CN114652853A

CN114652853A - anti-IL-4R antibody-drug conjugate and medical application thereof

Info

Publication number: CN114652853A
Application number: CN202111576520.3A
Authority: CN
Inventors: 王欢; 林�源; 唐昱澄; 柯可; 林侃; 廖成
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Shengdi Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Shengdi Pharmaceutical Co Ltd
Priority date: 2020-12-22
Filing date: 2021-12-22
Publication date: 2022-06-24

Abstract

The present disclosure relates to anti-IL-4R antibody-drug conjugates and medical uses. In particular, antibody-drug conjugates of an antibody or antigen-binding fragment thereof that specifically binds IL-4R and a drug molecule, such as MMAE or a derivative thereof, irinotecan or a derivative thereof, eribulin or a derivative thereof, pharmaceutical compositions containing the antibody-drug conjugates, and their use in the manufacture of a medicament for the treatment of an IL-4R-mediated disease or disorder, particularly for use in the manufacture of an anti-cancer medicament, are provided.

Description

anti-IL-4R antibody-drug conjugate and medical application thereof

Technical Field

The present disclosure relates to an antibody-drug conjugate comprising an anti-IL-4R antibody or antigen binding fragment thereof, and its use as an anti-cancer drug.

Background

Malignant tumors (cancers) are the second cause of death worldwide, ranking only after heart disease. Glioblastomas (GBMs) are the most common malignant primary brain tumors, with median survival of glioblastoma patients undergoing standard treatment being less than 15 months. Despite the advances in tumor biology, transformation has not been achieved to benefit patients, and there is an urgent need to develop effective methods for treating glioblastoma.

IL-4R is an interleukin 4 receptor, expressed on glioblastoma tumor cells and infiltrating immunosuppressive cells (e.g., tumor-associated macrophages (TAM), myeloid-derived immunosuppressive cells (MDSC), etc.). The treatment of the target IL-4R can not only kill tumor cells, but also improve the tumor microenvironment. In preclinical animal models of glioblastoma and in early clinical trials, targeted IL-4R therapy has been shown to be superior to conventional therapies.

MDNA55 developed by Mediconna is a fusion protein of IL-4 and Pseudomonas exotoxin A (PE38KDEL), which is a bacterial toxin, and has shown positive therapeutic effects in clinical development experiments (see WO9527732, biochem. J. (1995)307,29-37, CANCER RESEARCH 55,3357, 3357-3363, August 1, 1995).

However, the first generation immunotoxin therapies had problems such as insufficient affinity for the target. Improving targeting and affinity of immunotoxins is one of the major and difficult points in the research of tumor immunotoxin therapy (see "clinical annual book of tumor in china 2009", edited by zhao hei, press of chinese consortium university of medical science, p.61 of 2010).

Antibody-drug conjugates (ADCs) aim to combine the selectivity of monoclonal antibodies (mAbs) with the cytotoxic potential of chemotherapeutic drugs. The antibody-drug conjugate is composed of three main components of "antibody", "linker" and "drug molecule (drug)". Compared to conventional fully or partially humanized antibodies or antibody fragments, antibody-drug conjugates are theoretically more effective because they release highly active cytotoxins in tumor tissues; higher tolerance or lower side effects than the fusion protein. The affinity of the antibody-drug conjugate with the target antigen affects the process of the antibody-drug conjugate permeating into tumor tissues to bind with the target antigen and directly affects the adhesion efficiency of the antibody-drug conjugate (refer to "biotechnology pharmacy", main edition of von Cynanchum, Chinese medicine science and technology Press, 2016 (month 01), p 209-.

One of the most critical factors in the design of antibody-drug conjugates is the choice of antibody, and importantly, high specificity for the antigen. Antibodies lacking high specificity and cross-reacting with other antigens may function unexpectedly, for example, by interacting with healthy tissue to cause off-target toxicity, or by causing premature clearance of the body before reaching the tumor site (see: Introduction to Antibody-Drug Conjugates (ADCs), Ilona pyz, Paul J. M. Jackson and David E. Thurston, CHAPTER 1: Introduction to Antibody-Drug Conjugates (ADCs), in Cytotoxic Payloads for Antibody-Drug Conjugates, 2019).

Currently, several pharmaceutical companies are developing monoclonal antibodies against IL-4R in various countries, related patent applications such as WO2010053751, WO2001092340, WO2008054606, WO2014031610, etc., and prior application WO2020038454 of the present inventors also relates to a new class of anti-IL-4R antibodies. However, IL-4R-directed antibody-drug conjugates have only been explicitly reported, e.g. WO2015188934, WO2014124227, WO2018217227, etc. all mention broadly that antibodies in antibody-drug conjugates can selectively target IL-4R, but none disclose specific anti-IL-4R antibody-drug conjugates.

The invention utilizes the anti-IL-4R antibody with high affinity to prepare the ADC medicine, achieves the aim of killing cancer cells strongly, and is used for meeting the requirements of treating cancer or delaying the progress of the cancer.

Disclosure of Invention

The present disclosure relates to conjugates (ADCs) of anti-IL-4R antibodies or antigen-binding fragments thereof and drugs and pharmaceutical uses, wherein ADC drugs conjugated to anti-IL-4R antibodies or antigen-binding fragments thereof and a cytotoxic agent MMAE or derivatives thereof, irinotecan or derivatives thereof, eribulin or derivatives thereof are provided.

Antibody-drug conjugates

The present disclosure provides an antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, comprising: an anti-IL-4R antibody or antigen-binding fragment thereof and one or more drug molecules; wherein the drug is selected from the group consisting of MMAE or a derivative thereof, irinotecan or a derivative thereof, and eribulin or a derivative thereof.

In some embodiments, anti-IL-4R antibodies or antigen-binding fragments thereof may be known, as described, for example, in WO2010053751, WO2001092340, WO2008054606, WO2014031610, WO2020038454 (each of which is incorporated herein by reference), or anti-IL-4R antibodies or antigen-binding fragments thereof.

In some embodiments, anti-IL-4R antibodies or antigen binding fragments thereof include, but are not limited to Dupixent, PRS-060, AK-120, 63IgG1, CBP201, AMG-317, or antigen binding fragments thereof.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof is an anti-human IL-4R antibody or antigen-binding fragment thereof.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises:

(I) respectively shown in SEQ ID NO: 3. SEQ ID NO: 4 and SEQ ID NO: 5 HCDR1, HCDR2 and HCDR 3; or

(II) are shown as SEQ ID NO: 11. SEQ ID NO: 12 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown at 13;

and/or the antibody light chain variable region comprises:

(I) respectively shown in SEQ ID NO: 6. SEQ ID NO: 7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in fig. 8; or

(II) are shown as SEQ ID NO: 14. the amino acid sequence of SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 16; or

(III) as shown in SEQ ID NO: 38. SEQ ID NO: 7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown at 40; or

(IV) are shown as SEQ ID NO: 42. SEQ ID NO: 39 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown in fig. 8.

Table 1 shows the CDR sequences of an anti-IL-4R antibody or antigen-binding fragment thereof.

TABLE 1 CDR sequences of anti-IL-4R antibodies or antigen-binding fragments thereof

Name (R)	Sequence of	Numbering
			HCDR1	GFTFSDYGMH	SEQ ID NO：3
HCDR2	FISSGSSIIYYADIVKG	SEQ ID NO：4
			HCDR3	GNKRGFFDY	SEQ ID NO：5
LCDR1	NASSSVSYMY	SEQ ID NO：6
			LCDR2	LTSNLAS	SEQ ID NO：7
LCDR3	QQWRSNPPMLT	SEQ ID NO：8
			HCDR1	GYTFTSYWMH	SEQ ID NO：11
HCDR2	LIHPNSDTTKFSENFKT	SEQ ID NO：12
			HCDR3	SKIITTIVARHWYFDV	SEQ ID NO：13
LCDR1	KASQSVDYGGDSYMN	SEQ ID NO：14
			LCDR2	AASNLES	SEQ ID NO：15
LCDR3	QHSNENPPT	SEQ ID NO：16
			LCDR1	RASSSVPYMY	SEQ ID NO：38
LCDR2	LASSRPS	SEQ ID NO：39
			LCDR3	QQWRAYPPMLT	SEQ ID NO：40
LCDR1	RASPGVPPLA	SEQ ID NO：42

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises any one selected from the following (I) to (IV):

(I) a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 3. SEQ ID NO: 4 and SEQ ID NO: 5 HCDR1, HCDR2 and HCDR 3; and

a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 6. SEQ ID NO: 7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in fig. 8;

(II) a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 11. SEQ ID NO: 12 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 13; and

a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 14. SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 16;

(III) a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 3. the amino acid sequence of SEQ ID NO: 4 and SEQ ID NO: 5 HCDR1, HCDR2 and HCDR 3; and

a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 38. SEQ ID NO: 7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown at 40;

(IV) a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 3. SEQ ID NO: 4 and SEQ ID NO: 5 HCDR1, HCDR2 and HCDR 3; and

a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 42. SEQ ID NO: 39 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown in fig. 8.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment is a murine antibody, a chimeric antibody, a fully human antibody, a humanized antibody, or a fragment thereof. In some specific embodiments, the anti-IL-4R antibody or antigen-binding fragment is humanized.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises the FR sequence derived from human germline light chain IGKV3-11 x 01(SEQ ID NO: 22 for antibody 25G7) or a back-mutated sequence that is at least 95% identical thereto. In some embodiments, the back-mutations are selected from one or more of L46P, L47W, F71Y. In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises the FR region sequence derived from human germline heavy chain IGHV3-48 x 01(SEQ ID NO: 21 for antibody 25G7) or a back-mutated sequence having at least 95% identity thereto. In some embodiments, the back-mutations are selected from one or more of S94A, F67S, a 93T. In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises the FR region sequence derived from human germline light chain IGKV2D-29 x 01(SEQ ID NO: 24 for antibody 7B10) or a back-mutated sequence that is at least 95% identical thereto. In some embodiments, the back-mutation is selected from M4L and/or V58I. In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises the FR region sequence derived from human germline heavy chain IGHV1-2 x 02(SEQ ID NO: 23 for antibody 7B10) or a back-mutated sequence having at least 95% identity thereto. In some embodiments, the back-mutations are selected from one or more of M69L, R71I, T73K, R94K.

Human germline heavy chain IGHV3-48 x 01:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR

SEQ ID NO：21

human germline light chain IGKV3-11 × 01:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP

SEQ ID NO：22

human germline heavy chain IGHV1-2 × 02:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR

SEQ ID NO：23

human germline light chain IGKV2D-29 x 01:

DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQKPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSIQLP

SEQ ID NO：24

in some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

the heavy chain variable region comprises:

(I) as shown in SEQ ID NO: 1 or a sequence corresponding to SEQ ID NO: 1, sequences having at least 70%, 80%, 90%, 95%, 98%, 99% identity; or

(II) as shown in SEQ ID NO: 9 or a sequence corresponding to SEQ ID NO: 9, sequences having at least 70%, 80%, 90%, 95%, 98%, 99% identity; or

(III) as shown in SEQ ID NO: 43 or a sequence identical to SEQ ID NO: 43 sequences having at least 70%, 80%, 90%, 95%, 98%, 99% identity; or

And/or the light chain variable region comprises:

(I) as shown in SEQ ID NO: 2 or a sequence similar to SEQ ID NO: 2, sequences having at least 70%, 80%, 90%, 95%, 98%, 99% identity; or

(II) as shown in SEQ ID NO: 10 or a sequence identical to SEQ ID NO: 10, sequences having at least 70%, 80%, 90%, 95%, 98%, 99% identity; or

(III) as shown in SEQ ID NO: 37 or a sequence identical to SEQ ID NO: 37, sequences having at least 70%, 80%, 90%, 95%, 98%, 99% identity; or

(IV) as shown in SEQ ID NO: 41 or a sequence corresponding to SEQ ID NO: 41 are sequences having at least 70%, 80%, 90%, 95%, 98%, 99% identity.

25G7 HCVR (25G7 heavy chain variable region)

EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYGMHWVRQAPEKGLEWVAFISSGSSIIYYADIVKGRSTISRDNAKNTLFLQMTSLRSEDTAMYYCTRGNKRGFFDYWGQGTILTVSS

SEQ ID NO：1

25G7 LCVR (25G7 light chain variable region)

QIVLTQSPALMSASPGEKVTMTCNASSSVSYMYWYQRKPRSSPKPWIYLTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWRSNPPMLTFGSGTKLEVK

SEQ ID NO：2

7B10 HCVR (variable region of heavy chain 7B10)

QVQLQQPGTELLKPGASVSLSCKASGYTFTSYWMHWVKQRPGQGLEWIGLIHPNSDTTKFSENFKTRATLTIDKSSSTAYMKLSSLTSEDSAVYYCAKSKIITTIVARHWYFDVWGTGTTVTVSS

SEQ ID NO：9

7B10 LCVR (7B10 light chain variable region)

DIVLTQSPPSLAVSLGQRATISCKASQSVDYGGDSYMNWYQQKLGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDVATYYCQHSNENPPTFGGGTKLEIK

SEQ ID NO：10

hu25G7-A LCVR (hu25G7-A light chain variable region)

EIVLTQSPATLSLSPGERATLSCRASSSVPYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRAYPPMLTFGGGTKVEIK

SEQ ID NO：37

hu25G7-B LCVR (hu25G7-B light chain variable region)

EIVLTQSPATLSLSPGERATLSCRASPGVPPLAWYQQKPGQAPRLLIYLASSRPSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRSNPPMLTFGGGTKVEIK

SEQ ID NO：41

hu25G7-VH (hu25G7 heavy chain variable region)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAFISSGSSIIYYADIVKGRSTISRDNAKNTLYLQMNSLRAEDTAVYYCTRGNKRGFFDYWGQGTLVTVSS

SEQ ID NO：43

In at least one embodiment, the heavy chain variable region of the anti-IL-4R antibody or antigen-binding fragment is as set forth in sequence SEQ ID NO: 1, and the light chain variable region is shown as a sequence SEQ ID NO: 2 is shown in the specification; or

The heavy chain variable region is shown as a sequence SEQ ID NO: 9, the light chain variable region is shown as a sequence SEQ ID NO: 10 is shown in the figure; or

The heavy chain variable region is shown as a sequence SEQ ID NO: 43, the light chain variable region is shown as sequence SEQ ID NO: 37 is shown in the figure; or

The heavy chain variable region is shown as a sequence SEQ ID NO: 43, the light chain variable region is shown as sequence SEQ ID NO: shown at 41.

the heavy chain variable region comprises:

(I) as shown in SEQ ID NO: 25-27 or a sequence as set forth in one of SEQ ID NOs: 25-27, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical; or

(II) as shown in SEQ ID NO: 31-33 or a sequence as set forth in one of SEQ ID NOs: 31-33, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical;

and/or the light chain variable region comprises:

(I) as shown in SEQ ID NO: 28-30 or a sequence as set forth in any one of SEQ ID NOs: 28-30, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical; or

(II) as shown in SEQ ID NO: 34-36 or a sequence as set forth in any one of SEQ ID NOs: 34-36 having at least 70%, 80%, 90%, 95%, 98%, or 99% identity.

hu25G7-VH-a：

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAFISSGSSIIYYADIVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGNKRGFFDYWGQGTLVTVSS

SEQ ID NO：25

hu25G7-VH-b：

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAFISSGSSIIYYADIVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTRGNKRGFFDYWGQGTLVTVSS

SEQ ID NO：26

hu25G7-VH-c：

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAFISSGSSIIYYADIVKGRSTISRDNAKNSLYLQMNSLRAEDTAVYYCTRGNKRGFFDYWGQGTLVTVSS

SEQ ID NO：27

hu25G7-VL-a：

EIVLTQSPATLSLSPGERATLSCNASSSVSYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRSNPPMLTFGGGTKVEIK

SEQ ID NO：28

hu25G7-VL-b：

EIVLTQSPATLSLSPGERATLSCNASSSVSYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWRSNPPMLTFGGGTKVEIK

SEQ ID NO：29

hu25G7-VL-c：

EIVLTQSPATLSLSPGERATLSCNASSSVSYMYWYQQKPGQAPRPWIYLTSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWRSNPPMLTFGGGTKVEIK

SEQ ID NO：30

hu7B10-VH-a：

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGLIHPNSDTTKFSENFKTRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSKIITTIVARHWYFDVWGQGTTVTVSS

SEQ ID NO：31

hu7B10-VH-b：

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGLIHPNSDTTKFSENFKTRVTMTIDTSISTAYMELSRLRSDDTAVYYCAKSKIITTIVARHWYFDVWGQGTTVTVSS

SEQ ID NO：32

hu7B10-VH-c：

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGLIHPNSDTTKFSENFKTRVTLTIDKSISTAYMELSRLRSDDTAVYYCAKSKIITTIVARHWYFDVWGQGTTVTVSS

SEQ ID NO：33

hu7B10-VL-a：

DIVMTQTPLSLSVTPGQPASISCKASQSVDYGGDSYMNWYLQKPGQPPQLLIYAASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSNENPPTFGGGTKVEIK

SEQ ID NO：34

hu7B10-VL-b：

DIVLTQTPLSLSVTPGQPASISCKASQSVDYGGDSYMNWYLQKPGQPPQLLIYAASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSNENPPTFGGGTKVEIK

SEQ ID NO：35

hu7B10-VL-c：

DIVMTQTPLSLSVTPGQPASISCKASQSVDYGGDSYMNWYLQKPGQPPQLLIYAASNLESGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSNENPPTFGGGTKVEIK

SEQ ID NO：36

In some specific embodiments, the heavy chain variable region is as set forth in SEQ ID NO: 25-27, and the light chain variable region is as shown in SEQ ID NO: one of 28-30;

the heavy chain variable region is shown as a sequence SEQ ID NO: 31-33 and the light chain variable region is as shown in SEQ ID NO: as shown in one of 34-36.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4, or a variant thereof. In some embodiments, the heavy chain constant region of human IgG1 or a variant thereof is included. In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a constant region of a human kappa, lambda chain or variant thereof.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof, wherein the antibody is a humanized antibody having a heavy chain sequence as set forth in SEQ ID NO: 17 or has at least 85% sequence identity thereto; the light chain sequence is shown as SEQ ID NO: 18 or has at least 85% sequence identity thereto.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof, wherein the antibody is a humanized antibody having a heavy chain sequence as set forth in SEQ ID NO: 19 or has at least 85% sequence identity thereto; the light chain sequence is shown as SEQ ID NO: 20 or has at least 85% sequence identity thereto.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof, wherein the antibody is a humanized antibody having a heavy chain sequence as set forth in SEQ ID NO: 44 or has at least 85% sequence identity thereto; the light chain sequence is shown as SEQ ID NO: 45 or has at least 85% sequence identity thereto.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof, wherein the antibody is a humanized antibody having a heavy chain sequence as set forth in SEQ ID NO: 44 or has at least 85% sequence identity thereto; the light chain sequence is shown as SEQ ID NO: 46 or has at least 85% sequence identity thereto.

