CN118667006A

CN118667006A - ROR 1-targeting antibody, antibody-coupled drug comprising same, preparation method and application

Info

Publication number: CN118667006A
Application number: CN202310271589.8A
Authority: CN
Inventors: 康小强; 赖寿鹏; 沈竞康; 孟韬; 马兰萍; 王昕�; 凌虹; 张鹏; 黄潇
Original assignee: Nanjing Wei Li Zhi Bo Biotechnology Co ltd; Shanghai Institute of Materia Medica of CAS
Current assignee: Nanjing Wei Li Zhi Bo Biotechnology Co ltd; Shanghai Institute of Materia Medica of CAS
Priority date: 2023-03-20
Filing date: 2023-03-20
Publication date: 2024-09-20
Also published as: WO2024193605A1

Abstract

The application provides an antibody targeting ROR1, an antibody coupling medicament containing the same, a preparation method and application. The ROR 1-targeting antibody or antigen binding fragment thereof has a heavy chain variable region, a light chain variable region, and a Complementarity Determining Region (CDR) as disclosed herein. The application also provides an antibody coupled drug comprising the ROR 1-targeting antibody or antigen binding fragment thereof, a preparation method and application thereof.

Description

ROR 1-targeting antibody, antibody-coupled drug comprising same, preparation method and application

Technical Field

The present invention relates to the field of medicine, and in particular relates to an antibody coupled drug targeting ROR1, a preparation method and application thereof.

Background

Receptor tyrosine kinase-like orphan receptor l (ROR 1) is a type I transmembrane receptor tyrosine kinase protein that is highly expressed during early embryonic development and plays an important role in a variety of physiological processes including regulating cell division, proliferation, migration, etc., involved in the generation of organs such as nerves, bones and blood vessels. During subsequent fetal development, the expression level of ROR1 gradually decreased, and the surface of normal pediatric and adult tissue cells was substantially free of ROR1 expression (SEMIN CANCER Biol 2014Vol.29, 21-31). However, among various tumor cells, solid tumor cells such as lung adenocarcinoma, breast cancer, ovarian cancer, melanoma, renal cancer, gastric cancer, colorectal cancer, pancreatic cancer (BMC cancer.2021Nov 11;21 (1): 1199.), and some hematological malignancies such as chronic lymphocytic leukemia (chronic lymphocytic leukemia, CLL), and Acute Lymphocytic Leukemia (ALL), the expression of ROR1 is greatly improved, and the expression of ROR1 is closely related to the progress of the disease and the therapeutic effect (CLIN CANCER Res.2017Jun 15;23 (12): 3061-3071;Front Oncol.2021May 28;11:680834), for example, the proportion of ROR ⁺ in CLL patients exceeds 90% (blood.2021Jun 17;137 (24): 3365-3377.). Expression of ROR1 in invasive malignancies initiates transcriptional processes similar to embryonic development. ROR1 can bind to wnt5a, participate in the signaling of wnt pathway, and interact with signaling pathways such as EGFR, met, etc., promoting growth, proliferation and metastasis of tumor cells. ROR1 plays an important role in the development of cancer stem cells and in the Epithelial Mesenchymal Transition (EMT) process. Cancer stem cells are cancer cells in tumors that are more stem cell-characteristic, and they are generally more resistant to chemotherapeutic agents. Whereas the EMT process converts the morphology of cells from that of epithelial cells to that of mesenchymal cells, making them more invasive, promoting metastasis of cancer.

The role of ROR1 in lung cancer includes inhibition of apoptosis, enhancement of EGFR signaling and induction of proliferation, while several documents demonstrate the important role of ROR1 in lung adenocarcinoma metastasis. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) targeted therapy plays a very important role in the combination therapy of non-SMALL CELL lung cancer (NSCLC), however, resistance inevitably occurs after a period of application in almost all patients. And EGFR T790M mutation and ROR1 expression are found to be increased in drug-resistant patients, suggesting that ROR1 targeted therapy may be applied to EGFR mutation and EGFR TKI resistant tumor patients (Transl Lung Cancer Res.2014jun;3 (3): 122-30.).

In conclusion, ROR1 can be used as a specific tumor marker and a tumor therapeutic drug target for drug development.

Disclosure of Invention

The Antibody conjugated drug (anti-drug conjugates, ADC) can consist of an Antibody (anti-body) targeting a tumor specific antigen or tumor associated antigen coupled to a different number of cytotoxic drugs (Payload) via a Linker (Linker), which is one of the fastest growing classes of drugs in oncology in recent years. Due to the dual advantages of high targeting of the monoclonal antibody drug and high activity of the cytotoxic drug in tumor tissues, the ADC drug can kill tumor cells with high efficiency, has lower side effect than the chemotherapeutic drug, has better curative effect than the traditional antibody tumor drug, and is called as a biological missile in the field of tumor treatment. Up to now, it is known that 14 ADC drugs are marketed in bulk worldwide, 7 for hematological tumors and 7 for solid tumors, the targets being CD33, CD30, CD22, CD79b, HER2, nectin-4, trop-2, BCMA, CD19 and TF. Wherein, 4 ADC drugs are marketed in China.

Currently, no antibody-conjugated drugs targeting ROR1 are marketed, and ROR1 ADC drugs in clinical stages include: VLS-101 (VLS 101), developed by the combination of the company Moesadong (MSD) and VelosBio, is currently being used in a phase 1 clinical trial and a phase 2 clinical trial, respectively, for the treatment of hematological cancers and solid tumors; antibody conjugated drug LCB71 of ROR1 commonly developed by kenite pharmaceutical industry and LegoChem Biosciences company, LCB71 carries tumor activated pyrrolobenzodiazepine(PBD) protoxins, whereas PBD, due to its own toxicity, exhibits an excessively narrow safety window during treatment of solid tumors (J Thorac Oncol.2021Sep;16 (9): 1429-1433.); in addition, the ROR1 iADC NBE-002 developed by Bolin and Yinghan/NBE Therapeutics, inc., coupled to the C-terminal end of the heavy and light chains of antibodies site-specifically and quantitatively by the transpeptidase Sortase A mediated specific reaction, PNU-159582 presents the same safety potential as the PBD toxoid. ROR1 differs from other ADC antigens, such as hundreds of thousands of copies of HER2 on tumor cells, and ROR1 is not expressed at very high levels in most tumor cells, not more than 5000 copies per cell. Therefore, there is an urgent need in the art to develop a novel ROR1 targeting antibody coupled drug, which can increase the drug concentration of ADC drugs in tumor cells and reduce the drug concentration in whole blood plasma, thereby improving the therapeutic effect of ADC drugs on tumors and reducing the side effects caused by toxin molecules.

To solve the problems of the prior art, a first aspect of the present invention provides an anti-ROR 1 antibody or antigen binding fragment thereof, comprising:

a heavy chain variable region which is capable of being altered, the heavy chain variable region comprises:

a) A complementarity determining region VH CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs 1, 17 and 33 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto;

b) A complementarity determining region VH CDR2 comprising the amino acid sequence shown in any one of SEQ ID NOs 3, 19 and 35 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto;

And

C) A complementarity determining region VH CDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs 5, 21 and 37 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and

A light chain variable region (light chain variable region), the light chain variable region comprises:

d) A complementarity determining region VL CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs 7, 23 and 39 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto;

e) A complementarity determining region VL CDR2 comprising the amino acid sequence shown in any one of SEQ ID NOs 9, 25 and 41 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and

F) A complementarity determining region VL CDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs 11, 27 and 43 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto.

