 And, Q ２ and Q ４ Each of these is independently either an oxygen atom or a sulfur atom.

27. The amino-substituted compound that controls STING activity is 【Transformation 33】 or a pharmaceutically acceptable salt thereof, in the formula, 【Transformation 34】 A compound according to any one of claims 1 to 21 and 24 to 26, wherein the bond site is L.

28. A compound of formula (VI) according to any one of claims 1 to 21 and 24 to 26, or a pharmaceutically acceptable salt thereof: 【Chemistry 35】 [In the formula, a is an integer between 1 and 6.]

29. Ab is an antibody or fragment that binds to human CCR2 or a part thereof, and chemokai A compound according to any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof, which is capable of blocking the binding of to CCR2 and inhibiting the function of CCR2.

30. The compound according to claim 29, or a pharmaceutically acceptable salt thereof, wherein the antibody is selected from the group consisting of monoclonal antibody 1D9, or human CCR2, or antibodies that can compete with 1D9 for binding to a portion of CCR2;MC-21;STI-B020X;UniTI-101; and 4.40A68G.

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

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

33. The compound according to claim 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 containing amino acids 24-39 of SEQ ID NO: 1; a light chain CDR2 containing amino acids 55-61 of SEQ ID NO: 1; a light chain CDR3 containing amino acids 94-102 of SEQ ID NO: 1; a heavy chain CDR1 containing amino acids 31-35 of SEQ ID NO: 2; a heavy chain CDR2 containing amino acids 50-68 of SEQ ID NO: 2; and a heavy chain CDR3 containing amino acids 101-106 of SEQ ID NO:

2.

34. The compound according to claim 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 containing the amino acid sequence of SEQ ID NO:

2.

35. The compound according to claim 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 containing the amino acid sequence of SEQ ID NO:

1.

36. The compound according to claim 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 claim 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, the light chain variable region comprising the amino acid sequence of SEQ ID NO:

1.

38. The compound according to claim 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 containing the amino acid sequence of SEQ ID NO: 2 and a light chain variable region, and the light chain variable region contains 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 A compound according to any one of claims 31 to 38, or a pharmaceutically acceptable salt thereof, further comprising a heavy chain steady region selected from the heavy chain steady region.

40. The anti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-binding fragment is used in human immunotherapy. A compound according to any one of claims 31 to 39, or a pharmaceutically acceptable salt thereof, further comprising a light chain constant region selected from the group consisting of epidemic globulin IgGκ and IgGλ light chain constant regions.

41. The compound according to claim 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 the 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 claim 31, or a pharmaceutically acceptable salt thereof, wherein the anti-CCR2 antibody comprises the heavy chain region of SEQ ID NO:

3.

43. The compound according to claim 31, or a pharmaceutically acceptable salt thereof, wherein the anti-CCR2 antibody comprises the light chain region of SEQ ID NO:

4.

44. The compound according to claim 31, or a pharmaceutically acceptable salt thereof, wherein the anti-CCR2 antibody comprises the heavy chain region of SEQ ID NO: 3 and the light chain region of SEQ ID NO:

4.

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

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

47. The pharmaceutical composition according to claim 46, wherein the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, semiprimab, pimivalimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, 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, pemplimab, pukotenlimab, CX-188, zimvalerimab, and teboterimab.

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

49. The pharmaceutical composition according to claim 48, wherein the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, durvalumab, cosiberimab, MSB-2311, ZKAB-001, FAZ-053, MDX-1105, CBT-502, IMC-001, RC-98, KL-A167, GR-1405, rodapolimab, sugemalimab, emvafolimab, opcolimab, and gariblimab.

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

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

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

53. Claim 50 or 51 further comprises administering an anti-PD-L1 antibody to the subject. Method of loading.

54. The method according to claim 52, wherein the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, semiprimab, pimivalimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, 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, pemplimab, pukotenlimab, CX-188, zimvalerimab, and teboterimab.

55. The aforementioned anti-PD-L1 antibodies include atezolizumab, avelumab, durvalumab, and kosibelli. The method according to claim 53, wherein a drug selected from the group consisting of mab, MSB-2311, ZKAB-001, FAZ-053, MDX-1105, CBT-502, IMC-001, RC-98, KL-A167, GR-1405, rodapolimab, sugemalimab, emvafolimab, opcolimab, and galiblimab.

56. The method according to any one of claims 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 claims 1 to 44.

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

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

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

60. The method according to claim 58 or 59, wherein the radiation is administered by external beam radiation.

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