In some embodiments, an isolated anti-IL-4R antibody or antigen-binding fragment thereof is provided that is characterized by competing with any of the anti-IL-4R antibodies or antigen-binding fragments thereof described above for binding to human IL-4R or an epitope thereof.

In some embodiments, a bispecific or multispecific antibody is provided comprising any of the anti-IL-4R antibodies or antigen-binding fragments thereof described above, a light chain variable region and/or a heavy chain variable region. In other embodiments, a single chain antibody is provided comprising the light chain variable region and/or the heavy chain variable region of any of the anti-IL-4R antibodies or antigen-binding fragments thereof described above.

In some embodiments, the humanized anti-IL-4R antibody, or antigen-binding fragment thereof, further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof. In at least one embodiment, included is a human IgG2 or IgG4 heavy chain constant region. Because IgG2 or IgG4 have no ADCC toxicity. In another embodiment, IgG1 that has no ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation is used. In at least one embodiment, the variant comprises a heavy chain constant region mutation with reduced or absent ADCC effector function, such as, but not limited to, N297A, L234A, L235A of IgG 1. In some embodiments, IgG1 comprises mutations E239D and M241L.

Unless otherwise indicated, the anti-IL-4R antibodies or antigen binding proteins thereof of the present disclosure are encoded according to Kabat.

In some embodiments, an anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises:

(I) as shown in SEQ ID NO: 17 or a sequence corresponding to SEQ ID NO: 17, a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity; or

(II) as shown in SEQ ID NO: 19 or a sequence corresponding to SEQ ID NO: 19, a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity; or

(III) as shown in SEQ ID NO: 44 or a sequence corresponding to SEQ ID NO: 44 is at least 70%, 80%, 90%, 95%, 98%, or 99% identical;

(IV as shown in SEQ ID NO: 47 or a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 47;

and/or the light chain comprises:

(I) as shown in SEQ ID NO: 18 or a sequence corresponding to SEQ ID NO: 18, at least 70%, 80%, 90%, 95%, 98%, or 99% identical; or

(II) as shown in SEQ ID NO: 20 or a sequence corresponding to SEQ ID NO: 20, a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity; or

(III) as shown in SEQ ID NO: 45 or a sequence identical to SEQ ID NO: 45 sequences having at least 90%, 95%, 98%, or 99% identity; or

(IV) as shown in SEQ ID NO: 46 or a sequence corresponding to SEQ ID NO: 46 are at least 90%, 95%, 98% or 99% identical.

hu25G7 HC

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAFISSGSSIIYYADIVKGRSTISRDNAKNSLYLQMNSLRAEDTAVYYCTRGNKRGFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK

SEQ ID NO：17

hu25G7 LC

EIVLTQSPATLSLSPGERATLSCNASSSVSYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRSNPPMLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：18

hu7B10 HC

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGLIHPNSDTTKFSENFKTRVTMTIDTSISTAYMELSRLRSDDTAVYYCAKSKIITTIVARHWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：19

hu7B10 LC

DIVLTQTPLSLSVTPGQPASISCKASQSVDYGGDSYMNWYLQKPGQPPQLLIYAASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSNENPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：20

hu25G7-IgG4 HC

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAFISSGSSIIYYADIVKGRSTISRDNAKNTLYLQMNSLRAEDTAVYYCTRGNKRGFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO：44

hu25G7-A LC

EIVLTQSPATLSLSPGERATLSCRASSSVPYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRAYPPMLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：45

hu25G7-B LC

EIVLTQSPATLSLSPGERATLSCRASPGVPPLAWYQQKPGQAPRLLIYLASSRPSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRSNPPMLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：46

hu25G7-24-IgG1(E239D，M241L)HC

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAFISSGSSIIYYADIVKGRSTISRDNAKNTLYLQMNSLRAEDTAVYYCTRGNKRGFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：47

In some embodiments, the heavy chain sequence is as set forth in SEQ ID NO: 17, and a light chain sequence as set forth in SEQ ID NO: 18 is shown in the figure; or

The heavy chain sequence is shown as SEQ ID NO: 19 and the light chain sequence is shown in SEQ ID NO: 20 is shown in the figure; or

The heavy chain sequence is shown as SEQ ID NO: 44 and a light chain sequence as shown in SEQ ID NO: 45 is shown; or

The heavy chain sequence is shown as SEQ ID NO: 44 and a light chain sequence as shown in SEQ ID NO: 46; or

The heavy chain sequence is shown as SEQ ID NO: 47, and a light chain sequence as set forth in SEQ ID NO: shown at 45.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof can be an antibody variant having 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes in the light chain and/or 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes in the heavy chain.

In some embodiments, the variant has the same or similar biological function or effect as a parent anti-IL-4R antibody or fragment thereof.

In some embodiments, antigen-binding fragments include, but are not limited to, Fab ', Fv, F (ab') 2, linear antibodies, scFv (single chain Fv antibodies), tandem di-scFv, tandem tri-scFv, diabody (diabody), triabody (triabody), tetrabody (tetrabody), sdAb (single domain or nanobody), sdFv, peptidobody (peptibody), domain antibodies, multispecific antibodies (e.g., bispecific, trispecific or tetraspecific antibodies), dsFv (disulfide stabilized Fv), ScdsFv (disulfide stabilized single chain Fv antibody).

In some embodiments, the antigen-binding fragment of an anti-IL-4R antibody in an antibody-drug conjugate of the disclosure, or a pharmaceutically acceptable salt or solvate thereof, is one that binds to the same IL-4R or epitope thereof as the antigen-binding fragment of an anti-IL-4R antibody described above.

In some embodiments, polynucleotides, such as DNA or RNA, are provided that encode an anti-IL-4R antibody or antigen-binding fragment thereof as described above.

In some embodiments, expression vectors, e.g., eukaryotic expression vectors, prokaryotic expression vectors, viral vectors, comprising the polynucleotides described above are provided.

In some embodiments, host cells, e.g., eukaryotic cells, prokaryotic cells, transformed with the above-described expression vectors are provided. In some embodiments, the host cell is a bacterium (e.g., e.coli), a yeast (e.g., pichia pastoris), a mammalian cell (e.g., a Chinese Hamster Ovary (CHO) cell or a Human Embryonic Kidney (HEK)293 cell).

In some embodiments, there is provided a method of making the above anti-IL-4R antibody or antigen-binding fragment thereof, comprising the steps of: expressing the antibody or antigen-binding fragment thereof in a host cell as described above, and isolating the antibody or antigen-binding fragment thereof from the host cell.

The three letter code and the one letter code for amino acids in antibodies against IL-4R or antigen binding fragments thereof of the present disclosure are as described in j.biol.chem, 243, p3558 (1968).

Antibody-drug (irinotecan derivative) conjugates

The present disclosure provides an antibody-irinotecan derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, represented by the general formula (Pc-L-Y-D) of formula (I):

wherein:

y is selected from-O- (CR)^aR^b)_m-CR¹R²-C(O)-、-O-CR¹R²-(CR^aR^b)_m-、-O-CR¹R²-、-NH-(CR^aR^b)_m-CR¹R²-C (O) -and-S- (CR)^aR^b)_m-CR¹R²-C(O)-；

R^aAnd R^bThe same or different, and each is independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, and a heterocyclic group; or, R^aAnd R^bTogether with the carbon atom to which they are attached form cycloalkyl and heterocyclyl radicals;

R¹selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, and heteroaryl; r²Selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, and heteroaryl; or, R¹And R²Together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group;

or, R^aAnd R²Together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group;

m is an integer of 0 to 4;

n is a decimal or integer from 1 to 10, for example n is from 2 to 8 or from 5 to 9;

l is a linker unit;

pc is an anti-IL-4R antibody or antigen-binding fragment thereof according to any of the preceding disclosure.

In some embodiments, the antibody-irinotecan derivative conjugate of the disclosure, or a pharmaceutically acceptable salt or solvate thereof, wherein-Y-is-O- (CR)^aR^b)m-CR¹R²-C(O)-；

R^aAnd R^bAre the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, or an alkyl group;

R¹is C_3-6Cycloalkylalkyl or C_3-6Cycloalkyl, hydrogen atom;

R²selected from hydrogen atoms, haloalkyl radicals or C_3-6A cycloalkyl group; such as a hydrogen atom;

or, R¹And R²Together with the carbon atom to which they are attached form C_3-6A cycloalkyl group;

m is 0 or 1.

In some embodiments, the antibody-irinotecan derivative conjugate of the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, wherein structural unit-Y-is-O- (CH)₂)m-CR¹R²-C(O)-；

R¹Is C_3-6Cycloalkylalkyl or C_3-6Cycloalkyl, hydrogen atom;

R²selected from hydrogen atoms, haloalkyl groups or C_3-6A cycloalkyl group;

m is 0 or 1.

R¹Is C_3-6Cycloalkylalkyl or C_3-6Cycloalkyl, hydrogen atom;

R²is a hydrogen atom;

m is 0 or 1.

R¹Is C_3-6Cycloalkylalkyl or C_3-6Cycloalkyl, hydrogen atom;

R²is a hydrogen atom;

m is 0.

R¹Is a hydrogen atom;

R²is a hydrogen atom;

m is 0.

In some embodiments, the antibody-irinotecan derivative conjugate of the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, Y is selected from:

wherein the O-terminal of Y is connected to the linker unit L.

In some embodiments, the antibody-irinotecan derivative conjugates of the present disclosure or pharmaceutically acceptable salts or solvates thereof.

In other embodiments, the present disclosure provides antibody-irinotecan derivative conjugates, or pharmaceutically acceptable salts or solvates thereof, of formula (II) having the general formula (Pc-L-D1):

wherein:

R¹is a hydrogen atom, C_3-6Cycloalkylalkyl or C_3-6A cycloalkyl group;

m is 0 or 1;

n is an integer or decimal of 1 to 10, for example n is a decimal or integer of 1 to 8 or 1 to 6, for example n is a decimal or integer of 1-5 or 2-4.

In some embodiments, n can be any value between 1 and 10, such as 2 and 8, or 2 and 6, or 1 and 6, or 4 and 6. In some embodiments, n is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2. 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.6, 7.8, 7.1, 8, 9.9.9, 8, 9.9.8, 8.9, 8, 9.0, 8.9.9, 8, 9.0, 9.9.0, 8, 9.0, 8, 9.0, 8.3, 9.3, 9.0, or the like.

In some embodiments, the present disclosure provides antibody-irinotecan derivative conjugates, or pharmaceutically acceptable salts or solvates thereof, wherein n is an integer or decimal number from 1 to 8; for example, an integer or decimal number of 1 to 6.

In some embodiments, the present disclosure providesConjugate of antibody-irinotecan derivative, wherein linker unit-L-is-L, or pharmaceutically acceptable salt or solvate thereof¹-L²-L³-L⁴-，

L¹Selected from- (succinimidin-3-yl-N) -W-C (O) -, -CH₂-C(O)-NR³-W-C (O) -or-C (O) -W-C (O) -, wherein W is selected from C_1-8Alkyl radical, C_1-8Alkyl-cycloalkyl or a linear heteroalkyl of 1 to 8 atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C_1-8Alkyl, cycloalkyl and linear heteroalkyl are each independently optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

L²is selected from-NR⁴(CH₂CH₂O)p¹CH₂CH₂C(O)-、-NR⁴(CH₂CH₂O)p¹CH₂C(O)-、-S(CH₂)p¹C (O) -or a chemical bond, wherein p¹Is an integer from 1 to 20;

L³is a peptide residue consisting of 2 to 7 amino acids, wherein said amino acids are selected from the group consisting of amino acid residues formed by amino acids selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, and optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl;

L⁴is selected from-NR⁵(CR⁶R⁷)_t-、-C(O)NR⁵、-C(O)NR⁵(CH₂)_t-or a chemical bond, wherein t is an integer from 1 to 6;

R³、R⁴and R⁵Are the same or different and are each independently selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

R⁶and R⁷Are the same or different and are each independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, deuteriumAlkyl and hydroxyalkyl.

In some embodiments, the present disclosure provides a conjugate of an antibody-irinotecan derivative, or a pharmaceutically acceptable salt or solvate thereof, wherein linker unit L is¹Selected from the group consisting of- (succinimid-3-yl-N) - (CH)₂)s¹-C (O) -, - (succinimid-3-yl-N) -CH₂-cyclohexyl-C (O) -, - (succinimid-3-yl-N) - (CH)₂CH₂O)s²-CH₂CH₂-C(O)-、-CH₂-C(O)-NR³-(CH₂)s³-C (O) -or-C (O) - (CH)₂)s⁴C (O) -, wherein s¹Is an integer from 2 to 8, s²Is an integer of 1 to 3, s³Is an integer from 1 to 8, s⁴Is an integer from 1 to 8; e.g. s¹Is 5.

In some embodiments, the present disclosure provides a conjugate of an antibody-irinotecan derivative, or a pharmaceutically acceptable salt or solvate thereof, wherein linker unit L is²Is selected from-NR⁴(CH₂CH₂O)p¹CH₂C (O) -or a chemical bond, p¹Is an integer from 6 to 12.

In some embodiments, the present disclosure provides a conjugate of an antibody-irinotecan derivative, or a pharmaceutically acceptable salt or solvate thereof, wherein L⁴Is selected from-NR⁵(CR⁶R⁷)t-，R⁵Selected from hydrogen atoms or alkyl radicals, R⁶And R⁷Identical or different and are each independently a hydrogen atom or an alkyl group, t is 1 or 2, for example 2; such as L⁴Is self-NR⁵CR⁶R⁷-; such as L⁴is-NHCH₂-。

In some embodiments, the present disclosure provides a conjugate of an antibody-irinotecan derivative, or a pharmaceutically acceptable salt or solvate thereof, wherein the linker unit-L-is-L¹-L²-L³-L⁴-，

L¹Is composed of

s¹Is a whole number of 2 to 8Counting;

L²is a chemical bond;

L³is a tetrapeptide residue;

L⁴is-NR⁵(CR⁶R⁷)t-，R⁵Selected from hydrogen atoms or alkyl radicals, R⁶And R⁷The same or different, and each independently is a hydrogen atom or an alkyl group, and t is 1 or 2.

L¹Is- (succinimidin-3-yl-N) -CH₂-cyclohexyl-c (o) -;

L²is-NR⁴(CH₂CH₂O)₉CH₂C(O)-；

L³Is a tetrapeptide residue;

In some embodiments, the present disclosure provides a conjugate of an antibody-irinotecan derivative, or a pharmaceutically acceptable salt or solvate thereof, wherein L is³The peptide residue of (a) is an amino acid residue formed of one, two or more amino acids selected from phenylalanine (E), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E), aspartic acid (N); for example, an amino acid residue formed from one, two or more amino acids selected from phenylalanine and glycine; for example, a tetrapeptide residue; such as a tetrapeptide residue of GGFG (glycine-phenylalanine-glycine).

In some embodiments, the present disclosure provides a conjugate of an antibody-irinotecan derivative, or a pharmaceutically acceptable salt or solvate thereof, wherein the linker unit-L-, wherein L is¹Terminal to antibody, L⁴Terminal andand Y is connected.

In some embodiments, the present disclosure provides an antibody-irinotecan derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein the-L-Y-is:

L¹selected from the group consisting of- (succinimidyl-3-yl-N) - (CH)₂)s¹-C (O) -or- (succinimid-3-yl-N) -CH₂-cyclohexyl-c (o) -;

L²is-NR⁴(CH₂CH₂O)p¹CH₂C (O) -or a chemical bond, p¹Is an integer from 6 to 12;

L³is a tetrapeptide residue of GGFG;

R¹is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group; e.g. C_3-6Cycloalkylalkyl or C_3-6A cycloalkyl group;

R⁵selected from hydrogen atoms or alkyl radicals, R⁶And R⁷The same or different, and each independently is a hydrogen atom or an alkyl group;

s¹is an integer from 2 to 8; such as 5;

m is an integer of 0 to 4.

In some embodiments, the disclosure provides a conjugate of an antibody-irinotecan derivative, or a pharmaceutically acceptable salt or solvate thereof,

wherein-L-Y-is:

for example:

L²is-NR⁴(CH₂CH₂O)₉CH₂C(O)-；

L³Tetrapeptide residues that are GGFG;

R⁵selected from hydrogen atoms or alkyl groups, R⁶And R⁷The same or different, and each independently is a hydrogen atom or an alkyl group;

m is an integer of 0 to 4.

In some embodiments, the present disclosure provides an antibody-irinotecan derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-irinotecan derivative conjugate comprises a structure of formula (III):

wherein:

L²is a chemical bond;

L³tetrapeptide residues that are GGFG;

R¹is a hydrogen atom, C_3-6Cycloalkylalkyl or C_3-6A cycloalkyl group;

R²selected from hydrogen atoms, haloalkyl radicals or C_3-6A cycloalkyl group;

R⁵is selected fromHydrogen atom or alkyl group, R⁶And R⁷The same or different, and each independently is a hydrogen atom or an alkyl group;

s¹is an integer from 2 to 8;

m is an integer of 0 to 4.