In some embodiments, the antibody or antigen binding fragment thereof comprises:

(1) A heavy chain variable region which is capable of being altered, the heavy chain variable region comprises:

a) A complementarity determining region VH CDR1 comprising the amino acid sequence set forth in SEQ ID No. 1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto;

b) A complementarity determining region VH CDR2 comprising the amino acid sequence shown in SEQ ID No. 3 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto;

And

C) A complementarity determining region VH CDR3 comprising the amino acid sequence shown in SEQ ID No. 5 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and

d) A complementarity determining region VL CDR1 comprising the amino acid sequence shown in SEQ ID No. 7 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto;

e) A complementarity determining region VL CDR2 comprising the amino acid sequence shown in SEQ ID No. 9 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and

F) A complementarity determining region VL CDR3 comprising the amino acid sequence shown in SEQ ID No. 11 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; or (b)

(2) A heavy chain variable region which is capable of being altered, the heavy chain variable region comprises:

a) A complementarity determining region VH CDR1 comprising the amino acid sequence shown in SEQ ID No. 17 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto;

b) A complementarity determining region VH CDR2 comprising the amino acid sequence shown in SEQ ID No. 19 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto;

And

C) A complementarity determining region VH CDR3 comprising the amino acid sequence set forth in SEQ ID No. 21 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and

d) A complementarity determining region VL CDR1 comprising the amino acid sequence shown in SEQ ID No. 23 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto;

e) A complementarity determining region VL CDR2 comprising the amino acid sequence shown in SEQ ID No. 25 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and

F) A complementarity determining region VL CDR3 comprising the amino acid sequence shown in SEQ ID NO 27 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; or (b)

(3) A heavy chain variable region which is capable of being altered, the heavy chain variable region comprises:

a) A complementarity determining region VH CDR1 comprising the amino acid sequence shown in SEQ ID NO 33 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto;

b) A complementarity determining region VH CDR2 comprising the amino acid sequence shown in SEQ ID No. 35 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto;

And

C) A complementarity determining region VH CDR3 comprising the amino acid sequence shown in SEQ ID NO 37 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and

d) A complementarity determining region VL CDR1 comprising the amino acid sequence shown in SEQ ID NO 39 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto;

e) A complementarity determining region VL CDR2 comprising the amino acid sequence shown in SEQ ID No. 41 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and

F) A complementarity determining region VL CDR3 comprising the amino acid sequence shown in SEQ ID No. 43 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto.

(1) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 13 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and a light chain variable region comprising the amino acid sequence shown in SEQ ID No. 14 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto;

(2) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 29 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 30 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; or (b)

(3) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 45 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 46 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto.

In some embodiments, the antibody or antigen binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO. 15-16, 31-32 or 47-48 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto.

In some embodiments, the antibody or antigen binding fragment thereof is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a single chain antibody, an Fv, a single chain Fv (scFv), fd, fab, fab ', and F (ab') 2.

In some embodiments, the antibody or antigen binding fragment thereof binds human ROR1 with a KD of 2.0 x 10 ^-9 M or less.

In some embodiments, the antibody, or antigen binding fragment thereof, does not cross-react with mouse ROR 1.

In a second aspect the invention provides a nucleic acid sequence encoding an antibody or antigen binding fragment thereof as described above.

In some embodiments, the nucleic acid sequence encoding the above antibody or antigen binding fragment thereof has at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to SEQ ID No. 2, 4, 6, 8, 10, 12, 18, 20, 22, 24, 26, 28, 34, 36, 38, 40, 42 or 44.

In a third aspect the invention provides a vector or plasmid comprising a nucleic acid sequence encoding an antibody or antigen binding fragment thereof as described above.

In a fourth aspect the invention provides a host cell comprising and expressing a nucleic acid sequence, plasmid or vector as described above.

In a fifth aspect, the present invention provides an antibody conjugated drug having a structure represented by formula I

Ab- (L-D) _n (formula I),

Wherein Ab is an antibody or antigen-binding fragment thereof of any one of the above;

L is a linker;

D is a cytotoxic drug; and is also provided with

N is a number between 1 and 10, preferably a value between 1 and 8; preferably n is 1, 2, 3, 4, 5, 6, 7, 8, and any value between any two values.

Further, L is selected from the group consisting of Maleimide Caproyl (MC), maleimide (MAL), succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate) (SMCC) linkers attached to the antibody moiety, disubstituted maleimides, and comprises a linker of one or more of valine-citrulline (VC), valine-alanine (VA), glycine-phenylalanine-glycine (GGFG), alanine-alanine (AAA), p-aminobenzyloxycarbonyl (PAB), polyethylene glycol (PEG).

Wherein D is selected from the group consisting of:

(i) Tubulin inhibitors, such as maytansinoid derivatives (DM 1, DM 4), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF);

(ii) Toxins for DNA, such as duocarmycin (duocarmycin), pyrrolobenzodiazepine (PBD);

(iii) Topoisomerase inhibitors, camptothecins, SN38, irinotecan, dxd.

The invention also provides a kit for diagnosing a disease comprising an anti-ROR 1 antibody or antigen binding fragment thereof of the invention, and instructions for use.

The invention also provides a composition comprising an antibody-conjugated drug as described above.

In another aspect, the invention provides the use of an anti-ROR 1 antibody or antigen binding fragment thereof of the invention in the manufacture of a kit for diagnosing a disease.

The invention also provides the use of an antibody-conjugated drug as described herein or a composition comprising the same in the manufacture of a medicament for the treatment of cancer.

In another aspect, the invention provides a method of treating a disease comprising administering to a subject in need thereof an antibody-conjugated drug or composition described herein.

The invention also provides a method for diagnosing a disease comprising applying an anti-ROR 1 antibody or antigen binding fragment thereof of the invention or a kit as described above.

Additional features and advantages of the application will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application. Other advantages of the application may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the specification. The contents of all references, genbank entries, patents and published patent applications cited throughout this disclosure are expressly incorporated herein by reference.

Drawings

The accompanying drawings are included to provide an understanding of the principles of the application, and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain, without limitation, the principles of the application.

FIG. 1 shows EC50 and representative binding curves for anti-ROR 1 antibodies binding to human ROR1, indicating that these clones specifically bind to human ROR1;

FIG. 2 shows representative binding curves of anti-ROR 1 antibodies to mouse ROR1, indicating that these clones did not bind to mouse ROR 1.

FIG. 3 shows representative binding curves for anti-ROR 1 antibodies to human ROR2, indicating that ROR1-14 and ROR1-1 bind to human ROR 2.

FIGS. 4-6 show the mass-spectral desugared profile of the humanized antibody ROR1-4 (FIG. 4) with the HIC of its antibody conjugate ROR1-4-BL20MMAE (abbreviated as ROR1-4-BL 20E) (FIG. 5) and the mass-spectral desugared profile (FIG. 6).

Figures 7-9 show the mass-spectral desugared profile of the humanized antibodies ROR1-12 (figure 7) with the HIC (figure 8) and mass-spectral desugared profile of their antibody conjugates ROR1-12-BL20MMAE (figure 9).

FIGS. 10-12 show the mass-spectral desugared pattern of the humanized antibody ROR1-12 (FIG. 10) with the HIC (FIG. 11) and mass-spectral desugared pattern of its antibody conjugate ROR1-12-GGFG-Dxd (abbreviated as ROR 1-12-DX) (FIG. 12).

FIG. 13 shows the effect of ROR1-12-BL20E, ROR1-12-DX on growth of human mantle cell lymphoma JeKo-1 subcutaneous graft.

FIG. 14 shows the effect of ROR1-12-BL20E, ROR1-4-BL20E, VLS-MMAE on the growth of human breast cancer MDA-MB-231 subcutaneous grafts.

FIG. 15 shows the effect of ROR1-12-BL20E, ROR1-4-BL20E, VLS-MMAE on body weight of human breast cancer MDA-MB-231 subcutaneously transplanted mice.

FIG. 16 shows the effect of ROR1-12-BL20E, ROR1-4-BL20E, VLS101-MMAE on growth of human lung carcinoma H1975 subcutaneous transplantation tumor.

FIG. 17 shows the effect of ROR1-12-BL20E, ROR1-4-BL20E, VLS-MMAE on body weight of human lung carcinoma H1975 subcutaneously transplanted mice.

Detailed Description

In order that the present disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The term "and/or" as used herein shall be taken to be a specific disclosure of each of two or more specified features or components, with or without other features or components. Thus, the term "and/or" as used in the phrase, e.g., "a and/or B" herein, is intended to include "a and B", "a or B", "a" (alone) and "B" (alone).

It will be understood that wherever aspects are described by the language "comprising" or "including," similar aspects are also provided that are described by the terms "consisting of" and/or "consisting essentially of.

The term "about" means an amount, level, value, number, frequency, percentage, size, quantity, weight, or length that varies at an acceptable level in the art. In some embodiments, such variations may be up to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a reference number, level, value, number, frequency, percentage, size, amount, weight, or length. When the term "about" is used in connection with a range of values, it modifies that range by extending the boundaries above and below the values.

The term "ROR1" refers to receptor tyrosine kinase-like orphan receptor 1, also known as neurotrophic tyrosine kinase receptor-related 1 (NTRKR 1). The term "ROR1" includes variants, isoforms, homologs, orthologs and paralogs. For example, in some cases, antibodies specific for human ROR1 proteins may cross-react with ROR1 proteins from non-human species (e.g., cynomolgus monkey). In other embodiments, antibodies specific for human ROR1 proteins may be fully specific for human ROR1 proteins and exhibit no cross-reactivity to other species or other types, or may cross-react with ROR1 from some other species but not all other species.

The term "human ROR1" refers to a human ROR1 sequence, such as the amino acid sequence of human ROR1 having NCBI reference sequence No. NP-005003.2.

The term "antibody" is used herein in a broad sense as understood by one of ordinary skill in the art, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), so long as they exhibit the desired antigen-binding activity. The antibodies can be whole antibodies (e.g., having two full length light chains and two full length heavy chains) of any type and subtype (e.g., igM, igD, igG, igG2, igG3, igG4, igE, igA1, and IgA 2). An "antibody" may also refer to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) that are linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, CH1, CH2 and CH 3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. VH and VL regions can be further subdivided into regions of higher variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxyl-terminus in the following order: FR-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable regions of the heavy and light chains contain binding domains that can interact with antigens. The constant regions of antibodies may mediate binding of immunoglobulins to host tissues or factors.

The term "antigen binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen bound by the entire antibody. Examples of binding fragments encompassed within the term "antigen binding fragment" of an antibody include, but are not limited to: (i) Fab fragments, i.e. monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) A F (ab') ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of the antibody single arm; (v) a dAb fragment consisting of a VH domain; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to form a single protein chain, in which the VL and VH regions pair to form monovalent molecules, known as single chain Fv (scFv).

The term "monoclonal antibody" refers to an antibody from a substantially homogeneous population of antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. In contrast, polyclonal antibodies generally include a number of antibodies directed against (or specific for) different epitopes. "monoclonal" refers to the characteristics of the antibody from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be prepared by hybridoma methods, or can be prepared by recombinant DNA methods. "monoclonal antibodies" can also be isolated from phage antibody libraries.

The term "multispecific antibody" refers to an antibody that has binding specificity for at least two different epitopes on the same antigen or different antigens. The term "bispecific antibody" means an antibody having binding specificity for two different epitopes.

The term "humanized antibody" as used herein refers to a form of antibody that comprises sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies comprise minimal sequences derived from non-human immunoglobulins. Generally, humanized antibodies comprise substantially all of at least one and typically 2 variable structures and, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin.

The term "human antibody" or "fully human antibody" includes antibodies having variable and constant regions (if present) derived from human germline immunoglobulin sequences. Human antibodies 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 "human antibody" does not include antibodies (i.e., humanized antibodies) in which CDR sequences derived from the germline of another mammal (e.g., a mouse) are grafted to human framework sequences. Fully human antibodies or human antibodies may be derived from transgenic mice carrying human antibody genes or from human cells.

The term "antibody derivative" refers to any modified form of an antibody, such as a conjugate of the antibody and other reagents or other antibodies.

As used herein, the term "anti-ROR 1 antibody", "anti-ROR 1", "ROR1 antibody" or "ROR1 binding antibody" refers to an antibody capable of binding ROR1 protein or fragment thereof with sufficient affinity such that the antibody may be used as a diagnostic and/or therapeutic agent targeting ROR 1.

As used herein, the term "epitope" refers to the portion of an antigen (e.g., ROR 1) that specifically interacts with an antibody molecule. This moiety (referred to herein as an epitope determinant) typically comprises elements such as amino acid side chains or sugar side chains or components thereof. Epitope determinants can be defined using methods known in the art. Some epitopes are linear epitopes while others are conformational epitopes.

As used herein, an "isolated antibody" refers to an antibody molecule that has been separated from components of its natural environment.

The terms "specific," "specifically binds," or "specific for …" refer to the number of different types of antigens or antigenic determinants that a particular antibody or antigen-binding fragment thereof can bind. Thus, an antibody or antigen binding fragment thereof as defined herein is said to be "specific" for a first target or antigen when it binds to the first antigen with an affinity (as described herein and suitably expressed, for example, as a KD value) that is at least 50-fold, such as at least 100-fold, preferably at least 1000-fold, and up to 10,000-fold or more better than the affinity of the amino acid sequence or polypeptide for binding to another target or polypeptide, as compared to a second target or antigen. Preferably, when an antibody or antigen-binding fragment thereof is "specific" for a target or antigen, it is capable of binding to the target or antigen, but not to the other target or antigen, as compared to the other target or antigen.

The term "K _assoc" or "K _a" as used herein refers to the rate of binding of a particular antibody-antigen interaction, while the term "K _dis" or "K _d" as used herein refers to the rate of dissociation of the antibody-antigen interaction.

The term "K _D" as used herein refers to the dissociation constant, which is obtained from the ratio of K _d to K _a (i.e., K _d/K_a) and is expressed as molar concentration (M). The K _D value of an antibody can be determined using methods established in the art. One preferred method of determining antibody K _D is to use surface plasmon resonance, preferably using a biosensor system, such as the Biacore system. K _D is a measure of the affinity of an antibody to an antigen.

"Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibodies and antigens).

The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to a nucleotide sequence comprising deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that normally contains the nucleic acid molecule but is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

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

The term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells" which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may include mutations. Included herein are mutant progeny having the same function or biological activity as screened or selected in the original transformed cell.