In some embodiments, the present disclosure provides an antibody-irinotecan derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-irinotecan derivative conjugate comprises a structure represented by the formula (-L-Y-):

it can be used to obtain antibody-drug conjugates by linking drugs to antibodies via linker fragments;

wherein:

L²is-NR⁴(CH₂CH₂O)p¹CH₂C (O) -or a chemical bond, p¹Is an integer from 1 to 20;

L³is a tetrapeptide residue of GGFG;

R¹is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group; such as C_3-6Cycloalkylalkyl or C_3-6A cycloalkyl group;

R⁵、R⁶or R⁷Are the same or different and are each independently a hydrogen atom or an alkyl group;

s¹is an integer from 2 to 8;

m is an integer of 0 to 4.

wherein:

L²is a chemical bond;

L³is a tetrapeptide residue of GGFG;

R⁵selected from hydrogen atoms or alkyl groups, R⁶And R⁷Are the same or different and are each independently a hydrogen atom or an alkyl group;

s¹is an integer from 2 to 8;

m is an integer of 0 to 4.

In some embodiments, the present disclosure provides antibody-irinotecan derivative conjugates of formula (IV) or pharmaceutically acceptable salts or solvates thereof, of the general formula (Pc-L)_a-Y-Dr) or a pharmaceutically acceptable salt or solvate thereof:

wherein:

w is selected from C_1-8Alkyl radical, C_1-8Alkyl-cycloalkyl or a linear 1 to 8 atom heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C is_1-8Alkyl, cycloalkylAnd linear heteroalkyl is each independently optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl;

L²is selected from-NR⁴(CH₂CH₂O)p¹CH₂CH₂C(O)-、-NR⁴(CH₂CH₂O)p¹CH₂C(O)-、-S(CH₂)p¹C (O) -or a chemical bond, p¹Is an integer from 1 to 20;

L³is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

R¹selected from hydrogen, halogen, cycloalkylalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl;

R²selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;

or, R¹And R²Together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group;

R⁴and R⁵The same or different, and each is independently selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

R⁶and R⁷The same or different, and each is independently selected from the group consisting of a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

m is an integer of 0 to 4;

n is an integer or decimal from 1 to 10;

pc is any anti-IL-4R antibody or antigen-binding fragment thereof provided by the present disclosure.

In some embodiments, the present disclosure provides antibody-irinotecan derivative conjugates of formula (V) or pharmaceutically acceptable salts or solvates thereof, of general formula (Pc-L)_b-Y-Dr) of an antibody-irinotecan derivativeThe conjugate or a pharmaceutically acceptable salt or solvate thereof:

wherein:

s¹is an integer from 2 to 8; such as 5;

Pc、R¹、R²、R⁵～R⁷m and n are as defined in formula (IV).

In some embodiments, the linking unit-L-Y-of the antibody-irinotecan derivative conjugates of polypeptides of the present disclosure include, but are not limited to:

in some embodiments, the antibody-irinotecan derivative conjugates provided by the present disclosure, or pharmaceutically acceptable salts or solvates thereof, include, but are not limited to:

wherein:

n is an integer or decimal from 1 to 10;

pc is any anti-IL-4R antibody or antigen-binding fragment thereof of the foregoing disclosure.

The present disclosure provides a process for preparing the compound of the formula (Pc-L)_a-Y-D) or a pharmaceutically acceptable salt or solvate thereof, comprising the steps of:

after reduction of Pc, with the general formula (L)_a-Y-D) coupling reaction to obtain the general formula (Pc-L)_a-Y-D);

wherein Pc is an anti-IL-4R antibody or antigen-binding fragment thereof of the present disclosure; w, L²、L³、R¹、R²、R⁵～R⁷M and n are as defined in formula (IV).

In the above embodiments, Pc is an anti-IL-4R antibody or antigen-binding fragment thereof of any of the present disclosure; for example, an anti-IL-4R antibody or antigen-binding fragment thereof of the examples; for example, a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 28 and the heavy chain as set forth in SEQ ID NO: 29, or an antibody comprising a light chain as set forth in SEQ ID NO: 30 and the heavy chain as set forth in SEQ ID NO: 31, or a light chain as set forth in seq id no.

In some embodiments, the antibody-irinotecan derivative conjugates of the present disclosure include tautomers, mesomers, racemates, enantiomers, diastereomers, deuterons, or mixtures thereof.

The compounds and processes for their preparation are incorporated herein in their entirety in WO 2020063673.

Antibody-drug (eribulin or derivative thereof) conjugates

The present disclosure provides a conjugate of an antibody-eribulin or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, as represented by formula (VI) having the general formula Pc- (L-D)_kShown in the figure:

wherein the antibody is Pc, said Pc being an anti-IL-4R antibody or antigen-binding fragment thereof of any of the present disclosure,

in formula (VI), L is a linker covalently attaching Pc to D, and k is 1 to 20 (including 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any number between any two),

-D is represented by the following formula (VI):

wherein R is^1aSelected from hydrogen, alkyl (e.g. C)_1-6Alkyl groups including, but not limited to, methyl, ethyl, isopropyl), cycloalkyl (e.g., C)_3-8Cycloalkyl groups, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, each independently optionally selected from alkyl (e.g., C)_1-6Alkyl including but not limited to methyl, ethyl, isopropyl), alkoxy (e.g., C)_1-6Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (e.g., fluoro, chloro, bromo), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, e.g., methyl; r is^1bSelected from hydrogen, alkyl (e.g. C)_1-6Alkyl including but not limited to methyl, ethyl, isopropyl), alkoxy, cycloalkyl (e.g., C)_3-8Cycloalkyl groups, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, each independently optionally selected from alkyl (e.g., C)_1-6Alkyl groups including, but not limited to, methyl, ethyl, isopropyl), alkoxy (e.g., C_1-6Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (e.g., fluoro, chloro, bromo), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, such as hydrogen; or R^1aAnd R^1bAtom attached theretoTogether form C_5-8Heterocycloalkyl optionally substituted by alkyl (e.g. C)_1-6Alkyl groups including, but not limited to, methyl, ethyl, isopropyl), alkoxy (e.g., C_1-6Alkoxy groups including, but not limited to, methoxy, ethoxy, propoxy, isopropoxy), halogen (e.g., fluoro, chloro, bromo), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl (e.g., C)_3-8Cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), heterocyclyl, aryl and heteroaryl, and R^1aAnd R^1bNot hydrogen at the same time.

In some embodiments, R in-D in the conjugate of antibody-eribulin or derivative thereof^1aSelected from methyl.

In some embodiments, the conjugate of antibody-eribulin or derivative thereof wherein-D is

In some embodiments, the conjugate of antibody-eribulin or derivative thereof Pc- (L-D)_kWherein k is selected from an integer or decimal from 1 to 10.

In another aspect, the present disclosure also provides an antibody-eribulin or derivative thereof conjugate having the structure shown in the following formula:

wherein, in the conjugate of the antibody-eribulin or derivative thereof, D is selected from

In some embodiments, the linker is stable extracellularly, such that the antibody-eribulin or derivative thereof conjugate remains intact when present in an extracellular environment, but is capable of cleavage upon internalization in a cell, such as a cancer cell. In some embodiments, when the antibody-eribulin or derivative thereof conjugate enters a cell expressing an antigen specific for the antibody portion thereof, the eribulin or derivative drug portion is cleaved from the antibody portion, and the cleavage releases an unmodified form of eribulin or a derivative thereof. In some embodiments, the linker comprises a cleavable moiety positioned such that no portion of the linker or the antibody moiety remains bound to the eribulin derivative after cleavage.

In some embodiments, the cleavable moiety in the linker is a cleavable peptide moiety. In some embodiments, the antibody-eribulin or derivative thereof conjugate comprising a cleavable peptide moiety exhibits a lower level of aggregation, an improved antibody-to-drug ratio, increased targeted killing of cancer cells, decreased off-target killing of non-cancer cells, and/or a higher drug loading (p) relative to a conjugate comprising the antibody-eribulin or derivative thereof in place of the cleavable moiety. In some embodiments, the addition of a cleavable moiety increases cytotoxicity and/or potency relative to a non-cleavable linker. In some embodiments, the increased potency and/or cytotoxicity is in cancers that express moderate levels of an antigen (e.g., IL-4R) targeted by the antibody moiety of the antibody-eribulin or derivative thereof conjugate. In some embodiments, the cleavable peptide moiety is capable of being cleaved by an enzyme, and the linker is a linker that is capable of being cleaved by the enzyme. In some embodiments, the enzyme is a cathepsin and the linker is a linker that is cleavable by a cathepsin. In certain embodiments, a linker that is cleavable by an enzyme (e.g., a linker that is cleavable by a cathepsin) exhibits one or more of the improved properties described above, as compared to alternative cleavage mechanisms.

In some embodiments, the linker comprises an amino acid unit, e.g., comprising a peptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, such as valine-citrulline (Val-Cit), alanine-asparagine (Ala-Asn), glycine-lysine (Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valine-phenylalanine (Val-Phe), or glycine-phenylalanine-glycine (Gly-Phe-Gly).

In some embodiments, the linker comprising an amino acid unit in the antibody-eribulin or derivative thereof of the present disclosure is selected from the group consisting of:

in some embodiments, the amino acid unit comprises valine-citrulline (Val-Cit). In some embodiments, an ADC comprising Val-Cit exhibits increased stability, reduced off-target cell killing, increased targeted cell killing, lower aggregation levels, and/or higher drug loading relative to an ADC comprising a replacement amino acid unit or a replacement cleavable moiety.

In another aspect, some embodiments provide linkers comprising a cleavable sulfonamide moiety, which linkers are capable of cleavage under reducing conditions.

In some embodiments, the linker comprises a cleavable disulfide moiety, the linker being cleavable under reducing conditions.

In another aspect, the linker in the antibody conjugates of the present disclosure comprises at least one spacer unit that attaches eribulin derivative D to the cleavable moiety. In some implementations, the connector includes a spacing unit attached to D.

In some embodiments, the spacer unit comprises p-aminobenzyloxycarbonyl (PAB),

in some embodiments, the spacer unit comprises:

wherein Z is₁～Z₅Optionally selected from carbon atoms or nitrogen atoms; r¹⁴Selected from the group consisting of alkyl, cycloalkyl, aryl and heteroaryl, each independently optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; r is¹¹And R¹²Each independently selected from hydrogen, deuterium and C_1-6Alkyl radical, C_3-6Cycloalkyl groups such as hydrogen; or, R¹¹And R¹²Together with the carbon atom to which they are attached form C_3-6A cycloalkyl group; x is selected from-O-or-NH-; l is selected from an integer between 1 and 4;

q is V-E, V-E providing a glycosidic bond cleavable by an intracellularly located glycosidase, E being selected from-O-, -S-or-NR¹³-，R¹³Selected from hydrogen or methyl, further, V is selected from

Wherein R is¹⁵Selected from-COOH or CH₂And (5) OH. In some embodiments, V is selected from-COOH.

In some embodiments, the spacer unit comprises:

in some embodiments, the spacer unit is selected from the following moieties:

-(CR^aR^b)_m1-O(CR^aR^b)_m2-CR⁸R⁹-C(O)-，

-(CR^aR^b)_m1NH-(CR^aR^b)_m2-CR⁸R⁹-C(O)-，

-(CR^aR^b)_m1O-CR⁸R⁹(CR^aR^b)_m2-，

-(CR^aR^b)_m1OCR⁸R⁹-C(O)-，

-(CR^aR^b)_m1-O-(CR^aR^b)_m2c (O) -or- (CR^aR^b)_m1-S-(CR^aR^b)_m2-CR⁸R⁹-C(O)-，

Wherein R is^aAnd R^bAre the same or different and are each independently selected from hydrogen, deuterium atoms, halogen or alkyl groups; r⁸Selected from hydrogen, C_3-6Cycloalkylalkyl or C_3-6A cycloalkyl group; r⁹Selected from hydrogen, haloalkyl or C_3-6Cycloalkyl groups such as hydrogen; or, R⁸And R⁹Together with the carbon atom to which they are attached form C_3-6A cycloalkyl group; m1 and m2 are each independently selected from 0, 1,2 or 3.

In some embodiments, the spacer unit is selected from:

-(CH₂)₃-C(O)-、-CH₂-O-CH₂-C(O)-、-(CH₂)₂-O-CH₂-C(O)-、

in another aspect, the antibody of the disclosure, eribulin or a derivative thereof, wherein L-D is a chemical moiety represented by the formula:

-Str-(Pep)-Sp-D

str is a stretcher unit covalently linked to Pc,

sp is a space unit, and Sp is a space unit,

pep is selected from an amino acid unit, a disulfide moiety, a sulfonamide moiety, or the following non-peptidic chemical moieties:

wherein W is-NH-heterocycloalkyl-or heterocycloalkyl; y is heteroaryl, aryl, -C (O) C_1-6Alkylene radical, C_2-6Alkenyl radical, C_1-6Alkylene or-C_1-6alkylene-NH-; each R²Is independently selected from C_1-10Alkyl radical, C_2-10Alkenyl, (C)_1-10Alkyl) NHC (NH) NH₂Or (C)_1-10Alkyl) NHC (O) NH₂；R³And R⁴Each independently is H, C_1-10Alkyl radical, C_2-10Alkenyl, arylalkyl, heteroarylalkyl, or R³And R⁴Together may form C_3-7A cycloalkyl group; r⁵And R⁶Each independently is C_1-10Alkyl radical, C_2-10Alkenyl, arylalkyl, heteroarylalkyl, (C)_1-10Alkyl) OCH₂-, or R⁵And R⁶Together may form C_3-7A cycloalkyl ring.

In some embodiments, Y in the antibody-eribulin or derivative thereof is selected from the group consisting of:

in another aspect, the antibody-eribulin or derivative thereof wherein Str is selected from a chemical moiety represented by the formula:

wherein R is⁷Selected from the group consisting of-W1-C (O) -, -C (O) -W1-C (O) -, (CH)₂CH₂O)_p1C(O)-、(CH₂CH₂O)_p1CH₂C(O)-、(CH₂CH₂O)_p1CH₂CH₂C (O) -, wherein W1 is selected from C_1-8Alkylene radical, C_1-8Alkylene-cycloalkyl or a linear 1 to 8 atom heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C is_1-8Each of alkyl, cycloalkyl, and linear heteroalkyl is independently optionally further substituted with one or more substituents selected from the group consisting of halogen, deuterium, hydroxy, cyano, amino, alkyl, haloalkyl, deuterated alkyl, alkoxy, and cycloalkyl;

L¹is selected from-NR¹⁰(CH₂CH₂O)_p1CH₂CH₂C(O)-、-NR¹⁰(CH₂CH₂O)_p1CH₂C(O)-、-S(CH₂)_p1C(O)-、-(CH₂)_p1C (o) -or a chemical bond, wherein p 1is an integer from 1 to 20, such as a chemical bond; p 1is an integer from 1 to 20, R¹⁰Selected from the group consisting of hydrogen atoms, alkyl groups, haloalkyl groups, deuterated alkyl groups, and hydroxyalkyl groups.

In some embodiments, C_1-8Alkylene-cycloalkyl radicals selected from methylene-cyclohexyl

Ethylene-cyclohexane radical

Methylene-cyclopentyl group

In some embodiments, the linker may comprise at least one polyethylene glycol (PEG) moiety. PEG moieties can, for example, comprise- (PEG)_p1-，

Wherein p 1is an integer from 1 to 20, e.g.

(PEG)₂；

(PEG)₄；

(PEG)₅。

In some embodiments, the spacer unit in the linker comprises (PEG)₂. In some embodiments, despite the shorter linker length, relative to the inclusion of a longer spacer unit: (a)For Example (PEG)₈) Comprising a shorter spacer unit (e.g. (PEG)₂) The ADCs of (a) show lower aggregation levels and/or higher drug loading.

In some embodiments, R in Str of the antibody-eribulin or derivative thereof⁷Is selected from C_1-6Alkylene radical C (O) -, - (CH)₂-CH₂O)₂C(O)-、-(CH₂-CH₂O)₂CH₂C(O)-、-(CH₂-CH₂O)₂CH₂CH₂C(O)-、-(CH₂-CH₂O)₃C (O) -and- (CH)₂-CH₂O)₄C(O)-。

In some embodiments, the linker L in the antibody-eribulin or derivative thereof comprises: maleimide- (PEG)₂-Val-Cit, maleimide- (PEG)₆-Val-Cit, maleimide- (PEG)₈-Val-Cit, maleimide- (PEG)₄-CH₂CH₂C (O) -Val-lys, maleimide- (CH)₂)₅-Val-Cit, maleimide- (CH)₂)₅-Val-lys, maleimide- (CH)₂)₅-Gly-Gly-Phe-Gly, maleimide- (PEG)₂-Ala-Ala-Asn, maleimide- (PEG)₆-Ala-Ala-Asn, maleimide- (PEG)₈-Ala-Ala-Asn, maleimide- (PEG)₄-triazole- (PEG)₃-sulfonamides, maleimides- (PEG)_2-CH₂CH₂C (O) -Val-lys, maleimide- (PEG)₄-triazole- (PEG)₃Sulfonamides or Mal- (PEG)₄-triazole- (PEG)₃-a disulfide.

In another aspect, some embodiments provide that the antibody-eribulin or derivative thereof wherein Str is selected from a chemical moiety represented by the formula:

wherein R is⁸Is selected from C_1-10Alkylene radical, C_2-10Alkenyl, (C)_1-10Alkylene) O-, N (R)^d)-(C_2-6Alkylene) -N (R)^d) And N (R)^d)-(C_2-6Alkylene groups); and each R^dIndependently is H or C₁-C₆An alkyl group.