IC50 (half maximal inhibitory concentration) refers to the half-inhibitory concentration of the antagonist being measured. It indicates that a certain drug or substance (inhibitor) is inhibiting half of a certain biological process (or a certain substance contained in the process, such as an enzyme, a cellular receptor or a microorganism). The IC50 measures the sensitivity of the antibody, the lower the IC50, the higher the sensitivity of the antibody.

EC50 (concentration for 50%of maximal effect) refers to the concentration that causes 50% of the maximum effect.

"Individual" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

"Therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the desired dosage and for the desired period of time. The therapeutically effective amount of the antibody or antibody fragment or conjugate or composition thereof may vary depending on a variety of factors such as the disease state, age, sex and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount of any toxic or detrimental effect of the antibody or antibody fragment or conjugate or composition thereof that is less than a therapeutically beneficial effect. The "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60% or 70% and still more preferably by at least about 80% or 90% relative to an untreated subject.

"Percent (%) sequence identity" with respect to a reference sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to residues in the reference sequence after aligning the candidate sequence to the reference sequence and introducing gaps, if necessary, to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. The alignment used to determine the percent sequence identity can be accomplished in a variety of ways in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the entire length of the sequences being compared. When referring to percentages of sequence identity in the present application, these percentages are calculated relative to the full length of the longer sequence unless explicitly stated otherwise. The calculation of the full length relative to longer sequences applies to both nucleic acid sequences and polypeptide sequences.

The term "pharmaceutical composition" refers to a formulation in a form that is effective for the biological activity of the active ingredient contained therein and that is free of additional components that have unacceptable toxicity to the subject to whom the formulation is administered.

The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, and the like, as known in the art. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), suitable mixtures thereof, and vegetable oils. Each carrier should be pharmaceutically and physiologically acceptable in terms of compatibility with the other ingredients and not deleterious to the subject. Unless any conventional medium or agent is incompatible with the active ingredient, its administration in a therapeutic composition is contemplated.

It is to be understood that the term "treating" as used herein means reducing, preventing, curing, reversing, ameliorating, weakening, alleviating, minimizing, inhibiting or delaying the onset of one or more clinical indications of a disease or condition.

The term "immunoconjugate" as defined herein refers to an antibody according to the present disclosure, or any antigen-binding fragment thereof, conjugated or linked to an additional agent. Immunoconjugates can be prepared by any method known to those of skill in the art, for example, by crosslinking additional agents with antibodies according to the disclosure or by recombinant DNA methods.

Anti-ROR 1 antibodies or antigen binding fragments thereof

In a first aspect the invention provides an anti-ROR 1 antibody or antigen binding fragment thereof,

And

TABLE 1 sequence composition of antibodies

Protein expression

Molecular biology techniques suitable for producing a polypeptide or protein of the invention (e.g., an antibody or antigen binding fragment thereof) in a cell are well known to those of ordinary skill in the art.

The polypeptide or protein may be expressed from a polynucleotide sequence. The polynucleotide sequence may be contained in a vector present in the cell, or may be incorporated into the genome of the cell.

The term "vector" as used herein is an oligonucleotide molecule (DNA or RNA) that serves as a vector to transfer exogenous genetic material into a cell. The vector may be an expression vector for expressing genetic material in a cell. Such vectors may include a promoter sequence operably linked to a polynucleotide sequence encoding the gene sequence to be expressed. The vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer and stop codon that is consistent with the art may be used to express a polypeptide from the vectors of the invention. Suitable vectors include plasmids, binary vectors, viral vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cellular system that can be used to produce the antibody molecules of the invention. The cell may be a prokaryotic or eukaryotic cell. Suitable prokaryotic cells include E.coli cells. Examples of suitable eukaryotic cells include yeast cells, plant cells, insect cells, or mammalian cells (e.g., chinese Hamster Ovary (CHO) cells). In some cases, the cells are not prokaryotic cells, as some prokaryotic cells do not allow the same post-translational modification as eukaryotic cells. Furthermore, very high expression levels are possible in eukaryotes, and proteins can be more easily purified from eukaryotes using appropriate tags. Specific plasmids can also be used, which enhance secretion of the protein into the culture medium. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissue or animal tissue.

Methods of producing a polypeptide of interest may include culturing or fermenting the modified cell to express the polypeptide. The cultivation or fermentation may be carried out in a bioreactor with a suitable nutrient supply, air/oxygen and/or growth factors. Secreted proteins can be collected by partitioning the medium/broth from the cells, extracting the proteins and isolating the individual proteins to isolate the secreted polypeptides. Culture, fermentation and isolation techniques are well known to those of ordinary skill in the art.

The bioreactor includes one or more vessels in which cells can be cultured. The culturing in the bioreactor may be performed continuously, with reactants flowing continuously in and cultured cells from the reactor flowing continuously. Alternatively, the cultivation may be performed batchwise. The bioreactor detects and controls environmental conditions such as pH, oxygen, inflow and outflow rates, and agitation within the vessel to provide optimal conditions for the cultured cells.

After culturing the cells expressing the polypeptide/protein of interest, the polypeptide/protein is preferably isolated. Any suitable method of isolating polypeptides/proteins from cell culture known to those of ordinary skill in the art may be used. In order to isolate a polypeptide/protein of interest from a culture, it may be necessary to first separate the cultured cells from the medium containing the polypeptide/protein of interest. If the polypeptide/protein of interest is secreted from the cells, the cells can be separated from the medium containing the secreted polypeptide/protein by centrifugation. If the polypeptide/protein of interest is aggregated within the cell, the cell must be destroyed prior to centrifugation, for example using sonication, rapid freeze thawing or osmotic lysis. Centrifugation will produce a pellet containing the cultured cells or cell debris of the cultured cells and a supernatant containing the culture medium and the polypeptide/protein of interest.

It may then be desirable to isolate the polypeptide/protein of interest from the supernatant or medium, which may contain other proteins and non-protein components. A common method of separating the polypeptide/protein component from the supernatant and the culture medium is by precipitation. Polypeptides/proteins of different solubilities are precipitated in different concentrations of precipitants such as ammonium sulfate. For example, water-soluble polypeptides/proteins are extracted at low concentrations of precipitants. Thus, by adding increasing concentrations of precipitants, polypeptides/proteins of different solubilities can be distinguished. Chromatography may then be used to remove ammonium sulfate from the isolated polypeptide/protein.

Other methods for distinguishing between different polypeptides/proteins are known to those of ordinary skill in the art, such as ion exchange chromatography and size chromatography. These may be used as alternatives to precipitation or may be performed after precipitation.

Once the polypeptide/protein of interest has been isolated from the culture, concentration may be required. Many methods of concentrating the polypeptide/protein of interest are well known to those of ordinary skill in the art, such as ultrafiltration or freeze drying.

Immunoconjugates

One example of an immunoconjugate is an antibody conjugated drug, also known as an antibody drug conjugate, an antibody drug conjugate. The antibody coupling medicine is formed by linking a monoclonal antibody targeting specific antigen and a small molecule cytotoxicity medicine through a linker, and has the strong killing effect of traditional small molecule chemotherapy and the tumor targeting of the antibody medicine. ADC is composed of three main parts: an antibody responsible for selectively recognizing a cancer cell surface antigen, a drug payload responsible for killing cancer cells, and a linker linking the antibody and the payload.