In some embodiments, L-D in the antibody-drug conjugate is represented by a formula selected from the group consisting of:

wherein R is²Is C₁-C₆Alkyl, (C)₁-C₆Alkyl) NHC (NH) NH₂Or (C)₁-C₆Alkyl) NHC (O) NH₂；

Wherein R is²Is C₁-C₆Alkyl radical, C₂-C₆Alkenyl, (C)₁-C₆Alkyl) NHC (NH) NH₂Or (C)₁-C₆An alkane;

wherein R is²Is C₁-C₆Alkyl, (C)₁-C₆Alkyl) NHC (NH) NH₂Or (C)₁-C₆Alkyl) NHC (O) NH₂(ii) a And R is⁴And R⁵Together form C₃-C₇A cycloalkyl ring.

In other embodiments, the antibody-eribulin or derivative thereof of the present disclosure is represented by the formula:

wherein R is²Is selected from C₁-C₆alkyl-NH₂、(C₁-C₆Alkyl) NHC (NH) NH₂Or (C)₁-C₆Alkyl) NHC (O) NH₂K is an integer or decimal from 1 to 10; p2 is selected from an integer between 2 and 6;

wherein R is²Is C₁-C₆alkyl-NH₂、(C₁-C₆Alkyl) NHC (NH) NH₂Or (C)₁-C₆Alkyl) NHC (O) NH₂And R is⁵And R⁶Form C₃-C₇A cycloalkyl ring, k being selected from an integer or decimal from 1 to 10; p2 is selected from an integer of 2-6.

In another aspect, some embodiments provide an antibody-eribulin or a derivative thereof represented by the formula:

wherein R is⁸Selected from hydrogen, C_3-6Cycloalkylalkyl or C_3-6Cycloalkyl groups such as hydrogen; r⁹Selected from hydrogen, haloalkyl or C_3-6Cycloalkyl groups such as hydrogen; or, R⁸And R⁹Together with the carbon atom to which they are attached form C_3-6A cycloalkyl group; k is an integer or decimal from 1 to 10; p2 is selected from an integer between 2 and 6;

wherein R is⁸Selected from hydrogen, C_3-6Cycloalkylalkyl or C_3-6Cycloalkyl groups such as hydrogen; r⁹Selected from hydrogen, haloalkyl or C_3-6Cycloalkyl groups such as hydrogen; or, R⁸And R⁹Together with the carbon atom to which they are attached form C_3-6A cycloalkyl group; and k is selected from an integer or decimal from 1 to 10; p1 is selected from 2,4, 6 or 8; p3 is selected from 0, 1 or 2;

k is selected from an integer or decimal from 1 to 10; p2 is selected from an integer between 2 and 6;

k is selected from an integer or decimal from 1 to 10; p1 is selected from 2,4, 6 or 8; p3 is selected from 0, 1 or 2;

k is selected from an integer or decimal from 1 to 10; p1 is selected from 2,4, 6 or 8;

k is selected from an integer or decimal from 1 to 10; p2 is selected from 2,4, 6 or 8;

k is an integer or decimal from 1 to 10; p2 is selected from 2,4, 6 or 8;

k is an integer or decimal from 1 to 10; p2 is selected from 2,4, 6 or 8;

k is an integer or less from 1 to 10Counting; p2 is selected from 2,4, 6 or 8;

k is an integer or decimal from 1 to 10; p1 is selected from 2,4, 6 or 8; p3 is selected from 0, 1 or 2;

wherein R is⁸Selected from hydrogen, haloalkyl or C_3-6Cycloalkyl groups such as hydrogen; r⁹Selected from hydrogen, haloalkyl or C_3-6Cycloalkyl groups such as hydrogen; or, R⁸And R⁹Together with the carbon atom to which they are attached form C_3-6A cycloalkyl group; k is selected from an integer or decimal from 1 to 10; p2 is selected from an integer between 2 and 6;

wherein R is⁸Selected from hydrogen, haloalkyl or C_3-6Cycloalkyl groups such as hydrogen; r⁹Selected from hydrogen, haloalkyl or C_3-6Cycloalkyl groups such as hydrogen; or, R⁸And R⁹Together with the carbon atom to which they are attached form C_3-6A cycloalkyl group; k is an integer or decimal from 1 to 10; p2 is selected from an integer between 2 and 6;

wherein R is⁸Selected from hydrogen, haloalkyl or C_3-6Cycloalkyl groups such as hydrogen;R⁹selected from hydrogen, haloalkyl or C_3-6Cycloalkyl groups such as hydrogen; or, R⁸And R⁹Together with the carbon atom to which they are attached form C_3-6A cycloalkyl group; and k is selected from an integer or decimal from 1 to 10; p1 is selected from 2,4, 6 or 8; p3 is selected from 0, 1 or 2;

wherein R is⁸Selected from hydrogen, haloalkyl or C_3-6Cycloalkyl groups such as hydrogen; r⁹Selected from hydrogen, haloalkyl or C_3-6Cycloalkyl groups such as hydrogen; or, R⁸And R⁹Together with the carbon atom to which they are attached form C_3-6A cycloalkyl group; and k is selected from an integer or decimal from 1 to 10; p1 is selected from 2,4, 6 or 8; p3 is selected from 0, 1 or 2.

In some such embodiments, the antibody-eribulin and derivatives thereof, are represented by the formula:

wherein K is selected from an integer or decimal from 1 to 10; further, R in D^1aFor example from methyl, R^1bFor example from hydrogen.

The present disclosure also provides compounds of formula d (eribulin),

or a tautomer, mesomer, racemate, enantiomer, diastereomer, deutero-isomer, or mixture thereof, or a pharmaceutically acceptable salt thereof,

wherein R is^1aSelected from hydrogen, alkyl (e.g. C)_1-6Alkyl groups including, but not limited to, methyl, ethyl, isopropyl), cycloalkyl (e.g., C)_3-8Cycloalkyl groups, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, each independently optionally selected from alkyl (e.g., C)_1-6Alkyl including but not limited to methyl, ethyl, isopropyl), alkoxy (e.g., C)_1-6Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (e.g., fluoro, chloro, bromo), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, for example, methyl; r^1bSelected from hydrogen, alkyl (e.g. C)_1-6Alkyl including but not limited to methyl, ethyl, isopropyl), alkoxy, cycloalkyl (e.g., C)_3-8Cycloalkyl groups, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, each independently optionally selected from alkyl (e.g., C)_1-6Alkyl groups including, but not limited to, methyl, ethyl, isopropyl), alkoxy (e.g., C_1-6Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (e.g., fluoro, chloro, bromo), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, e.g., hydrogen, methyl; or R^1aAnd R^1bTogether with the carbon atom to which they are attached form C_5-8Heterocycloalkyl optionally substituted by alkyl (e.g. C)_1-6Alkyl groups including, but not limited to, methyl, ethyl, isopropyl), alkoxy (e.g., C_1-6Alkoxy radicals, including but not limited to methoxyAlkyl, ethoxy, propoxy, isopropoxy), halogen (e.g. fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl (e.g. C)_3-8Cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), heterocyclyl, aryl and heteroaryl, and R^1aAnd R^1bNot hydrogen at the same time.

In some embodiments, R in the compound of formula D (Eribulin)^1aAnd R^1bEach independently selected from C_1-6Alkyl groups including but not limited to methyl, ethyl, isopropyl.

In some embodiments, R in the compound of formula D (Eribulin)^1aIs selected from C_1-6Alkyl groups including but not limited to methyl, ethyl, isopropyl; r^1bSelected from hydrogen.

In some embodiments, R in the compound of formula D (Eribulin)^1aAnd R^1bTogether with the carbon atom to which they are attached form C_6-8A heterocycloalkyl group.

In some embodiments, the compound of formula d (eribulin) is:

in some embodiments, the compound of formula d (eribulin) is:

in some embodiments, the compound of formula d (eribulin) is:

the present disclosure also provides compounds of formula DZ (Eribulin),

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof,

wherein R is^1aSelected from hydrogen, alkyl (e.g. C)_1-6Alkyl groups including, but not limited to, methyl, ethyl, isopropyl), cycloalkyl (e.g., C)_3-8Cycloalkyl groups, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, each independently optionally selected from alkyl (e.g., C)_1-6Alkyl groups including, but not limited to, methyl, ethyl, isopropyl), alkoxy (e.g., C_1-6Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (e.g., fluoro, chloro, bromo), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, such as methyl; r^1bSelected from hydrogen, alkyl (e.g. C)_1-6Alkyl groups including, but not limited to, methyl, ethyl, isopropyl), cycloalkyl (e.g., C)_3-8Cycloalkyl groups, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), alkoxy, aryl and heteroaryl, each independently optionally selected from alkyl (e.g., C)_1-6Alkyl groups including, but not limited to, methyl, ethyl, isopropyl), alkoxy (e.g., C_1-6Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (e.g., fluoro, chloro, bromo), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, such as hydrogen; or R^1aAnd R^1bTogether with the carbon atom to which they are attached form C_5-8Heterocycloalkyl optionally substituted by alkyl (e.g. C)_1-6Alkyl groups including, but not limited to, methyl, ethyl, isopropyl), alkoxy (e.g., C_1-6Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (e.g., fluoro, chloro, bromo), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl (e.g., C)_3-8CycloalkanesGroups including, but not limited to, cyclopropyl, cyclopentyl, or cyclohexyl), heterocyclyl, aryl, and heteroaryl; and R is^1aAnd R^1bNot hydrogen at the same time;

y is selected from-O (CR)^aR^b)_m2-CR⁸R⁹-C(O)-、-NH-(CR^aR^b)_m2-CR⁸R⁹-C(O)-、-O-CR⁸R⁹(CR^aR^b)_m2-、-OCR⁸R⁹-C(O)-、-O(CR^aR^b)_m2C (O) -or-S- (CR)^aR^b)_m2-CR⁸R⁹-C (O) -, wherein R^aAnd R^bAre the same or different and are each independently selected from hydrogen, deuterium atoms, halogen or alkyl groups; r⁸Selected from hydrogen, C_3-6Cycloalkylalkyl or C_3-6A cycloalkyl group; r⁹Selected from hydrogen, haloalkyl or C_3-6Cycloalkyl groups such as hydrogen; or, R⁸And R⁹Together with the carbon atom to which they are attached form C_3-6A cycloalkyl group; m2 is selected from 0, 1,2 or 3.

In some embodiments, R in the compound of formula DZ (Eribulin)^1aAnd R^1bEach independently selected from C_1-6Alkyl groups including but not limited to methyl, ethyl, isopropyl.

In some embodiments, R in the compound of formula DZ (Eribulin)^1aIs selected from C_1-6Alkyl groups including but not limited to methyl, ethyl, isopropyl; r^1bSelected from hydrogen.

In some embodiments, R in the compound of formula DZ (Eribulin)^1aAnd R^1bTogether with the carbon atom to which they are attached form C_6-8A heterocycloalkyl group.

In some embodiments, the compound represented by dz (eribulin) is:

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereofThe compound (I) or a pharmaceutically acceptable salt thereof,

wherein: r is⁸Selected from hydrogen, C_3-6Cycloalkylalkyl or C_3-6A cycloalkyl group; r is⁹Selected from hydrogen, haloalkyl or C_3-6Cycloalkyl groups such as hydrogen; or, R⁸And R⁹Together with the carbon atom to which they are attached form C_3-6A cycloalkyl group; m2 is selected from 0, 1,2 or 3.

In some embodiments, the compound dz (eribulin) is selected from the group consisting of:

in another aspect, some embodiments provide compounds represented by DZ (Eribulin) that may contain one or more asymmetric centers, e.g.

Can be that

In the above embodiments, Pc is an anti-IL-4R antibody or antigen-binding fragment thereof of any of the present disclosure; for example, an anti-IL-4R antibody or antigen-binding fragment thereof as described in the examples; for example, a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 28 and the heavy chain as set forth in SEQ ID NO: 29, or an antibody comprising a light chain as set forth in SEQ ID NO: 30 and the heavy chain as set forth in SEQ ID NO: 31, an antibody to the light chain shown in seq id no.

In some embodiments, the conjugate of the antibody-eribulin or derivative thereof of the present disclosure includes a tautomer, mesomer, racemate, enantiomer, diastereomer, deuteron, or mixture thereof.

Compounds and methods for their preparation in CN202010073671.6 are incorporated herein in their entirety.

Antibody-drug (MMAE or derivatives thereof) conjugates

The present disclosure provides a novel MMAE analog/derivative, which is a compound represented by the general formula (d (MMAE)):

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein:

R¹-R⁶selected from the group consisting of hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy, and cycloalkyl;

R⁷selected from the group consisting of hydrogen atoms, alkyl groups, alkoxy groups, and cycloalkyl groups;

R⁸-R¹¹any two of which form a cycloalkyl group, the remaining two groups being optionally selected from hydrogen atoms, alkyl groups and cycloalkyl groups;

R¹²selected from a hydrogen atom or an alkyl group;

R¹³-R¹⁵selected from hydrogen, hydroxyl, alkyl, alkoxy or halogen;

R¹⁶selected from aryl or heteroaryl, said aryl or heteroaryl being optionally further substituted by substituents selected from hydrogen atoms, halogen, hydroxy, alkyl, alkoxy and cycloalkyl.

In some embodiments of the disclosure, the compound of formula (D) (MMAE) is of formula (D) (MMAE)₁) A compound shown in the formula (I):

R⁹and R¹⁰Form a cycloalkyl group;

R²-R⁸，R¹¹-R¹⁶as defined in formula (D).

In some embodiments of the present disclosure, the compound of formula (d) (mmae)) is:

another aspect of the present disclosure relates to an antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate comprises a structure represented by the formula (-d (mmae)):

R²-R⁶selected from the group consisting of hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy, and cycloalkyl;

R¹²selected from a hydrogen atom or an alkyl group;

R¹³-R¹⁵selected from hydrogen, hydroxyl, alkyl, alkoxy or halogen;

R¹⁶selected from aryl or heteroaryl, said aryl or heteroaryl being optionally further substituted by a substituent selected from the group consisting of hydrogen, halogen, hydroxy, alkyl, alkoxy and cycloalkyl;

the wavy line indicates a hydrogen atom, either covalently attached to a linker unit or to an antibody that binds to an antigen expressed by the target cell.

In some embodiments of the disclosure, the antibody-MMAE or derivative thereof conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate packageContaining formula (-D (MMAE)₁) The structure shown is as follows:

wherein:

R⁹and R¹⁰Form a cycloalkyl group;

wave line, R²-R⁸，R¹¹-R¹⁶As defined in general formula (D) (MMAE)).

In some embodiments of the disclosure, the conjugate of antibody-MMAE or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the conjugate of antibody-MMAE or a derivative thereof comprises a structure of formula:

In some embodiments of the present disclosure, the antibody-MMAE or its derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is an antibody-drug conjugate represented by the general formula (Pc-L-d (MMAE)):

wherein:

R²-R¹⁶as defined in general formula (D) (MMAE);

n is an integer or decimal from 1 to 10;

pc is an anti-IL-4R antibody or antigen-binding fragment thereof of the disclosure; l is a linker unit.

In some embodiments of the present disclosure, the conjugate of the antibody-MMAE or the derivative thereof, or the pharmaceutically acceptable salt or solvate thereof, is a conjugate of the antibody-MMAE or the derivative thereof represented by the general formula (Pc-L-D1), or a pharmaceutically acceptable salt or solvate thereof:

wherein:

R²-R¹⁶as defined in the general formula (-D (MMAE));

pc, L, n are as defined in the general formula (Pc-L-D) (MMAE).

In some embodiments of the disclosure, the conjugate of antibody-MMAE or derivative thereof, or pharmaceutically acceptable salt or solvate thereof, is a conjugate of antibody-MMAE or derivative thereof represented by the general formula:

pc, L, n are as defined in the general formula (Pc-L-D) (MMAE).

In some embodiments of the disclosure, the conjugate of antibody-MMAE or derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein n is an integer or decimal from 1 to 8; for example, an integer or decimal number from 1 to 6.

In some embodiments of the disclosure, the conjugate of antibody-MMAE or derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the linker unit-L-is-Y-L¹-L²-L³-L⁴，

Y is a stretching unit selected from

Or a chemical bond, X₁Selected from hydrogen, alkyl, alkoxy, aryl or halogen, X₂Selected from the group consisting of alkylene optionally further selected from one or more of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkylSubstituted with a plurality of substituents;

L¹is a stretching unit selected from- (succinimide-3-yl-N) -W-C (O) -, -CH₂-C(O)-NR¹⁷-W-C (O) -or-C (O) -W-C (O) -, wherein W is selected from C_1-8Alkyl radical, C_1-8Alkyl-cycloalkyl or a linear 1 to 8 atom heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C is_1-8Alkyl, cycloalkyl and linear heteroalkyl are each independently optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

L²is selected from-NR¹⁸(CH₂CH₂O)p¹CH₂CH₂C(O)-、-NR¹⁸(CH₂CH₂O)p¹CH₂C(O)-、-S(CH₂)p¹C (O) -or a chemical bond, wherein p¹Is an integer from 1 to 20; for example, a chemical bond;

L³is a peptide residue consisting of 2 to 7 amino acids, for example from valine, citrulline, methylvaline; wherein the amino acid is optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuteroalkyl, alkoxy, and cycloalkyl;

R¹⁷and R¹⁸Are the same or different and are each independently selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

L⁴is an extender unit, such as PAB.

In some embodiments of the disclosure, the conjugate of antibody-MMAE or derivative thereof, or pharmaceutically acceptable salt or solvate thereof, wherein Y is selected from

In some embodiments of the disclosure, the conjugate of antibody-MMAE or derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein L¹Selected from the group consisting of- (succinimidyl-3-yl-N) - (CH)₂)s¹-C (O) -, wherein s¹Is an integer from 2 to 8; for example, is

In some embodiments of the disclosure, the conjugate of antibody-MMAE or derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein L³Is a dipeptide amino acid unit, for example from valine-citrulline.