The invention provides a novel class of antibody-conjugated drugs having the formula Ab- (L-D) _n, wherein: ab is an antibody or antigen binding fragment thereof that specifically binds to human receptor tyrosine kinase-like orphan receptor 1 (ROR 1); l is a linker; d is a cytotoxic drug; and n is a number from 1 to 10.

Connector

According to the mechanism of drug release in cells, "linkers" or "linkers of antibody drug conjugates" can be divided into two classes: non-cleavable linkers and cleavable linkers.

For antibody drug conjugates containing non-cleavable linkers, the mechanism of drug release is: after the conjugate is combined with antigen and endocytosed by cell, the antibody is enzymolyzed in lysosome to release active molecule comprising small molecule medicine, connector and antibody amino acid residue. The resulting change in the structure of the drug molecule does not impair its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate into neighboring cells. Thus, such active agents cannot kill tumor cells adjacent to those that do not express the targeted antigen (antigen negative cells) (bystander effect ).

Cleavable linkers, as the name suggests, can cleave and release the active agent (the small molecule drug itself) within the target cell. Cleavable linkers can be divided into two main categories: chemically labile linkers and enzymatically labile linkers. Chemically labile linkers can be selectively cleaved due to differences in plasma and cytoplasmic properties. Such properties include pH, glutathione concentration, etc. pH sensitive linkers, also commonly referred to as acid-cleavable linkers. Such linkers are relatively stable in the neutral environment of blood (pH 7.3-7.5), but will be hydrolyzed in the weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). The first generation of antibody drug conjugates mostly used such linkers, e.g. hydrazones, carbonates, acetals, ketals. Antibody drug conjugates based on such linkers typically have a short half-life (2-3 days) due to the limited plasma stability of the acid-cleavable linker. This short half-life limits to some extent the use of pH-sensitive linkers in new generation antibody drug conjugates.

For glutathione-sensitive linkers, also known as disulfide linkers. Drug release is based on the difference between the high concentration of intracellular glutathione (millimolar range) and the relatively low concentration of glutathione in the blood (micromolar range). This is especially true for tumor cells, where low oxygen content leads to an increased activity of the reductase and thus to higher glutathione concentrations. Disulfide bonds are thermodynamically stable and thus have better stability in plasma.

Enzyme labile linkers, such as peptide linkers, can better control drug release. Peptide linkers can be effectively cleaved by an intra-lysosomal protease, such as cathepsin (CATHEPSIN B) or plasmin (an increase in such enzyme content in some tumor tissues). This peptide linkage is believed to be very stable in the plasma cycle because extracellular unfavorable pH values and serum protease inhibitors result in proteases that are generally inactive. In view of the high plasma stability and good intracellular cleavage selectivity and availability, enzyme labile linkers are widely used as cleavable linkers for antibody drug conjugates. Typical enzymatically labile linkers include Val-Cit (VC), phe-Lys, and the like.

The self-releasing linker is typically chimeric between the cleavable linker and the active agent or is itself part of the cleavable linker. The mechanism of action of the self-releasing linker is: when the cleavable linker is cleaved under suitable conditions, the self-releasing linker is capable of spontaneously undergoing structural rearrangement, thereby releasing the active agent attached thereto. Common suicide linkers include p-aminobenzyl alcohols (PAB) and beta-glucuronides (beta-Glucuronide), among others.

The present invention provides a linker or coupling agent comprising a diarylthiomaleimide unit and a coupling group. The diarylthiomaleimide units are used to crosslink thiol groups between antibody chains (after reduction), while the coupling groups are used to couple with small molecule drugs or drug-linker units. Because the diarylthiomaleimide units bind to the two disulfide atoms of the open cysteine-cysteine disulfide bond in the antibody (bidentate binding), these ADCs are homogeneous and have greater stability than ADCs containing single-tooth junctions. They will therefore have an increased in vivo half-life, reduced amounts of cytotoxins released systemically, and safer pharmaceutical properties than ADCs with single tooth junctions.

In another aspect, the resulting drug-linker unit is coupled to the antibody via the linker, resulting in a partially interchain crosslinked conjugate. Compared with the traditional antibody drug conjugate, the antibody drug conjugate prepared by the method has narrower drug/antibody ratio (DAR) distribution, thereby greatly improving the uniformity of products and the uniformity of pharmacological characteristics. The antibody drug conjugates are useful for targeted delivery of drugs to a target cell population, such as tumor cells. The antibody drug conjugate can specifically bind to a cell surface protein, and the resulting conjugate is then endocytosed by the cell. Within the cell, the drug is released as an active drug to produce efficacy. Antibodies include chimeric antibodies, humanized antibodies, human antibodies; an antibody fragment that binds to an antigen; or an antibody Fc fusion protein; or a protein. A "drug" is a highly active drug (see definition section) which in some cases may be polyethylene glycol.

In one embodiment, linker L may be a cleavable linker or a non-cleavable linker. In one embodiment, the linker is a chemical linker. In one embodiment, the linker comprises a covalent bond, such as an ester bond, an ether bond, an amine bond, an amide bond, a disulfide bond, an imide bond, a sulfone bond, a phosphate bond (phosphorus ester bond), a peptide bond, a hydrazone bond, or a combination thereof. In one embodiment, the linker comprises a hydrophobic poly (ethylene glycol) linker. In one embodiment, the linker comprises a peptide bond.

In one embodiment, cytotoxic drug D may be any cytotoxic, cytostatic or immunosuppressive drug. In embodiments, the linker connects the antibody and the drug, and the drug has a functional group that can bond to the linker. For example, the drug may have an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, or a ketone group that may be bonded to the linker. In the case of a drug directly attached to a linker, the drug has reactive groups prior to attachment to the antibody.

Useful classes of cytotoxic drugs D may include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful classes of cytotoxic drugs D include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs include, for example, auristatins (auristatins), camptothecins (camptothecins), duocarmycin/carcinomycin (duocarmycins), etoposide (etoposides), maytansinoids (maytansines) and maytansinoids (maytansinoids) (e.g., DM1 and DM 4), taxanes (taxanes), benzodiazepines (benzodiazepines) or benzodiazepine-containing drugs (benzodiazepine containing drugs) (e.g., bello [1,4] benzodiazepines (PBDs), indoline benzodiazepines (indolinobenzodiazepines) and java benzodiazepines (oxazolidinobenzodiazepines)) and vinca alkaloids (vinca alkaloids).

In one embodiment, the cytotoxic drug D may be selected from a chemotherapeutic agent, a growth inhibitory agent, a cali Li Ji-mycin-like drug unit, an antimitotic agent, a radioisotope, or a combination thereof. In one embodiment, cytotoxic drug D comprises kar Li Jimei, ozagrel, monomethyl auristatin E, maytansinone (emtansine), 7-ethyl-10-hydroxycamptothecin (SN-38), an isatecan derivative, or a combination thereof. In one embodiment, cytotoxic drug D comprises monomethyl auristatin E, maytansine, 7-ethyl-10-hydroxycamptothecin (SN-38), irinotecan, alpha-amanitine, sesqui-carcinomycin, pyrrolobenzodiazepine(PBD), PNU-159582 and pharmaceutically acceptable salts, esters and analogues thereof.

Other common L-D moieties of antibody conjugated drugs are, for example

In some preferred embodiments, the linker L is a maleimide-based linker, preferably L is a disubstituted maleimide-based linker. The linker can be fully/partially cross-coupled to cysteine sulfhydryl groups reduced by disulfide bonds of the light chain-heavy chain and the heavy chain-heavy chain of the antibody, and the targeting ROR1 antibody drug conjugate obtained by the coupling method has relatively more uniform drug/antibody ratio (DAR) distribution compared with the traditional antibody drug conjugate. The structure of the ROR1 antibody coupling drug with the disubstituted maleimide linker is shown as formulas Ia and Ib:

Wherein,

Ar' is selected from the group consisting of: a substituted or unsubstituted C6-C10 arylene group, a substituted or unsubstituted 5-12 membered heteroarylene group;

L ₁ is-O (CH ₂CH₂O)_n -, wherein n is selected from any integer from 1 to 20, preferably any integer from 1 to 10;

L ₂ is a bond or an AA-PAB structure; wherein AA is a polypeptide fragment consisting of 2-4 amino acids, and PAB is p-aminobenzyl carbamoyl;

CTD is a cytotoxic drug bonded to L ₂ through an amide linkage and/or a drug to treat autoimmune diseases and anti-inflammatory;

m is 1.0 to 5.0, preferably 3.0 to 4.2; more preferably 3.5 to 4.5; still more preferably 3.8-4.2, still more preferably 3.9-4.1, most preferably 4.0;

ab is the ROR 1-targeted antibody or antigen binding fragment thereof.

In another preferred embodiment, the formula Ib is the ring-opened product of N-phenylmaleimide of formula Ia.

In another preferred embodiment, the conjugate is covalently linked to one or more pharmaceutical components.

In another preferred embodiment, the conjugate is one in which the antibody and the drug are coupled covalently (e.g., by covalent attachment to a linker, respectively).

In another preferred embodiment, the closed or open loop maleimide group is attached to the reduced disulfide chain thiol group of the antibody hinge region.

In another preferred embodiment, the antibody-drug conjugate is obtained by reducing the disulfide chain of the hinge region of the antibody or antibody fragment to form a pair of cysteine residues, and by substitution reaction of the thiol group in the cysteine residues with the aryl sulfide in the substituted maleimide-based linker-drug conjugate of formula Ic.

In another preferred embodiment, the closed or open loop maleimide groups are attached to the fully reduced antibody, i.e. the 4-pair disulfide chains of the hinge region are fully opened, preferably m is 3.8-4.2, more preferably 3.9-4.1, most preferably 4.0.

In another preferred embodiment, the ROR-targeting antibody is selected from the group consisting of: monoclonal antibodies, chimeric antibodies, single chain antibodies, fv, single chain Fv (scFv), fd, fab, fab 'and F (ab') 2.

In another preferred embodiment, the antibody fragment is an antibody Fab fragment.

In another preferred embodiment, the antibody is an antibody capable of binding to ROR.

In another preferred embodiment, the ROR-targeting antibody or antibody fragment is an antibody or antigen binding fragment thereof according to the first aspect of the invention.

In another preferred embodiment, the Ar' is selected from the group consisting of: phenyl, halobenzene, C1-C4 alkylphenyl, C1-C4 alkoxyphenyl, 2-pyridyl, 2-pyrimidinyl, 1-methylimidazol-2-yl, wherein: C1-C4 alkylphenyl is further preferably 4-methylphenyl; the C1-C4 alkoxyphenyl group is more preferably a 4-methoxyphenyl group.

In another preferred embodiment, ar' is selected from substituted or unsubstituted phenylene or pyridinyl, wherein the hydrogen atoms of the groups are replaced by one or more substituents selected from the group consisting of: halogen, C1-C4 alkyl, C1-C4 alkoxy, trifluoromethyl, nitrile group, amide group.

In another preferred embodiment, the AA is selected from the group consisting of: val-Cit (valine-citrulline), val-Ala (valine-alanine), phe-Lys (phenylalanine-lysine), ala-Ala-Asn (alanine-asparagine), D-Ala-Phe-Lys (D-alanine-phenylalanine-lysine), gly-Gly-Phe-Gly (glycine-phenylalanine-glycine).

In some preferred embodiments, methods of coupling a cytotoxic drug to an ROR 1-targeting antibody or antigen binding fragment thereof via a maleimide linker are known to those skilled in the art, for example, see chinese patent applications CN201611093699.6 and CN201711169847.2.

The antibody drug conjugate provided by the invention, although still a mixture, has a narrow DAR distribution range compared with the antibody drug conjugate obtained by coupling in a traditional way. Its average DAR value is near 4, approaching the range of optimal antibody drug conjugate average DAR values (2-4). In addition, antibody drug conjugates rarely contain a bare antibody (dar=0), a component that does not contribute to cytotoxic killing. At the same time, the antibody drug conjugate also contained no heavy conjugate (dar=8), and this component cleared rapidly in vivo relative to the low DAR component. Therefore, the antibody drug conjugate provided by the invention is greatly improved in non-uniformity.

Preparation of ROR1 antibody coupling medicine

The preparation route of the antibody drug conjugate is shown below. The inter-chain disulfide bond of the antibody is reduced, yielding 2n (n is a number between 1 and 4) sulfhydryl groups. The substituted maleimide linker-drug conjugate (compound of formula Ic) of the invention is crosslinked with reduced antibody sulfhydryl groups to generate corresponding antibody drug conjugates, wherein the antibody drug conjugates exist in one or two forms as shown in the following.

Wherein the compound of formula Ic is selected from the group consisting of:

Etc.

An exemplary method of preparation comprises: diluting the stock solution of the antibody to 2-10mg/mL with a reaction buffer, adding Dithiothreitol (DTT) in an excess molar ratio of 140-200 times, or adding tris (2-carboxyethyl) phosphine hydrochloride (TCEP) in an excess molar ratio of 6.0-20 times, and stirring the reaction solution at 10-35 ℃ for 2-48 hours. The reaction buffer may be a buffer prepared according to the following ratio: 50mM potassium dihydrogen phosphate-sodium hydroxide (KH ₂PO₄ -NaOH)/150 mM sodium chloride (NaCl)/1 mM diethylenetriamine pentaacetic acid (DTPA), pH 6-9;50mM disodium hydrogen phosphate-citric acid/150 mM sodium chloride (NaCl)/1 mM diethylenetriamine pentaacetic acid (DTPA), pH 6-9;50mM boric acid-borax/150 mM sodium chloride (NaCl)/1 mM diethylenetriamine pentaacetic acid (DTPA), pH 6-9;50mM histidine-sodium hydroxide/150 mM sodium chloride (NaCl)/1 mM diethylenetriamine pentaacetic acid (DTPA), pH 6-9 and PBS//1mM diethylenetriamine pentaacetic acid (DTPA), pH 6-9.

Cooling the reaction liquid to 0-10 ℃, if adopting DTT reduction, removing excessive DTT by a desalting column or ultrafiltration after the reduction reaction is completed, adding substituted maleimide compounds (10 mg/ml of which are dissolved in Acetonitrile (ACN), dimethyl sulfoxide (DMSO), dimethylformamide (DMF) or diethyl acetamide (DMA) in advance), ensuring that the volume ratio of organic solvent in the total reaction liquid is not more than 15%, and stirring the coupling reaction at 0-37 ℃ for 2-4 hours. If TCEP reduction is adopted, the residual TCEP is not required to be removed, and the substituted maleimide compound can be directly added for coupling.