In some embodiments of the disclosure, the conjugate of antibody-MMAE or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the linker unit-L-is selected from the group consisting of:

wherein the a-terminal is connected with the antibody and the b-terminal is connected with the drug.

In some embodiments of the disclosure, the conjugate of antibody-MMAE or derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, is selected from the following structural formulae:

wherein:

n is an integer or decimal from 1 to 10;

pc is an anti-IL-4R antibody or antigen-binding fragment thereof of the disclosure; for example, an anti-IL-4R antibody or antigen-binding fragment thereof of the examples, for example, an antibody comprising an amino acid sequence as set forth in SEQ ID NO: 17 and the heavy chain as set forth in SEQ ID NO: 18, or an antibody comprising a light chain as set forth in SEQ ID NO: 47 and the heavy chain as set forth in SEQ ID NO: 45, or a light chain as set forth in seq id no.

Another aspect of the present disclosure relates to a method of preparing a compound of formula (d) (mmae)) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, comprising the steps of:

deprotection reaction of the general formula (DA) (MMAE)) to obtain a compound represented by the general formula (D) (MMAE)),

wherein: r²-R¹⁶As defined in formula (D).

Another aspect of the present disclosure relates to a compound as shown below:

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, are useful as intermediates for preparing antibody-drug conjugates of the present disclosure.

Another aspect of the present disclosure relates to a method of preparing compound 2(MMAE) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, comprising the steps of:

the compound 1(MMAE) and the compound 2a (MMAE) are subjected to condensation reaction to obtain a compound 2 (MMAE).

Another aspect of the present disclosure relates to a method of preparing an antibody-drug conjugate represented by the general formula (Pc-L-d (mmae)) or a pharmaceutically acceptable salt or solvate thereof, comprising the steps of:

after reducing Pc, carrying out coupling reaction with a compound to obtain a compound shown as a general formula (ADC) (MMAE) -1);

wherein Pc and n are defined in the general formula (Pc-L-D) (MMAE).

For the purpose of synthesizing the MMAE and the diffractometer thereof, the following synthesis technical scheme is adopted in the disclosure:

the first scheme is as follows:

a method of treating a compound of the general formula (d) (mmae)) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, comprising the steps of:

deprotection reaction of the general formula (DA) (MMAE) under basic condition to obtain a compound represented by the general formula (D) (MMAE),

wherein: r²-R¹⁶As defined in the general formula (D) (MMAE)).

The reagents that provide basic conditions include organic bases including, but not limited to, triethylamine, diethylamine, N-methylmorpholine, pyridine, piperidine, N-diisopropylethylamine, N-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide, or potassium tert-butoxide, and inorganic bases including, but not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, and lithium hydroxide; such as diethylamine.

Scheme II:

a process for the preparation of compound 2(MMAE) of the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, comprising:

the compound (1(MMAE)) and the compound (2 a) (MMAE)) are subjected to a condensation reaction under an alkaline condition by adding a condensing agent, thereby obtaining a compound 2.

The reagents that provide basic conditions include organic bases including, but not limited to, triethylamine, diethylamine, N-methylmorpholine, pyridine, piperidine, N-diisopropylethylamine, N-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide, or potassium tert-butoxide, and inorganic bases including, but not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, and lithium hydroxide, such as N, N-diisopropylethylamine.

The condensing agent is selected from 4- (4, 6-dimethoxy-1, 3, 5-triazine-2-yl) -4-methyl morpholine chloride salt, 1-hydroxybenzotriazole and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N, N '-dicyclohexylcarbodiimide, N, N' -diisopropylcarbodiimide, O-benzotriazol-N, N, N ', N' -tetramethyluronium tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazol, O-benzotriazol-N, N, N ', N' -tetramethyluronium hexafluorophosphate, 2- (7-azobenzotriazol) -N, N, n ', N' -tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, for example 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt or 1-hydroxybenzotriazole and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, for example 1-hydroxybenzotriazole.

And a third scheme is as follows:

the disclosed method for an antibody-drug conjugate represented by the general formula (Pc-L-D) or a pharmaceutically acceptable salt or solvate thereof, comprises the steps of:

after reducing Pc, carrying out coupling reaction with a compound 2(MMAE) to obtain a compound shown as a general formula (ADC (MMAE) -1); reducing agents such as TCEP, in particular, for example, reducing disulfide bonds on antibodies;

wherein Pc and n are defined in the general formula (Pc-L-D) (MMAE).

EXAMPLES Compounds

The present disclosure provides ligand-drug conjugates, or pharmaceutically acceptable salts or solvates thereof, selected from the group consisting of:

wherein Pc is any anti-IL-4R antibody or antigen-binding fragment thereof of the present disclosure, and n is an integer or decimal from 1 to 10.

In some embodiments, Pc is an anti-IL-4R antibody or antigen-binding fragment thereof in embodiments of the disclosure, e.g., an antibody comprising an amino acid sequence as set forth in SEQ ID NO: 17 and the heavy chain as set forth in SEQ ID NO: 18, or an antibody comprising a light chain as set forth in SEQ ID NO: 47 and the heavy chain as set forth in SEQ ID NO: 45, n is an integer or decimal between 1 and 6.

In some embodiments, the drug is one or more small molecule cytotoxic agents. Cytotoxic agents, also known as cytotoxic drugs, are chemical molecules that strongly destroy normal growth of tumor cells. Examples of cytotoxic agents include, but are not limited to, toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin; alkaloids, such as paclitaxel, vinorelbine, docetaxel, camptothecins; metabolites such as gemcitabine, cytarabine, tegafur, methotrexate; antibiotics, such as epirubicin, pirarubicin, idarubicin, mitomycin, mitoxantrone; alkylating agents, such as ifosfamide, dacarbazine; platinum agents, such as cisplatin, oxaliplatin; radioisotope (e.g. At)²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²And radioactive isotopes of Lu); a nucleolytic enzyme.

Pharmaceutical composition

The present disclosure further provides a pharmaceutical composition, the active ingredient of which comprises a drug conjugate, or a compound, or a pharmaceutically acceptable salt or solvate thereof, of an anti-IL-4R antibody or antigen-binding fragment thereof as described above, for use as a medicament.

The pharmaceutical compositions of the present disclosure may contain, in addition to the active ingredient, one or more pharmaceutically acceptable excipients, diluents or carriers (vehicles). The pharmaceutical composition may contain 0.1 to 99% by weight of the active ingredient.

The present disclosure further provides the use of any one or a combination of the following in the manufacture of a medicament: an anti-IL-4R antibody or antigen-binding fragment thereof according to the present disclosure, an antibody-drug conjugate of an anti-IL-4R antibody or antigen-binding fragment thereof, a pharmaceutical composition according to the present disclosure; wherein the antibody or antigen-binding fragment thereof or drug conjugate thereof is used for treating and/or preventing or delaying progression of a proliferative disease; the proliferative disorder may be a cancer or a tumor; for example, wherein the cancer or tumor is a cancer or tumor associated with IL-4R expression. The cancer or tumor is selected from the group consisting of prostate cancer, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphoma (e.g., hodgkin's lymphoma, non-hodgkin's lymphoma, or recurrent anaplastic large cell lymphoma), lung cancer, kidney cancer, liver cancer (e.g., small cell lung cancer and non-small cell lung cancer), colorectal cancer, pancreatic cancer, gastric cancer, leukemia (e.g., acute lymphocytic leukemia, acute myelocytic leukemia, acute promyelocytic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), and brain cancer, central nervous system tumors.

In some embodiments, the central nervous system tumor comprises a glioma, a glioblastoma, an astrocytoma, a medulloblastoma, a craniopharyngioma, an ependymoma, a pinealoma, a hemangioblastoma, an acoustic neuroma, an oligodendroglioma, a hemangioma, a meningioma, a neuroblastoma, a retinoblastoma, an adult blastoma.

In some embodiments, the central nervous system tumor is a glioblastoma.

In some embodiments, the glioblastoma is a relapsed or refractory glioblastoma.

In some embodiments, the glioblastoma expresses O6-methylguanine-DNA methyltransferase (O6-methylguanine-DNA methylhhransferase, MGMT) as positive or negative.

The dosage of a compound (such as an antibody-drug conjugate or composition of the disclosure) used in the treatment and/or prevention of the disclosure will generally vary with the severity of the disease, the weight of the subject, and the relative efficacy of the compound. As a general guide, a suitable unit dose may be 0.1mg to 1000 mg.

As is well known to those skilled in the art, the dosage of a drug administered depends on a variety of factors, including, but not limited to: the activity of the particular compound used, the age of the subject, the weight of the subject, the health of the subject, the behavior of the subject, the diet of the subject, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, and the like, can be verified according to well-known treatment protocols.

Description of the drawings:

FIG. 1is a schematic diagram of the structure of an exemplary antibody drug conjugate 25G7-C1 (anti-IL-4R antibody 25G7+ an analog of irinotecan).

FIG. 2 flow cytometry is used to detect IL-4R expression level in glioblastoma multiforme (GBM) cell line. Constructing human IL-4R stable-transfer LN229, U251 and U87 glioma cell strains by using a lentivirus system, detecting the IL-4R overexpression level by using a flow cytometry method, and using a contrast as a corresponding cell which is not transfected with a human IL-4R construct.

FIG. 3.25G 7-C1 results of in vitro cytotoxicity assays on LN229-IL4R, U251-IL4R, and control glioma cells.

FIG. 4 in vitro cytotoxicity assay results of (DAR3.52, DAR5.86)25G7-C1 at HEK293-IL4R (HEK 293 cells overexpressing IL 4R) and control cells for different DAR values.

FIG. 5.25G 7-C1 shows the results of comparison of in vivo efficacy in U87-MG subcutaneously inoculated nude mouse tumor model. The dose administered was 1, 3mpk (i.t.) or 1mpk administered intravenously (i.v.).

FIG. 6.25G 7-C1 shows the results of comparison of in vivo efficacy in LN229-IL4R (LN 229 cells overexpressing IL-4R) subcutaneously inoculated nude mouse tumor model. The dose administered was 1, 3mpk (i.t.) or 1mpk administered intravenously (i.v.).

Detailed Description

Term(s) for

In order that the disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

This disclosure incorporates the disclosure of patent document WO2020/038454A1 in its entirety.

"human IL 4R" (hIL-4R) means a human cytokine receptor that specifically binds to interleukin-4 (IL-4), IL-4R α. hIL-4R is intended to encompass various forms of molecules of IL-4R at various stages in vivo, such as, but not limited to, molecules produced by the IL-4R gene during amplification, replication, transcription, splicing, processing, translation, modification, e.g., precursor IL-4R, mature IL-4R, naturally occurring IL-4R splice variants, modified IL-4R, or fragments thereof.

"antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., IgM, IgD, IgG, IgA and IgE, with their corresponding heavy chains being the μ, δ, γ, α and ε chains, respectively. The same class of Ig can be divided into different subclasses according to the differences of amino acid composition of the hinge region and the number and position of disulfide bonds of heavy chains, for example, IgG can be divided into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. Each of the five classes of Ig may have either a kappa chain or a lambda chain.

The antibody light chain may further comprise a light chain constant region comprising a human or murine kappa, lambda chain light chain constant region or variant thereof.

The antibody heavy chain may further comprise a heavy chain constant region comprising a human or murine IgG1, IgG2, IgG3, IgG4 heavy chain constant region or a variant thereof.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, being variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region). The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) is composed of 3 CDR regions and 4 FR regions, arranged sequentially from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain refer to light chain complementarity determining region 1(LCDR1), light chain complementarity determining region 2(LCDR2), and light chain complementarity determining region 3(LCDR 3); the 3 CDR regions of the heavy chain refer to heavy chain complementarity determining region 1(HCDR1), heavy chain complementarity determining region 2(HCDR2) and heavy chain complementarity determining region 3(HCDR 3).

Antibodies include murine, chimeric, humanized, fully human antibodies, which may be recombinantly obtained, e.g., may be affinity matured recombinant fully human antibodies.

"recombinant fully human antibodies" include fully human antibodies prepared, expressed, created or isolated by recombinant methods, the techniques and methods involved being well known in the art, such as (1) antibodies isolated from transgenes of human immunoglobulin genes, transchromosomal animals (e.g., mice), or hybridomas prepared therefrom; (2) antibodies isolated from host cells transformed to express the antibodies, such as transfectomas; (3) antibodies isolated from a library of recombinant combinatorial human antibodies; and (4) antibodies prepared, expressed, created or isolated by splicing human immunoglobulin gene sequences to other DNA sequences, and the like. Such recombinant human antibodies comprise variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as occur during antibody maturation.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria susceptible to transformation include members of the family Enterobacteriaceae (Enterobacteriaceae), such as strains of Escherichia coli (Escherichia coli) or Salmonella (Salmonella); bacillaceae (Bacillus) such as Bacillus subtilis; pneumococcus (Pneumococcus); streptococcus (Streptococcus) and Haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (chinese hamster ovary cell line) and NS0 cells.

"murine antibody" is herein a monoclonal antibody to human IL-4R prepared according to the knowledge and skill in the art. Preparation is accomplished by injecting the subject with the IL-4R antigen (or epitope-containing polypeptide), and then isolating hybridomas that express antibodies having the desired sequence or functional properties. In some embodiments, the murine antibody that binds human IL-4R, or an antigen binding fragment thereof, may further comprise a light chain constant region of a murine kappa, lambda chain, or variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3, or IgG4, or variant thereof.

"fully human antibodies" include antibodies having variable and constant regions of human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "fully human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., "humanized antibodies").

"humanized antibodies," also known as CDR-grafted antibodies (CDR-grafted antibodies), refer to antibodies produced by grafting non-human CDR sequences into the framework of the variable regions of a human antibody. Can overcome the strong immune response reaction induced by the chimeric antibody because of carrying a large amount of heterologous protein components. To avoid a decrease in activity associated with a decrease in immunogenicity, the human antibody variable regions may be subjected to minimal back-mutation to maintain activity.

"chimeric antibody" is an antibody obtained by fusing a variable region of a murine antibody and a constant region of a human antibody, and can reduce an immune response induced by the murine antibody. Establishing a chimeric antibody, selecting and establishing a hybridoma secreting a mouse-derived specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to needs, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into a human vector, and finally expressing a chimeric antibody molecule in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of the human antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, for example comprising the heavy chain constant region of human IgG2 or IgG4, or IgG1 that is free of ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

"antigen-binding fragment" refers to Fab fragments, Fab 'fragments, F (ab') 2 fragments, Fv fragments that bind to human IL-4R, scFv fragments, and polypeptides or proteins comprising the fragments, which have antigen-binding activity. The "antigen binding fragment" comprises one or more CDR regions of an antibody described herein. The Fv fragment contains the variable regions of the antibody heavy and light chains, but lacks the constant region, and has the smallest antibody fragment with the entire antigen-binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. Two antibody variable regions can also be joined into a single polypeptide chain using different linkers, called single chain antibodies (scFv) or single chain fv (scFv).

The term "single chain antibody", "single chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) joined by a linker (or connecting peptide). Such scFv molecules can have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, e.g.variants comprising 1-4 repeats (Holliger et al (1993), Proc. Natl. Acad. Sci. USA90: 6444-. Other linkers useful in the present disclosure are described by Alfthan et al (1995), Protein Eng.8: 725-.

The term "antibody framework" refers to a portion of a variable domain, VL or VH, that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain. It is essentially a variable domain without CDRs.

"binding to IL-4R" refers to the ability to interact with human IL-4R (or epitopes, fragments thereof). The term "antigen binding site" herein refers to a three-dimensional spatial site recognized by an antibody or antigen binding fragment herein.

An "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed by adjacent amino acids, or non-adjacent amino acids by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after denaturing solvent treatment. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods of determining what epitope is bound by a given antibody are well known in the art and include immunoblot and immunoprecipitation detection assays, and the like. Methods of determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.

By "specifically binds," "selectively binds," and "specifically binds" is meant that the antibody binds to an epitope on a predetermined antigen. Typically, when using recombinant human IL-4R as the analyte and an antibody as the antibody, as determined by Surface Plasmon Resonance (SPR) techniques in an instrument, the antibody is present at a level of about less than 10^-7M or an even smaller equilibrium dissociation constant (KD) binds to a predetermined antigen and its affinity for binding to the predetermined antigen is that it is in or close phase with the predetermined antigenNon-specific antigens other than the antigen of interest (e.g., BSA, etc.) bind with at least twice the affinity. The term "antibody recognizing an antigen" is used interchangeably herein with the term "specifically binding antibody".

"Cross-reactive" refers to the ability of an antibody herein to bind to IL-4R from a different species. For example, an antibody herein that binds human IL-4R may also bind IL-4R of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens, or binding or functional interactions with cells that physiologically express IL-4R in binding assays (e.g., SPR and ELISA). Methods of determining cross-reactivity include standard binding assays as described herein, such as Surface Plasmon Resonance (SPR) analysis, or flow cytometry.

By "neutralizing" or "blocking" antibody is meant an antibody whose binding to hIL-4R results in inhibition of hIL-4 and/or hIL-13 biological activity. Such inhibition of hIL-4 and/or IL-13 bioactivity can be assessed by measuring one or more hIL-4 and/or hIL-13 bioactivity indicators well known in the art, such as hlL-4 and/or hIL-13-induced cell activation and hIL-4 binding to hIL-4R. See, for example, CN 103739711A. "inhibiting growth" (e.g., reference to a cell) is intended to include any measurable decrease in cell growth.

"inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to stimulation (i.e., passive or adaptive) of an immune response to a particular antigen. The term "induction" with respect to induction of CDC or ADCC refers to stimulation of a specific direct cell killing mechanism.

"ADCC (antibody-dependent cell-mediated cytotoxicity)", antibody-dependent cell-mediated cytotoxicity (Fc-mediated cytotoxicity), refers to the direct killing of Fc receptor-expressing cells by recognition of the Fc fragment of an antibody against antibody-coated target cells. The ADCC effector function of an antibody may be reduced or eliminated by modification of the Fc-fragment of the IgG. The modification refers to mutation in the heavy chain constant region of an antibody, such as N297A, L234A, L235A selected from IgG 1; IgG2/4 chimeras, F235E for IgG4, or L234A/E235A mutations.

Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as the antibody test technical guide of cold spring harbor, chapters 5-8 and 15. For example, mice can be immunized with human IL-4R or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods. The antibodies or antigen-binding fragments described herein are genetically engineered to incorporate one or more human FRs in CDR regions of non-human origin. Human FR germline sequences can be obtained from the website of ImmunoGeneTiCs (IMGT) or from the immunoglobulin journal, 2001ISBN 012441351.

The engineered antibody or antigen-binding fragment can be prepared and purified using conventional methods. As an example, cDNA sequences encoding the heavy and light chains may be cloned and recombined into a GS expression vector. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. The sequence of the humanized antibody is inserted into a corresponding expression vector by using a molecular cloning technology, and the corresponding humanized antibody can be obtained by using an HEK293 cell expression system for expression and production. As an example, mammalian-like expression systems can lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones were obtained by expressing antibodies that specifically bind to antigens of human origin. Positive clones were expanded in bioreactor serum-free medium to produce antibodies. The antibody-secreting culture medium can be purified and collected by conventional techniques. The antibody can be concentrated by filtration by a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

The antibody may be a monoclonal antibody (mAb) which refers to an antibody obtained from a single clonal cell line, not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombinant technology, phage display technology, synthetic techniques (e.g., CDR-grafting), or other known techniques.

The antibody may be a monospecific, bispecific or multispecific antibody. Multispecific antibodies may exhibit specificity for different epitopes of a target peptide, or may also comprise antigen binding domains that exhibit specificity for more than one target peptide. The human anti-IL-4R antibody may be linked to, or co-expressed with, another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding, or other means) to one or more other molecules (e.g., another antibody or antigen-binding fragment) to produce a bispecific or multispecific antibody having at least one binding specificity.

"administration," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administering," and "treating" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering", "administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo by an agent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treating" is meant administering a therapeutic agent, such as a composition comprising any one of the antibody-drug conjugates herein, either internally or externally to a subject having (suspected of having, susceptible to) one or more disease symptoms for which the therapeutic agent has a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the subject or population being treated, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically useful degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce a desired therapeutic effect in the subject. Whether a disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although the present embodiments (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of the target disease in a single subject, they should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test method known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckhere-Terpsra test, and Wilcoxon test.

The term "naturally occurring" as applied to an object refers to the fact that the object may be found in nature. For example, a polypeptide sequence or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man is naturally-occurring.

An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the subject, the method and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

"exogenous" refers to a substance produced outside an organism, cell, or human body depending on the background. "endogenous" refers to a substance produced in an organism, cell, or human body according to background.

"homology" or "identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, two sequences are 60% homologous if there are 6 matches or homologies at 10 positions in the two sequences when the sequences are optimally aligned. In general, comparisons are made when aligning two sequences to obtain the greatest percentage of homology. As used herein, "at least 85% sequence identity" means that the variant has at least 85% homology, in some embodiments at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology to the parent sequence; in some specific embodiments, it has 90%, 95%, or 99% or more; in other embodiments, it has at least 95% sequence homology. The amino acid sequence having at least 85% sequence identity comprises a sequence obtained by one or more amino acid deletion, insertion or substitution mutations in a parent sequence.

As used herein, the terms "cell," "cell line," "cell strain," and "cell culture" are used interchangeably and all such terms include progeny thereof. Thus, the terms "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of passages. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. The term includes mutant progeny that have the same function or biological activity as the parent cell, which was selected from the originally transformed cell.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that antibody heavy chain variable regions may, but need not, be present.

"pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof and other ingredients, such as a physiologically/pharmaceutically acceptable carrier (vehicle) or excipient. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. The vectors disclosed herein are capable of autonomous replication in a host cell into which they have been introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of a host cell upon introduction into the host cell so as to be replicated along with the host genome (e.g., non-episomal mammalian vectors).

The term "excipient" is an addition in a pharmaceutical formulation other than the active ingredient, such as a binder, filler, disintegrant, lubricant in a tablet; base portion in semisolid formulations ointments, creams; preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, tonicity adjusting agents, colorants and the like in liquid preparations can all be referred to as excipients.

The term "diluent" is also known as filler, and its primary purpose is to increase the weight and volume of the tablet. The addition of the diluent not only ensures a certain volume size, but also reduces the dosage deviation of the main components, improves the compression moldability of the medicament, and the like. When the tablet contains oily components, absorbent is added to absorb the oily components to keep the oily components in a dry state, so as to facilitate the preparation of the tablet. Such as starch, lactose, inorganic salts of calcium, microcrystalline cellulose, and the like.

The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to salts of the antibody-drug conjugates of the disclosure, or salts of the compounds described in the disclosure, which salts are safe and effective for use in the body of a mammal and possess the requisite biological activity, and the antibody-antibody drug conjugate compounds of the disclosure contain at least one amino group and thus can form salts with acids, non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulphate, hydrogen sulphate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate.

The term "solvate" refers to an antibody-drug conjugate compound of the present disclosure that forms a pharmaceutically acceptable solvate with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, DMSO, ethyl acetate.

The compounds of the present disclosure may contain one or more asymmetric centers and may therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms which may be defined in terms of absolute stereochemistry as (R) -or (S) -or (D) -or (L) -for amino acids. The present disclosure includes all possible isomers as well as racemic and optically pure forms thereof. Optically active (+) and (-), (R) -and (S) -or (D) -and (L) -isomers can be prepared using chiral synthons or chiral reagents, or can be prepared using conventional methods such as chromatography and fractional crystallization. Conventional methods for the preparation/separation of the individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral High Pressure Liquid Chromatography (HPLC). When a compound described herein contains an olefinic double bond or other center of geometric asymmetry, unless otherwise indicated, it is meant that the compound includes both E and Z geometric isomers. Moreover, all tautomeric forms are also intended to be included.

"stereoisomers" refers to compounds of the same atomic composition bonded by the same bond but having different three-dimensional structures, which are not interchangeable. Various stereoisomers and mixtures thereof are contemplated in the present disclosure and include "enantiomers," which refers to two stereoisomers whose molecules are non-superimposable mirror images of each other.

"tautomer" refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. Tautomers of any of the compounds are included in the present disclosure.

The term "linker, linker unit, linker or linking fragment" refers to a chemical structure fragment or bond that is linked to a ligand at one end and a drug at the other end, and may be linked to a drug after linking to other linkers.

The joint may comprise one or more joint members. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), and those derived from conjugation to a linker reagent: N-Succinimidyl 4- (2-pyridylthio) valerate ("SPP"), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1 carboxylate ("SMCC", also referred to herein as "MCC"), and N-succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB"). Linkers may include stretcher units, spacer units, amino acid units and extender units, and may be synthesized by methods known in the art, such as those described in US2005-0238649A 1. The linker may be a "cleavable linker" that facilitates release of the drug in the cell. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, Cancer Research 52: 127-.

The term "amino acid unit" means that the following formula Y can be substituted if an extender unit is present_RThe carbonyl group in (2) is linked to an extender unit, and if there is no extender unit, Y may be linked_RAn amino acid directly linked to a cytotoxic drug, in the embodiments of the disclosure the amino acid unit is represented as-K_k-：

-K_kIs a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or decapeptide, each of the K-units independently has the following structural formula K_aOr K_bK is an integer between 0 and 10Number:

wherein:

r in the above amino acid unit₂₃is-H or methyl;

R₂₄is H, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH₂OH、-CH(OH)CH₃、-CH₂CH₂SCH₃、-CH₂CONH₂、-CH₂COOH、-CH₂CH₂CONH₂、-CH₂CH₂COOH、-(CH₂)₃NHC(＝NH)NH₂、-(CH₂)₃NH₂、-(CH₂)₃NHCOCH₃、-(CH₂)₃NHCHO、-(CH₂)₄NHC(＝NH)NH₂、-(CH₂)₄NH₂、-(CH₂)₄NHCOCH₃、-(CH₂)₄NHCHO、-(CH₂)₃NHCONH₂、-(CH₂)₄NHCONH₂、-CH₂CH₂CH(OH)CH₂NH₂2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,

R₂₅is-aryl-, -alkyl-aryl-, -cycloalkyl-, -alkyl-cycloalkyl-, -cycloalkyl-alkyl-, -alkyl-cycloalkyl-alkyl-, -heterocyclyl-, -alkyl-heterocyclyl-, -heterocyclyl-alkyl-, -alkyl-heterocyclyl-alkyl-, -aryl-, -alkyl-aryl-, -aryl-alkyl-, -alkyl-aryl-alkyl-, -heteroaryl-, -alkyl-heteroaryl-, -heteroaryl-alkyl-, -alkyl-heteroaryl-alkyl-.

In one embodiment, -K_k-is a dipeptide, preferably-valine-citrulline-, -phenylalanineAmino acid-lysine-or-N-methylvaline-citrulline-, more preferably-valine-citrulline-.

The term "stretch unit" refers to a chemical moiety having one end covalently linked to a ligand through a carbon atom and the other end linked to a cytotoxic drug through a sulfur atom.

The term "spacer unit" is a bifunctional structural fragment that can be used to couple a linker unit to a cytotoxic drug to form a ligand-cytotoxic drug conjugate in a manner that selectively links the cytotoxic drug to the linker unit.

The term "amino acid" refers to an organic compound that contains both amino and carboxyl groups in the molecular structure, and both the amino and carboxyl groups are directly attached to the-CH-structure. Is of the formula H₂NCHRCOOH. Amino acids are classified as alpha, beta, gamma, delta, epsilon … … -amino acids, depending on the position of the carbon atom to which the amino group is attached in the carboxylic acid. In the biological world, amino acids constituting natural proteins have their specific structural characteristics, that is, amino groups thereof are directly linked to α -carbon atoms, that is, α -amino acids including Glycine (Glycine), Alanine (Alanine), Valine (Valine), Leucine (leucin), Isoleucine (isoleucin), Phenylalanine (Phenylalanine), Tryptophan (Tryptophan), Tyrosine (Tyrosine), Aspartic acid (Aspartic acid), Histidine (Histidine), Asparagine (aspargine), Glutamic acid (Glutamic acid), Lysine (Lysine), Glutamine (Glutamine), Methionine (Methionine), Arginine (Arginine), Serine (Serine), Threonine (Threonine), Cysteine (Cysteine), Proline (Proline), and the like.

The term "extender unit" means that the amino acid unit, when present, can be conjugated to a cytotoxic drug, or ammoniaThe chemical structure that can be coupled to a cytotoxic drug via the carbonyl group on the YR in the absence of an amino acid unit. In the disclosed embodiments, the extension unit is represented by-Q_q-q is selected from 0, 1, 2.

In the disclosure, the extension unit is PAB, the structure is 4-iminobenzyl carbamoyl fragment, the structure is shown in the following formula, and the extension unit is connected with D,

abbreviations

Joint assemblies include, but are not limited to:

MC ═ 6-maleimidocaproyl, the structure is as follows:

Val-Cit or "vc" ═ valine-citrulline (exemplary dipeptides in protease cleavable linkers)

Citrulline ═ 2-amino-5-ureidopentanoic acid

PAB ═ p-aminobenzyloxycarbonyl ("exemplary self-immolative" linker component)

Me-Val-Cit ═ N-methyl-valine-citrulline (in which the linker peptide bond has been modified to prevent its cleavage by cathepsin B)

Mc (peg)6-OH ═ maleimidocaproyl-polyethylene glycol (attachable to the antibody cysteine)

SPP-N-Succinimidyl 4- (2-pyridylthio) pentanoate

SPDP ═ N-succinimidyl 3- (2-pyridyldithio) propionate

SMCC ═ succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate

IT is iminothiolane

PBS (phosphate buffered saline)

The term "antibody-drug conjugate," means that the antibody is linked to the biologically active drug via a stable linking unit. In the present disclosure, "antibody drug conjugate" (ADC) refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug via a stable linking unit.

The term "drug loading" refers to the average number of cytotoxic drugs loaded per ligand in the ADC and can also be expressed as the ratio of drug amount to antibody amount, and the drug loading can range from 1 to 20, preferably 1 to 10 cytotoxic drugs (D) attached per antibody (Pc). In embodiments of the present disclosure, the drug load is expressed as n or k, illustratively 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or an average of any two values. Preferably 1 to 10, more preferably 1 to 8, or 1 to 7, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 3, or 1 to 2, or 2 to 4, or 1 to 5, or 1 to 6, or 3 to 8, or 3 to 7, or 3 to 6, or 4 to 7, or 4 to 6, or 4 to 5. The average amount of drug per ADC molecule after the conjugation reaction can be identified by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assays, monoclonal antibody molecular size variant assays (CE-SDS) and HPLC characterization.

The monoclonal antibody molecular size variant determination method (CE-SDS) disclosed by the invention can be used for quantitatively determining the purity of a recombinant monoclonal antibody product by adopting a sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method according to the molecular weight under reducing and non-reducing conditions and according to a hair electrophoresis method (2015 edition Chinese pharmacopoeia 0542).

In one embodiment of the present disclosure, the cytotoxic drug is coupled to the N-terminal amino group of the ligand and/or the epsilon-amino group of the lysine residue via a linker unit, and generally, the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than the theoretical maximum.

The loading of the ligand cytotoxic drug conjugate can be controlled by the following non-limiting methods, including:

(1) controlling the molar ratio of the connecting reagent to the monoclonal antibody,

(2) the reaction time and the temperature are controlled,

(3) different reagents were selected.

Although the drug-to-antibody ratio has an exact value for a particular conjugate molecule (e.g., n in formula (I)), it is understood that when used to describe a sample containing many molecules, the value will often be an average value due to some degree of non-uniformity typically associated with the conjugation step. The average loading of the immunoconjugate (antibody-drug conjugate), referred to herein as the drug-to-antibody ratio or "DAR", is denoted by n, the average number of drug molecules coupled per antibody molecule. In some embodiments, n can be any value between 1 and 10, such as 2 and 8, or 2 and 6, or 1 and 6, or 4 and 6. In some embodiments, the DAR is about 1 to about 6, for example about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0. In some embodiments, at least 50% by weight of the sample is a compound having an average DAR ± 2, and in some embodiments, at least 50% of the sample is a conjugate containing an average DAR ± 1. In some embodiments, a DAR that is "about the value x" means that the measurement of the DAR can vary within ± 20% of the value x. In some embodiments, the antibody-drug conjugates of the present disclosure include a collection of antibody-drug conjugates within certain of the foregoing ranges.

Detection methods for DAR, for example, DAR values were extrapolated from LC-MS data for reduced and deglycosylated samples. LC/MS allows quantification of the average number of payload (drug moiety) molecules attached to the antibody in the ADC. HPLC separates the antibody into light and heavy chains, and also separates the Heavy (HC) and Light (LC) chains according to the number of linker-payload groups per chain. Mass spectral data enables identification of component species in a mixture, e.g., LC +1, LC +2, HC +1, HC +2, etc. From the average loading of the LC and HC chains, the average DAR of the ADC can be calculated. The DAR for a given immunoconjugate sample represents the average number of drug (payload) molecules attached to a tetrameric antibody containing two light chains and two heavy chains. Such as the DAR detection method in WO 2018142322.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing 1 to 20 carbon atoms, for example an alkyl group containing 1 to 12 carbon atoms, for example an alkyl group containing 1 to 10 carbon atoms, for example an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-dimethylpentyl, 2, 4-dimethylpentyl, 2-dimethylpentyl, 3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2, 3-dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 2-dimethylhexyl, 3-dimethylhexyl, 4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-dimethylpentyl, 2-dimethylhexyl, 3-dimethylpentyl, 2-ethylhexyl, 3-dimethylhexyl, 2-ethylhexyl, 2-dimethylhexyl, 2-ethylhexyl, 2-dimethylhexyl, 2-dimethylhexyl, 2-dimethylhexyl, 2-ethylhexyl, 2-ethyl, 2-2, 2-2, 2-2, or, 2, 2-diethylpentyl, n-decyl, 3-diethylhexyl, 2-diethylhexyl, and various branched isomers thereof. For example, lower alkyl having 1 to 6 carbon atoms, non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl and the like. Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, such as one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halo, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.

The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.

The term "alkylene" refers to a saturated straight or branched chain aliphatic hydrocarbon group having 2 residues derived from the parent alkane by removal of two hydrogen atoms from the same carbon atom or two different carbon atoms, and is a straight or branched chain group containing 1 to 20 carbon atoms, for example, an alkylene group containing 1 to 12 carbon atoms, for example, 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH)₂-), 1-ethylidene (-CH (CH)₃) -), 1, 2-ethylene (-CH)₂CH₂) -, 1-propylene (-CH (CH)₂CH₃) -), 1, 2-propylene (-CH)₂CH(CH₃) -), 1, 3-propylene (-CH)₂CH₂CH₂-) 1, 4-butylene (-CH₂CH₂CH₂CH₂-) and 1, 5-butylene (-CH)₂CH₂CH₂CH₂CH₂-) and the like. The alkylene groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, for example, with one or more substituents independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

The term "alkoxy" refers to-O- (alkyl) and-O- (unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituents are, for example, one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing from 3 to 20 carbon atoms, for example from 3 to 12 carbon atoms, for example from 3 to 10 carbon atoms, for example from 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups.

The term "heterocyclyl" refers to a saturated or partially unsaturated mono-or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms wherein one or more of the ring atoms is selected from nitrogen, oxygen, or S (O)_m(wherein m is an integer from 0 to 2) but excludes the ring moiety of-O-O-, -O-S-, or-S-S-, the remaining ring atoms being carbon. For example, 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; for example cycloalkyl rings contain 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclic groups include spiro, fused and bridged heterocyclic groups.

The term "spiroheterocyclyl" refers to a 5-to 20-membered polycyclic heterocyclic group in which one atom (referred to as the spiro atom) is shared between monocyclic rings, and in which one or more ring atoms is selected from nitrogen, oxygen, or S (O)_m(wherein m is an integer of 0 to 2) and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a completely conjugated pi-electron system. For example 6 to 14, for example 7 to 10. Spiro heterocyclic groups are classified into a mono-spiro heterocyclic group, a di-spiro heterocyclic group or a multi-spiro heterocyclic group according to the number of spiro atoms shared between rings, for example, a mono-spiro heterocyclic group and a di-spiro heterocyclic group. For example, a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered mono spiroheterocyclyl group. Non-limiting examples of spiro heterocyclic groups include:

the term "fused heterocyclyl" refers to a5 to 20 membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system, one or more rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron system in which one or more ring atoms is selected from nitrogen, oxygen or S (O)_m(wherein m is an integer of 0 to 2) and the remaining ring atoms are carbon. For example 6 to 14, for example 7 to 10. They may be classified as bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups, for example as bicyclic or tricyclic, for example as 5-or 6-membered bicyclic fused heterocyclic groups, depending on the number of constituent rings. Non-limiting examples of fused heterocyclic groups include:

the term "bridged heterocyclyl" refers to a5 to 14 membered polycyclic heterocyclic group in which any two rings share two atoms not directly attached which may contain one or more double bonds, but none of the rings have a fully conjugated pi-electron system in which one or more of the ring atoms is selected from nitrogen, oxygen or S (O)_m(wherein m is an integer of 0 to 2) and the remaining ring atoms are carbon. For example 6 to 14, for example 7 to 10. They may be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic groups, for example bicyclic, tricyclic or tetracyclic, for example bicyclic or tricyclic, depending on the number of constituent rings. Non-limiting examples of bridged heterocyclic groups include:

the heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring to which the parent structure is attached is heterocyclyl, non-limiting examples of which include:

and the like.

The heterocyclyl group may be optionally substituted or unsubstituted, and when substituted, the substituents are, for example, one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.

The term "aryl" refers to a 6 to 14 membered all carbon monocyclic or fused polycyclic (i.e., rings which share adjacent pairs of carbon atoms) group having a conjugated pi-electron system, for example, 6 to 10 membered, such as phenyl and naphthyl, such as phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, non-limiting examples of which include:

aryl groups may be substituted or unsubstituted, and when substituted, the substituents are, for example, one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl is, for example, 5 to 10 membered, for example 5 or 6 membered, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring joined together with the parent structure is a heteroaryl ring, non-limiting examples of which include:

heteroaryl groups may be optionally substituted or unsubstituted, and when substituted, substituents are, for example, one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

The term "amino protecting group" is intended to protect an amino group with a group that can be easily removed in order to keep the amino group unchanged when the rest of the molecule is subjected to a reaction. Non-limiting examples include 9-fluorenylmethyloxycarbonyl, t-butyloxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl and the like. These groups may be optionally substituted with 1 to 3 substituents selected from halogen, alkoxy or nitro. The amino protecting group is, for example, 9-fluorenylmethyloxycarbonyl.

The term "cycloalkylalkyl" means an alkyl group substituted with one or more cycloalkyl groups, for example with one cycloalkyl group, wherein alkyl is as defined above, and wherein cycloalkyl is as defined above.

The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.

The term "hydroxy" refers to an-OH group.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "amino" refers to-NH₂。

The term "nitro" means-NO₂。

The term "amido" refers to-C (O) N (alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl are as defined above.

The term "carboxylate" refers to-C (O) O (alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl are as defined above.

The disclosure also includes various deuterated forms of the compounds of formula (I). Each available hydrogen atom attached to a carbon atom may be independently replaced by a deuterium atom. The person skilled in the art is able to synthesize the deuterated forms of the compounds of the formula (I) with reference to the relevant literature. Commercially available deuterated starting materials can be used in preparing the deuterated forms of the compounds of formula (I), or they can be synthesized using conventional techniques using deuterated reagents including, but not limited to, deuterated boranes, trideuteroborane tetrahydrofuran solutions, deuterated lithium aluminum hydrides, deuterated iodoethanes, deuterated iodomethanes, and the like.

In the chemical structure of the compounds described in the present disclosure, a bond

Denotes an unspecified configuration, i.e. if chiral isomers are present in the chemical structure, the bond

Can be made of

Or

Or at the same time comprise

And

two configurations. In the chemical structure of the compounds described in the present disclosure, a bond

Not specifying a configuration, i.e. a bond

Can be in E or Z configuration, or contain both E and Z configurations.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "a heterocyclic group optionally substituted with an alkyl" means that an alkyl may, but need not, be present, and the description includes the case where the heterocyclic group is substituted with an alkyl and the heterocyclic group is not substituted with an alkyl.

The immunohistochemical scoring is a histological scoring method for processing immunohistochemical results, and the number of positive cells in each section and the staining intensity of the positive cells are converted into corresponding numerical values, so that the aim of semi-quantifying tissue staining is fulfilled. A score of 0 indicates no staining observed or membrane staining observed in less than 10% of the tumor cells; score +1 indicates that faint/barely detectable membrane staining was detected in greater than 10% of the tumor cells; for score +2, moderate complete membrane staining was observed in greater than 10% of tumor cells; a score of +3 indicates that strong complete membrane staining was observed in greater than 10% of the tumor cells. In the present disclosure, those samples with IL-4R expression of a score of 0 or +1 may be considered to not overexpress IL-4R, while those with a score of +2 or +3 may be considered to overexpress IL-4R.

Detailed Description

The following examples are presented for further description and are not intended to limit the scope of the invention.

The experimental methods in examples or test examples, in which specific conditions are not specified, are generally performed under conventional conditions or under conditions recommended by manufacturers of raw materials or commercial products. See Sambrook et al, molecular cloning, A laboratory Manual, Cold spring harbor laboratory; contemporary molecular biology methods, Ausubel et al, Greene publishing Association, Wiley Interscience, NY. Reagents of specific sources are not indicated, and are conventional reagents purchased in the market.

Example 1 preparation of exemplary ADC

The antibody was expressed and purified according to the method described in WO2020038454a1, and the heavy chain sequence of antibody 25G7 is as set forth in SEQ ID NO: 47, and the light chain sequence is shown as SEQ ID NO: shown at 45. The ADC of an irinotecan analogue with 25G7 (also known as 25G7-C1) (drug-antibody ratio DAR ═ 4) was prepared according to the method described in WO2020063676a1, the structure of 25G7-C1 being:

n is an integer or decimal from 1 to 10, for example, 25G7-C1 with n being 3.52 and 5.86 was synthesized in example 5. FIG. 1is a schematic structural diagram of 25G 7-C1. The foregoing patent application is incorporated herein by reference in its entirety.

Example 2 in vitro killing efficiency assay in GBM target cells

To determine the killing efficiency of 25G7-C1 in GBM target cells in vitro, we over-expressed human IL-4R in GBM cell lines U87, U251 and LN229 by using a lentivirus system in the case of relatively low expression of endogenous IL-4R in the GBM cell lines U87, U251 and LN229, and after screening by G418, we identified the effect of IL-4R over-expression by using flow cytometry (FIG. 2), which shows that IL-4R is stably over-expressed in the three cell lines.

The small molecule drug-antibody conjugate 25G7-C1 was tested for in vitro killing toxicity by the CTG (CELL TITER-GLO) method against IL-4R-overexpressed LN229, U251 glioma cells. Complete medium was prepared using DMEM medium with 10% FBS and 1% penicillin/streptomycin at 37 deg.C with 5% CO₂The incubator of (1) was used to culture the U251-IL4R and LN229-IL4R stable transgenic cell line and the U251 and LN229 control cells. Plating was performed in 96-well plates at a density of 4000 cells per well and 130ul of medium. After overnight, 20ul of gradient drug containing 25G7-C1, respectively, was added to allow cells to adhere completely and cultured in an incubator for 3 days. After three days, the 96-well Plate was taken out of the incubator and equilibrated to room temperature, 50ul of CTG reagent (CellTiter-Glo Luminescence, Promega, # G7573) was added to the cell suspension, mixed by a shaker for 2 minutes, allowed to stand at room temperature for 10 minutes, and the cell viability was measured by a Luminescence pattern built in an EnVision2105 Multimode Plate Reader (Perkinelmer). The absorbance results were converted to percentages and analyzed by Prism 8. The experimental results are shown in FIG. 3, compared with the control IL-4R naked antibody (antibody without connected small molecule drug), in LN229 and U251 with high IL-4R expression, 25G7-C1 has good in vitro killing activity; compared with naked toxin, 25G7-C1 pairsLN229 and U251 highly expressed by IL-4R exhibit specific killing activity.

In addition, we synthesized a small molecule toxin antibody conjugate drug 25G7-C1 with a DAR (drug-antibody ratio) value of 4 or 6 for interest, actually measured 3.52 and 5.86, and over-expressed human IL-4R in HEK293, and compared the in vitro toxicity of the different DAR values 25G7-C1 in HEK293-IL4R cells as well as control cells. The results of the experiment, as shown in fig. 4, show that increasing DAR values can increase the killing efficiency on target cells for 25G7-C1 drugs in a range of 0.458nM for the IC50 of 5.86 DAR 25G7-C1 drug in HEK293-IL4R, while 3.289nM for the IC50 of 3.52 DAR at the same time.

Example 3 in vivo efficacy of Targeted IL-4R drugs in GBM tumor models

To determine the in vivo efficacy of 25G7-C1 in the GBM tumor model, we first constructed a model of subcutaneous inoculation of U87-MG with IL-4R expression of 1+ (immunohistochemical score). Experimental mice were inoculated subcutaneously on the right back with 2X 10⁶U87-MG cells, which were resuspended in 1:1 PBS and matrigel (0.1 ml/cell), and tumor growth was observed periodically. When the average size of the subcutaneous inoculation tumor reaches about 100-150mm³Groups were made, 8 mice per group. Multiple site intratumoral administration, 25G7-C1 doses were 1mpk (i.t.), 3mpk (i.t.), or 1mpk (i.v.), administered once a week for a total of four times. After the start of the administration, the body weight and tumor size of the mice were measured twice a week, and the tumor volume was calculated by the formula: tumor volume (mm)³)＝1/2×(a×b²) (wherein a represents a long diameter and b represents a short diameter).

As shown in fig. 5 and table 2, 25G7-C1 showed tumor suppression effect at each dose, with the efficacy of intratumoral administration of 3mpk being significantly better and the tumor growth inhibition rate TGI being 65.4%.

TABLE 2 in vivo efficacy summary of targeted IL-4R drugs in the mouse glioma model

Note: s.c. subcutaneously; mpk mg/kg (milligrams per kilogram); i.t. intratumoral injection; i.v. intravenous injection; QW once per week. Denotes p <0.01, denotes p <0.001, ns denotes not significant.

Example 4 in vivo efficacy of Targeted IL-4R drugs in GBM tumor model (IL-4R 3+)

To further determine the in vivo efficacy of 25G7-C1 in the GBM tumor model, we modeled subcutaneous vaccination using an IL-4R overexpressed LN229 cell line (labeled LN229-IL4R, immunohistochemical score 3 +). Experimental mice were inoculated subcutaneously on the right back with 1X 10⁷LN229-IL4R cells, resuspended in 1:1 PBS (0.1 ml/cell), and the tumor growth was observed periodically. When the average size of the subcutaneous inoculation tumor reaches about 100-150mm³Groups were made, 6-7 mice per group. 25G7-C1 was administered at a dose of 1mpk (i.t.), 3mpk (i.t.), or 1mpk (i.v.), once a week for a total of four times, with no administration to the control group; tumor growth monitoring and calculation were the same as in example 3.

The results of the experiments, as shown in FIG. 6 and Table 3, show that in LN229-IL4R tumor model with high IL-4R expression (immunohistochemical score of 3+), TGI reached 99% at dose 3mpk (i.t.) for 25G7-C1, TGI reached 75% at dose 1mpk (i.t.) for 25G7-C1, and TGI reached 55.6% at dose 1mpk (i.v.) for 25G7-C1, indicating that the 25G7-C1 drug exhibited dose effects in glioma model with IL-4R 3+, and that intratumoral effects were superior to those of intravenous administration.

TABLE 3 summary of in vivo efficacy of targeted IL-4R drugs in the mouse glioma model

Note: s.c. subcutaneously; mpk mg/kg (milligrams per kilogram); i.t. intratumoral injection; i.v. intravenous injection; QW once per week. Denotes p <0.01, denotes p <0.001, and ns denotes not significant.

The use and welfare of the experimental animals in the present disclosure are performed in compliance with the provisions of the international committee for evaluation and approval of experimental animals (AAALAC). The health and death of the animals are monitored daily and routine examinations include observations of the effects of the test substances and drugs on the daily performance of the animals, such as behavioral activities, weight changes, physical signs of appearance, etc.

Although specific embodiments of the present disclosure have been described above, it will be appreciated by those skilled in the art that these are merely illustrative and that various changes or modifications may be made without departing from the principles and spirit of the invention. Accordingly, the scope of the disclosure is defined by the appended claims.

SEQUENCE LISTING

<110> Hengrui pharmaceutical Co., Ltd, Jiangsu

SHANGHAI SHENGDI PHARMACEUTICAL Co.,Ltd.

<120> anti-IL-4R antibody-drug conjugate and medical application

<150> CN202011529845.1

<151> 2020-12-22

<160> 47

<170> PatentIn version 3.5

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

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Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

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Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

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Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Leu Ile His Pro Asn Ser Asp Thr Thr Lys Phe Ser Glu Asn Phe

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Lys Thr Arg Ala Thr Leu Thr Ile Asp Lys Ser Ser Ser Thr Ala Tyr

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

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Ala Lys Ser Lys Ile Ile Thr Thr Ile Val Ala Arg His Trp Tyr Phe

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

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

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

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

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

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Ala Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val

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

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Thr Arg Gly Asn Lys Arg Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr

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

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Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

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Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

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Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

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

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

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Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

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

325 330 335

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

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Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

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Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

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Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

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Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

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His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

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

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

20 25 30

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

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

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

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Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Arg Ser Asn Pro Pro Met

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Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

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Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

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Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

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Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

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Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

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

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

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

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Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Leu Ile His Pro Asn Ser Asp Thr Thr Lys Phe Ser Glu Asn Phe

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Lys Thr Arg Val Thr Met Thr Ile Asp Thr Ser Ile Ser Thr Ala Tyr

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

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Ala Lys Ser Lys Ile Ile Thr Thr Ile Val Ala Arg His Trp Tyr Phe

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Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

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

130 135 140

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

145 150 155 160

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

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

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

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

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Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

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Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

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Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

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

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Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

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Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

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Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

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

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

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Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

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Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

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

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Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

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Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

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Leu Ser Leu Ser Pro Gly Lys

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

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Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Gly

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

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Gln Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

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

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Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Asn

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Glu Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

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

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

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

145 150 155 160

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

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

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

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

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Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val

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

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Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro

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

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

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Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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

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Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

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

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Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser

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Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Pro

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Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro

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

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

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Ile Gln Leu Pro

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

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

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

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Ala Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val

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

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

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

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

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

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Ala Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val

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

65 70 75 80

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

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

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

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Ala Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val

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

65 70 75 80

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

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115

<210> 28

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (hu25G7-VL-a)

<400> 28

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

1 5 10 15

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

20 25 30

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

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Arg Ser Asn Pro Pro Met

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 29

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (hu25G7-VL-b)

<400> 29

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

1 5 10 15

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

20 25 30

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

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Arg Ser Asn Pro Pro Met

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 30

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (hu25G7-VL-c)

<400> 30

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

1 5 10 15

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

20 25 30

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

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Arg Ser Asn Pro Pro Met

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 31

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (hu7B10-VH-a)

<400> 31

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile His Pro Asn Ser Asp Thr Thr Lys Phe Ser Glu Asn Phe

50 55 60

Lys Thr Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Ser Lys Ile Ile Thr Thr Ile Val Ala Arg His Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 32

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (hu7B10-VH-b)

<400> 32

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile His Pro Asn Ser Asp Thr Thr Lys Phe Ser Glu Asn Phe

50 55 60

Lys Thr Arg Val Thr Met Thr Ile Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Lys Ser Lys Ile Ile Thr Thr Ile Val Ala Arg His Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 33

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (hu7B10-VH-c)

<400> 33

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile His Pro Asn Ser Asp Thr Thr Lys Phe Ser Glu Asn Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Lys Ser Lys Ile Ile Thr Thr Ile Val Ala Arg His Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 34

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (hu7B10-VL-a)

<400> 34

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

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Gly

20 25 30

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

35 40 45

Gln Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

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

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Asn

85 90 95

Glu Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 35

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (hu7B10-VL-b)

<400> 35

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

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Gly

20 25 30

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

35 40 45

Gln Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

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

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Asn

85 90 95

Glu Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 36

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (hu7B10-VL-c)

<400> 36

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

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Gly

20 25 30

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

35 40 45

Gln Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Asp

50 55 60

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

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Asn

85 90 95

Glu Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 37

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (hu25G7-A LCVR)

<400> 37

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

1 5 10 15

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

20 25 30

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

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Arg Ala Tyr Pro Pro Met

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 38

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (LCDR1)

<400> 38

Arg Ala Ser Ser Ser Val Pro Tyr Met Tyr

1 5 10

<210> 39

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (LCDR2)

<400> 39

Leu Ala Ser Ser Arg Pro Ser

1 5

<210> 40

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (LCDR3)

<400> 40

Gln Gln Trp Arg Ala Tyr Pro Pro Met Leu Thr

1 5 10

<210> 41

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (hu25G7-B LCVR)

<400> 41

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Pro Gly Val Pro Pro Leu

20 25 30

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

35 40 45

Leu Ala Ser Ser Arg Pro Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Arg Ser Asn Pro Pro Met

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 42

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (LCDR1)

<400> 42

Arg Ala Ser Pro Gly Val Pro Pro Leu Ala

1 5 10

<210> 43

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (hu25G7-VH)

<400> 43

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val

50 55 60

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

65 70 75 80

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

85 90 95

Thr Arg Gly Asn Lys Arg Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 44

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (hu25G7 HC)

<400> 44

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val

50 55 60

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

65 70 75 80

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

85 90 95

Thr Arg Gly Asn Lys Arg Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

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

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

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

325 330 335

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

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 45

<211> 215

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (hu25G7-A LC)

<400> 45

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

1 5 10 15

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

20 25 30

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

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Arg Ala Tyr Pro Pro Met

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

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

180 185 190

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

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 46

<211> 215

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (hu25G7-B LC)

<400> 46

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Pro Gly Val Pro Pro Leu

20 25 30

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

35 40 45

Leu Ala Ser Ser Arg Pro Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Arg Ser Asn Pro Pro Met

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

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

180 185 190

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

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 47

<211> 448

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesized sequence (hu25G7-24-IgG1)

<400> 47

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val

50 55 60

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

65 70 75 80

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

85 90 95

Thr Arg Gly Asn Lys Arg Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

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

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

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

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

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

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

Claims

1. An antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, comprising:

anti-IL-4R antibody or antigen-binding fragment thereof, and

-one or more drug molecules;

wherein: the anti-IL-4R antibody or antigen-binding fragment thereof comprises any one selected from the group consisting of the following (I) to (IV):

(I) a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 3. the amino acid sequence of SEQ ID NO: 4 and SEQ ID NO: 5 HCDR1, HCDR2 and HCDR 3; and

a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 38. the amino acid sequence of SEQ ID NO: 7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown at 40;

(II) a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 11. the amino acid sequence of SEQ ID NO: 12 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown at 13; and

a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 42. SEQ ID NO: 39 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in fig. 8;

the drug molecule is selected from MMAE or a derivative thereof, irinotecan or a derivative thereof, and eribulin or a derivative thereof.