The coupling reaction mixture was purified by filtration through sodium succinate/NaCl buffer or histidine-acetic acid/sucrose gel using a desalting column, and peak samples were collected based on UV280 UV absorbance, or ultrafiltered several times. Then filtering and sterilizing, and preserving the obtained product at low temperature. Preferably at a temperature of-100 ℃ to-20 ℃ and the pore size of the filtration device is preferably 0.15-0.3 microns.

The DAR value of the obtained antibody drug conjugate is relatively uniform. When the invention is used to replace maleimide linkers (linker fragments) differently, the ADC product uniformity is very high (typically DAR dominant products (e.g., DAR values of about 4) accounting for at least 60%, at least 70%, at least 80%, at least 90% or higher of all ADCs). For ADCs with certain differences in DAR, if a sample with better uniformity is required to be obtained, the following methods can be further used for separation and purification, but are not limited to: hydrophobic Interaction Chromatography (HIC), size Exclusion Chromatography (SEC), ion Exchange Chromatography (IEC).

The antibodies or antigen-binding portions thereof of the invention may also be encoded by or used in combination with oncolytic viruses.

Kit for detecting a substance in a sample

Such a test kit may, for example, comprise a disposable test device configured to generate a detectable signal related to the presence or amount of ROR1 in a biological sample. Or such test kits may be formulated for measurement in a clinical analyzer that does not utilize a disposable testing device. Preferably, the test kit is an in vitro diagnostic agent. As used herein, the term "in vitro diagnosis" refers to a medical device that is a reagent, reagent product, calibrator, control material, kit, instrument, device, apparatus, or system, whether used alone or in combination, that a manufacturer intends to examine in vitro specimens from the human body (including blood and tissue donations), specifically provide or pay attention to provide information about physiological or pathological conditions or about congenital anomalies or to determine the compatibility of safety with potential recipients, or to monitor therapeutic measures.

Composition and method for producing the same

The invention also provides compositions comprising the antibody drug conjugates of the invention and a pharmaceutically acceptable carrier.

In some embodiments, the antibody drug conjugates of the invention may be administered as a single pharmaceutically active ingredient, or may be administered in combination with other anticancer drugs.

In some embodiments, the antibody drug conjugates of the invention, or compositions comprising the same, are administered in an effective amount or effective dose, e.g., in the range of 1ng/kg to about 20-100mg/kg body weight per day. The antibody-conjugated drug of the present invention may be administered once, twice, three times or more daily for a period of 1-4 weeks or more.

In the present application, the "effective amount" or "effective dose" refers to an amount sufficient to affect the beneficial or desired symptoms of the disease, its complications, or a pathological marker intermediate in the course of disease progression.

The antibody drug conjugates of the invention or compositions comprising the same may be administered by any conventional route, such as intravenous, intra-arterial, intramuscular, intraperitoneal administration, and the like.

Therapeutic and diagnostic methods

In another aspect, the invention provides a method of treating or ameliorating cancer in a subject comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the invention. The cancer may be a hematologic cancer or a solid tumor selected from lymphoma, CLL, small lymphocytic lymphoma, marginal cell B-cell lymphoma, berkette lymphoma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell cancer, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, and head and neck cancer. In some embodiments, at least one additional anti-cancer antibody may be administered with an antibody of the invention or antigen-binding fragment thereof, e.g., an anti-PD-1 antibody, an anti-LAG-3 antibody, and/or an anti-CTLA-4 antibody. In yet another embodiment, the antibodies, or antigen-binding portions thereof, of the invention are conjugated to a cytokine (e.g., IL-2 and/or IL-21) or a co-stimulatory antibody (e.g., an anti-CD 137 and/or anti-GITR antibody). The antibodies of the invention may be, for example, mouse, human, chimeric or humanized antibodies.

In another aspect, the invention provides a method for diagnosing or prognosing cancer in a subject, comprising collecting a tissue sample of interest from the subject, and contacting the tissue sample with an antibody or antigen-binding portion thereof, the invention. If an amount of ROR1 is detected, the subject may be diagnosed with cancer, and an increase/decrease in ROR1 expression indicates cancer progression/improvement. The cancer may be a hematologic cancer or a solid tumor selected from lymphoma, CLL, small lymphocytic lymphoma, marginal cell B-cell lymphoma, berkette lymphoma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell cancer, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, and head and neck cancer.

Examples

Example 1: phage library panning and screening of ROR1 monoclonal antibodies

An antibody single chain phage display library was generated by cloning a repertoire of light chain variable regions (VL) and heavy chain variable regions (VH). The heavy and light chain repertoires were generated by PCR amplification from human lymphocytes collected mainly from peripheral blood and neonatal umbilical cord blood. VL and VH libraries were mixed and PCR was performed using overlapping primers. The final form of the antibody is a single chain Fv (scFv) in which the VH and VL fragments are linked by a flexible linker peptide (SGGSTITSYNVYYTKLSSSGT (SEQ ID NO: 46)). The primary library was further amplified by the LoxP-cre system.

Selection of phage particles displaying specific scFv fragments was performed on an immuno96 MicroWellTM plate (Nunc company, denmark). First, 50. Mu.g/ml of human ROR1 recombinant protein (catalog number: RO1-H522y, acrobiosystems Co.) in Phosphate Buffered Saline (PBS) was coated on the plate overnight at 4 ℃. After blocking with 2% (w/v) of milk powder in PBS (2% MPBS), a library containing about 10 ¹¹ phage particles was added and the plates were incubated for 2 hours at room temperature (RT; 25 ℃ -28 ℃). Unbound phage were eliminated by washing 10-20 times with PBS (PBS-T) containing 0.1% Tween 20 followed by 10-20 washes with PBS. Bound phage were eluted by incubation with 50. Mu.l of 1. Mu.g/. Mu.l trypsin for 10min followed by 50. Mu.l of 50mM glycine hydrochloride (pH 2.0) (immediately after 10min neutralization with 50. Mu.l of 200mM Na ₂HPO₄ (pH 7.5)). The eluted phage were used to infect exponentially growing E.coli TG1 cells by incubation at 37℃for 30 min. Infected cells were plated on TYE plates containing ampicillin (ampicillin) (100. Mu.g/mL) and glucose (1% w/v) and the plates were then incubated overnight at 37 ℃. Individual phage-infected colonies were picked and grown in 96-well plates to produce phagemid particles. Cultures were rescued using M13KO7 or KM13 helper phages. The rescued phage particles were used to trigger subsequent rounds of selection using similar conditions. Three rounds of selection were performed for ROR1 protein.

To test ROR1 binding in an enzyme-linked immunosorbent assay (ELISA), individual clones from the last panning were selected and grown at 37 ℃ and rescued with M13K07 helper phage. Amplified phage preparations were blocked for 1 hour at 37℃with 5% skim milk in PBS and added to 96-well microwell plates (Nunc Co.) coated with ROR1 (catalog number: RO1-H522y, acrobiosystems Co.) (0.5. Mu.g/ml). After an additional 1 hour incubation at 37 ℃, the plates were washed 3 times with PBST and incubated with mouse horseradish peroxidase (HRP) -conjugated anti-M13 phage antibody (Amersham). After careful washing 3,30,5,50-tetramethylbenzidine (TMB, sigma) was added as substrate. The chromogenic reaction was measured at 450nm using a Thermo multiskan ELISA reader (MA, USA) in the united states.