2. The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-IL-4R antibody is selected from any one of the following: murine, chimeric, fully human, and humanized antibodies;

preferably, the anti-IL-4R antibody or antigen-binding fragment thereof comprises FRs derived from human germline light chain IGKV3-11 x 01 or FRs at least 95% identical thereto; the FR preferably comprises a back mutation selected from one or more of 46P, 47W and 71Y; and/or

Comprises the FR derived from human germline heavy chain IGHV3-48 x 01 or a FR having at least 95% identity therewith; the FR preferably comprises a back mutation selected from one or more of 49A, 67S and 93T;

or preferably, said anti-IL-4R antibody or antigen-binding fragment thereof comprises FRs derived from human germline light chain IGKV2D-29 x 01 or FRs at least 95% identical thereto; preferably, the FR comprises a back mutation selected from 4L and/or 58I; and/or

Comprises the FR derived from human germline heavy chain IGHV1-2 x 02 or a FR having at least 95% identity therewith; the FR preferably comprises a back mutation selected from one or more of 69L, 71I, 73K and 94K.

3. The antibody-drug conjugate of any one of claims 1-2, or a pharmaceutically acceptable salt or solvate thereof, wherein: the anti-IL-4R antibody or antigen-binding fragment thereof comprises any one selected from the group consisting of the following (i) to (vi):

(i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 43 or an amino acid sequence substantially identical to SEQ ID NO: 43 amino acid sequence having at least 90%, 95%, 98%, 99% identity; and

a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 37 or an amino acid sequence substantially identical to SEQ ID NO: 37, amino acid sequence having at least 90%, 95%, 98%, 99% identity;

(ii) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence corresponding to SEQ ID NO: 9 an amino acid sequence having at least 90%, 95%, 98%, 99% identity; and

a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence corresponding to SEQ ID NO: 10 amino acid sequences having at least 90%, 95%, 98%, 99% identity;

(iii) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence substantially identical to SEQ ID NO: 1 amino acid sequence having at least 90%, 95%, 98%, 99% identity; and

a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence corresponding to SEQ ID NO: 2, an amino acid sequence having at least 90%, 95%, 98%, 99% identity;

(iv) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 43 or an amino acid sequence substantially identical to SEQ ID NO: 43 amino acid sequence having at least 90%, 95%, 98%, 99% identity; and

a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 41 or an amino acid sequence substantially identical to SEQ ID NO: 41 having at least 90%, 95%, 98%, 99% identity;

(v) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 25-27 or a sequence identical to SEQ ID NO: 25-27, or a sequence having at least 90%, 95%, 98%, or 99% identity; and

a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 28-30 or a sequence identical to SEQ ID NO: 28-30, having at least 90%, 95%, 98%, or 99% identity;

(vi) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31-33 or a sequence identical to SEQ ID NO: 31-33, or a sequence having at least 90%, 95%, 98%, or 99% identity; and

a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34-36 or a sequence identical to SEQ ID NO: 34-36, or a sequence that is at least 90%, 95%, 98%, or 99% identical;

preferably, the anti-IL-4R antibody or antigen-binding fragment comprises any one selected from the following (vii) to (xii):

(vii) the sequence is shown as SEQ ID NO: 43, and the sequence of the heavy chain variable region is shown as SEQ ID NO: 37;

(viii) the sequence is shown as SEQ ID NO: 9, and the sequence of the heavy chain variable region is shown as SEQ ID NO: 10;

(ix) the sequence is shown as SEQ ID NO: 1, and the sequence of the heavy chain variable region is shown as SEQ ID NO: 2;

(x) The sequence is shown as SEQ ID NO: 43, the sequence of which is shown in SEQ ID NO: 41;

(xi) The sequence is shown as SEQ ID NO: 25-27, and the sequence is as shown in SEQ ID NO: a light chain variable region as set forth in one of claims 28-30;

(xii) The sequence is shown as SEQ ID NO: 31-33, and the sequence is as shown in SEQ ID NO: 34-36.

4. The antibody-drug conjugate of any one of claims 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein: the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain constant region that is a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof; and/or

The antigen binding fragment is Fab, Fv, scFv, Fab 'or F (ab') 2;

preferably, the anti-IL-4R antibody or antigen-binding fragment thereof comprises an antibody selected from any one of (a) - (d):

(a) a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 44 or an amino acid sequence substantially identical to SEQ ID NO: 44, an amino acid sequence having at least 90%, 95%, 98%, or 99% identity; and

a light chain comprising the amino acid sequence set forth as SEQ ID NO: 45 or an amino acid sequence substantially identical to SEQ ID NO: 45, having at least 90%, 95%, 98%, or 99% identity;

(b) a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 19 or an amino acid sequence substantially identical to SEQ ID NO: 19, an amino acid sequence having at least 90%, 95%, 98%, or 99% identity; and

a light chain comprising the amino acid sequence set forth as SEQ ID NO: 20 or an amino acid sequence substantially identical to SEQ ID NO: 20 an amino acid sequence having at least 90%, 95%, 98% or 99% identity;

(c) a heavy chain comprising the amino acid sequence set forth as SEQ ID NO: 17 or an amino acid sequence corresponding to SEQ ID NO: 17, an amino acid sequence having at least 90%, 95%, 98%, or 99% identity; and

a light chain comprising the amino acid sequence set forth as SEQ ID NO: 18 or an amino acid sequence substantially identical to SEQ ID NO: 18 having at least 90%, 95%, 98% or 99% identity;

(d) a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 44 or an amino acid sequence substantially identical to SEQ ID NO: 44 having at least 90%, 95%, 98%, or 99% identity; and

a light chain comprising the amino acid sequence set forth as SEQ ID NO: 46 or an amino acid sequence substantially identical to SEQ ID NO: 46 have at least 90%, 95%, 98%, or 99% identity;

more preferably, the anti-IL-4R antibody or antigen-binding fragment thereof comprises any one selected from (e) - (i):

(e) the sequence is shown as SEQ ID NO: 44, the sequence of the heavy chain is shown as SEQ ID NO: 45, a light chain;

(f) the sequence is shown as SEQ ID NO: 47, the sequence of which is shown in SEQ ID NO: 45, a light chain;

(g) the sequence is shown as SEQ ID NO: 17, the sequence of the heavy chain is shown as SEQ ID NO: 18, a light chain;

(h) the sequence is shown as SEQ ID NO: 19, the sequence of the heavy chain is shown as SEQ ID NO: 20, a light chain;

(i) the sequence is shown as SEQ ID NO: 44, the sequence of the heavy chain is shown as SEQ ID NO: 46, or a light chain as shown.

5. The antibody-drug conjugate of any one of claims 1 to 4, which is represented by the general formula (Pc-L-Y-D) of formula (I):

wherein:

l is a linker unit;

pc is an anti-IL-4R antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 4;

y is selected from-O- (CR)^aR^b)_m-CR¹R²-C(O)-、-O-CR¹R²-(CR^aR^b)_m-、-O-CR¹R²-、-NH-(CR^aR^b)_m-CR¹R²-C (O) -and-S- (CR)^aR^b)_m-CR¹R²-C(O)-；R^aAnd R^bThe same or different, and each is independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, and a heterocyclic group; or, R^aAnd R^bTogether with the carbon atom to which they are attached form cycloalkyl and heterocyclyl radicals; r¹Selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, and heteroaryl; r²Selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, and heteroaryl; or, R¹And R²Together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group; or, R^aAnd R²Together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group; m is an integer of 0 to 4; n is 1 to 10, n is a decimal or integer, preferably, n is 2 to 8 or 5 to 9;

preferably, Y is-O- (CR)^aR^b)_m-CR¹R²-C(O)-；R^aAnd R^bAre the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, or an alkyl group; r¹Is a hydrogen atom, a haloalkyl group or C_3-6A cycloalkyl group; r²Selected from hydrogen atoms, haloalkyl radicals or C_3-6A cycloalkyl group; or, R¹And R²To which carbon atom is attachedTogether form C_3-6A cycloalkyl group; m is 0 or 1;

more preferably, Y is selected from:

wherein the O-terminal of Y is connected to the linker unit L.

6. The antibody-drug conjugate of claim 5, or a pharmaceutically acceptable salt or solvate thereof,

which is represented by the general formula (Pc-L-D1) of formula (II):

wherein:

R¹is a hydrogen atom, C_3-6Cycloalkylalkyl or C_3-6A cycloalkyl group;

R²selected from hydrogen atoms, haloalkyl radicals or C_3-6A cycloalkyl group; preferably a hydrogen atom;

m is 0 or 1;

n is an integer or decimal from 1 to 10, preferably n is a decimal or integer from 2 to 8 or from 3 to 8;

pc is an anti-IL-4R antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 4; l is a linker unit;

preferably, the linker unit-L-is-L¹-L²-L³-L⁴-，

L¹Selected from- (succinimidin-3-yl-N) -W-C (O) -, -CH₂-C(O)-NR³-W-C (O) -or-C (O) -W-C (O) -, wherein W is selected from C_1-8Alkyl radical, C_1-8Alkyl-cycloalkyl orA linear heteroalkyl group of 1 to 8 atoms, said heteroalkyl group containing 1 to 3 heteroatoms selected from N, O or S, wherein said C_1-8Alkyl, cycloalkyl and linear heteroalkyl are each independently optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

R⁶and R⁷Are the same or different and are each independently selected from the group consisting of hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuterated alkyl groups, and hydroxyalkyl groups;

more preferably, L¹Selected from the group consisting of- (succinimidyl-3-yl-N) - (CH)₂)s¹-C (O) -, - (succinimid-3-yl-N) -CH₂-cyclohexyl-C (O) -, - (succinimid-3-yl-N) - (CH)₂CH₂O)s²-CH₂CH₂-C(O)-、-CH₂-C(O)-NR³-(CH₂)s³-C (O) -or-C (O)-(CH₂)s⁴C (O) -, wherein s¹Is an integer from 2 to 8, s²Is an integer of 1 to 3, s³Is an integer from 1 to 8, s⁴Is an integer from 1 to 8;

L²is selected from-NR⁴(CH₂CH₂O)p¹CH₂C (O) -or a chemical bond, p¹Is an integer from 6 to 12;

L⁴is selected from-NR⁵(CR⁶R⁷)_t-，R⁵Selected from hydrogen atoms or alkyl radicals, R⁶And R⁷Identical or different and are each independently a hydrogen atom or an alkyl group, t is 1 or 2, preferably 2; and/or

L³Is a peptide residue consisting of 2 to 7 amino acids selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid; preferably a tetrapeptide residue; more preferably a glycine-phenylalanine-glycine tetrapeptide residue.

7. The antibody-drug conjugate of any one of claims 5-6, or a pharmaceutically acceptable salt or solvate thereof, wherein: the linker unit-L-is-L¹-L²-L³-L⁴-，

L¹Is composed of

s¹Is an integer from 2 to 8;

L²is a chemical bond;

L³is a tetrapeptide residue;

L⁴is-NR⁵(CR⁶R⁷)_t-，R⁵Selected from hydrogen atoms or alkyl radicals, R⁶And R⁷The same or different and each independently is a hydrogen atom or an alkyl group, t is 1 or 2;

preferably, the linker unit-L-is-L¹-L²-L³-L⁴-，

L¹Is- (succinimidin-3-yl-N) -CH₂-cyclohexyl-c (o) -;

L²is-NR⁴(CH₂CH₂O)₉CH₂C(O)-；

L³Is a tetrapeptide residue;

L⁴is-NR⁵(CR⁶R⁷)t-，R⁵Selected from hydrogen atoms or alkyl radicals, R⁶And R⁷The same or different and each independently is a hydrogen atom or an alkyl group, t is 1 or 2;

more preferably, said linker unit-L-, L thereof¹Terminal to antibody, L⁴And the end is connected with Y.

8. The antibody-drug conjugate of any one of claims 5 to 7, or a pharmaceutically acceptable salt or solvate thereof, wherein: -L-Y-is:

wherein L is¹Selected from the group consisting of- (succinimidyl-3-yl-N) - (CH)₂)s¹-C (O) -or- (succinimid-3-yl-N) -CH₂-cyclohexyl-c (o) -;

L³tetrapeptide residues that are GGFG;

R¹is a hydrogen atom, C_3-6Cycloalkylalkyl or C_3-6A cycloalkyl group;

R²selected from hydrogen atoms, haloalkyl radicals or C_3-6A cycloalkyl group;

s¹is an integer from 2 to 8;

m is an integer of 0 to 4;

preferably, -L-Y-is:

L²is-NR⁴(CH₂CH₂O)₉CH₂C(O)-；

L³Is a tetrapeptide residue of GGFG;

R¹is a hydrogen atom, C_3-6Cycloalkylalkyl or C_3-6A cycloalkyl group;

R²selected from hydrogen atoms, haloalkyl radicals or C_3-6A cycloalkyl group;

m is an integer of 0 to 4;

more preferably, -L-Y-is

L²Is a chemical bond;

L³is a tetrapeptide residue of GGFG;

R¹is a hydrogen atom, C_3-6Cycloalkylalkyl or C_3-6A cycloalkyl group;

R²selected from hydrogen atoms, haloalkyl radicals or C_3-6A cycloalkyl group;

s¹is an integer from 2 to 8; preferably 5;

m is an integer of 0 to 4.

9. The antibody-drug conjugate of any one of claims 1 to 8, which is of the general formula (Pc-L) of formula (IV), or a pharmaceutically acceptable salt or solvate thereof_a-Y-Dr) or a pharmaceutically acceptable salt or solvate thereof:

wherein:

w is selected from C_1-8Alkyl radical, C_1-8Alkyl-cycloalkyl or a linear 1 to 8 atom heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C is_1-8Alkyl, cycloalkyl and linear heteroalkyl are each independently optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

or, R¹And R²Together with the carbon atom to which they are attached formCycloalkyl or heterocyclyl;

R⁴and R⁵Are the same or different and are each independently selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

m is an integer of 0 to 4;

n is an integer or decimal from 1 to 10;

preferably, the antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof is of the general formula (Pc-L) of formula (V)_b-Y-Dr) or a pharmaceutically acceptable salt or solvate thereof:

wherein:

s¹is an integer from 2 to 8; preferably 5;

Pc、R¹、R²、R⁵～R⁷m and n are as defined in claim 8.

10. The antibody-drug conjugate of any one of claims 5 to 9, or a pharmaceutically acceptable salt or solvate thereof, wherein-L-Y-is selected from:

11. the antibody-drug conjugate of any one of claims 1-10, or a pharmaceutically acceptable salt or solvate thereof, selected from the following structural formulae:

wherein:

n is an integer or decimal from 1 to 10, preferably n is an integer or decimal from 1 to 6;

preferably, the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof is:

wherein Pc is the anti-IL-4R antibody or antigen-binding fragment thereof of claims 1-4, n is an integer or decimal from 1 to 10; preferably, Pc is the anti-IL-4R antibody or antigen-binding fragment thereof of claim 4, and n is an integer or decimal from 1 to 6.

12. Preparation of the general formula (Pc-L)_a-Y-D) or a pharmaceutically acceptable salt or solvate thereof, comprising reacting a compound of formula (i) with a pharmaceutically acceptable acid or baseThe following steps:

wherein:

W、L²、L³、R¹、R²、R⁵～R⁷m and n are as defined in claim 9.

13. A pharmaceutical composition comprising a therapeutically effective amount of an antibody-drug conjugate according to any one of claims 1 to 11, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

14. Use of an antibody-drug conjugate according to any one of claims 1-11, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to claim 13, in the manufacture of a medicament for the treatment and/or prevention of cancer or tumour;

preferably, the cancer or tumor is selected from any one or a combination of: prostate cancer, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer, kidney cancer, liver cancer, colorectal cancer, pancreatic cancer, gastric cancer, leukemia, and central nervous system tumors;

more preferably, the central nervous system tumor is selected from any one or a combination of the following: glioma, glioblastoma, neuroblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, meningioma, retinoblastoma;

further preferably, the central nervous system tumor is a glioblastoma, e.g. the central nervous system tumor is a recurrent or refractory glioblastoma, or a glioblastoma that is positive or negative for O6-methylguanine-DNA methyltransferase expression.