In a further test, 3 clones were screened: ROR1-4, ROR1-12, and ROR1-14.

EXAMPLE 2 expression and purification of full Length antibodies

Methods for producing full length human IgG1 antibodies from scFv were established. Genes encoding VH and VL regions of an anti-ROR 1 antibody were sequentially inserted into an expression vector pIgG containing genes for the hIgG1 heavy chain constant region and kappa light chain constant region. To express soluble antibodies in mammalian cells, recombinant pIgG was transiently transfected into human 293T cells using lipofectamine. Transfected cells were maintained in 293SFM for 8 days at 37 ℃. During this time, the medium was changed 2 times and the culture supernatant was collected. The full length antibody secreted into the medium was purified using protein a affinity chromatography (Pharmacia). Purified antibodies were concentrated to 1mg/ml, sterile filtered, and characterized by SDS-PAGE, ELISA, and Isothermal Titration Calorimetry (ITC).

EXAMPLE 3 physical and chemical analysis

Clones ROR1-4, ROR1-12, ROR1-14 were further tested in size exclusion chromatography. Specifically, 20 μg samples were injected onto a TSK G3000SWXL column using 100mM sodium phosphate+100 mM Na ₂SO₄ (pH 7.0) as running buffer. The run time was 29 minutes. All measurements were performed on an Agilent 1220 HPLC. Data were analyzed using OpenLAB software. The main peak of ROR1-12 is higher than 95% in SEC, indicating that the purified antibody has high purity and integrity.

Example 4 specific binding of anti-ROR 1 antibodies to human ROR1

The relative binding activity of the antibodies to recombinant human ROR1 was determined using ELISA assay.

Human ROR1 protein (catalog number: RO1-H522y, acrobiosystems Co.) was immobilized on a 96-well plate by incubation overnight at4 ℃. The plates were then blocked by incubation with 1% BSA in PBS for 1 hour at 37 ℃. After blocking, the plates were washed 3 times with PBST (PBS containing 0.05% Tween 20). Serial dilutions of anti-ROR 1 antibodies were prepared in binding buffer (PBS containing 0.05% Tween20 and 0.5% BSA) and incubated with immobilized protein for 1 hour at 37 ℃. After binding, the plates were washed 3 times with PBST, incubated with peroxidase-labeled donkey anti-human IgG (jackson immunoresearch company (JacksonImmuno Research)) diluted 1/15,000 in binding buffer for 1 hour at 37 ℃, washed again, developed with TMB and terminated with 1M H ₂SO₄. The absorbance at 450nm-620nm was measured.

The EC50 and representative binding curves for anti-ROR 1 antibodies binding to human ROR1 are shown in fig. 1 (fig. 1, below and where reference to VLS101 in the figures refers to ROR1 antibodies in VLS101 ADC) indicating that these clones specifically bind to human ROR1.

Example 5 affinity of anti-ROR 1 antibodies for human ROR1

5.1

Buffer solution is added into the prewetted disc, and the AHC sensor is pre-balanced for 10min. The baseline was equilibrated with 1 XPBS, 0.02% Tween-20 for 100s prior to loading. ROR1 antibody-coupled AHC sensor: ROR1 antibody binding sensors were prepared by diluting the ROR1 antibody to 5 μg/mL with 1 XPBS, 0.02% Tween-20, and stopping the curing time for 200 seconds. Running buffer (1 XPBS, 0.02% Tween-20) equilibrates the baseline for 150s. The analytes ROR1 Protein (ARCO, cat. No. RO1-H522 y) were diluted with Running buffer at 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.13nM and 1.56nM. The analyte binds for 200s and dissociates for 600s. The regenerated liquid is regenerated for 30s. The resulting data were fit to a 1:1 binding model using an Octet software and calculated using Global fitting.

TABLE 2 affinity parameters for ROR1-4 and ROR1-14

Name of the name	K_a(M^-1S^-1)	K_d(S^-1)	K_D(M)
				ROR1-4	3.28E+05	3.15E-04	9.62E-10
ROR1-14	2.57E+05	1.75E-04	6.81E-10

5.2

The kinetic binding activity of anti-ROR 1 antibodies to human ROR1 (Acro biosystems) was measured by surface plasmon resonance using the Biacore T200 system (Biacore, GE HEALTHCARE).

Goat anti-human fcγ antibody (Jackson ImmunoReaserch, cat No. 109-005-098) at about 7000RU was immobilized on CM5 sensor chip by amino acid coupling chemistry. ROR1 antibodies bind to the surface of immobilized goat anti-human IgG antibodies. HBS-EP+ buffer was used as running buffer. Different concentrations of human ROR1 protein (from 6.25nM to 200 nM) were injected into the antibody surface. After each cycle, CM5 chip surface was regenerated by injection of 10mM glycine (ph 1.5). The binding rate Ka and dissociation Kd are analyzed using a background subtraction binding sensorgram, and the equilibrium dissociation constant K _D. The resulting dataset was fitted to a 1:1langmuir binding model using Biacore T200 evaluation software.

TABLE 3 affinity parameters for ROR1-12 and ROR1-14

Name of the name	K_a(M^-1S^-1)	K_d(S^-1)	K_D(M)
				ROR1-12	7.75E+5	1.37E-3	1.76E-9
ROR1-14	3.58E+5	3.68E-4	1.02E-9

Example 6 no cross-reaction of anti-ROR 1 antibodies with mouse ROR1

ELISA assays were used to determine the relative binding activity of antibodies to mouse ROR 1.

The mouse ROR1 protein (catalog number: RO1-M5221, acrobiosystems company) was immobilized on a 96-well plate by incubation overnight at 4 ℃. The plates were then blocked by incubation with 1% BSA in PBS for 1 hour at 37 ℃. After blocking, the plates were washed 3 times with PBST (PBS containing 0.05% Tween 20). Serial dilutions of anti-ROR 1 antibodies were prepared in binding buffer (PBS containing 0.05% Tween20 and 0.5% BSA) and incubated with immobilized protein for 1 hour at 37 ℃. After binding, the plates were washed 3 times with PBST, incubated with peroxidase-labeled donkey anti-human IgG (jackson immunoresearch company (JacksonImmuno Research)) diluted 1/15,000 in binding buffer for 1 hour at 37 ℃, washed again, developed with TMB and terminated with 1M H ₂SO₄. The absorbance at 450nm-620nm was measured.

Representative binding curves for these antibodies are shown in figure 2, indicating that these clones did not bind to mouse ROR 1.

Example 7 Cross-reaction experiments of anti-ROR 1 antibodies with human ROR2

ELISA assays were used to determine the relative binding activity of antibodies to human ROR 2.

Human ROR2 (catalog number: RO2-H52E5, acrobiosystems) was immobilized on a 96-well plate by incubation overnight at 4 ℃. The non-specific binding sites were blocked by incubation with 1% BSA in PBS for 1 hour at 37 ℃. After blocking, the plates were washed 3 times with PBST (PBS containing 0.05% Tween 20). Serial dilutions of anti-ROR 1 antibodies and human IgG controls were prepared in binding buffer (PBS containing 0.05% Tween20 and 0.5% BSA) and incubated with immobilized protein for 1 hour at 37 ℃. After binding, plates were washed 3 times with PBST, incubated with peroxidase-labeled donkey anti-human IgG (Jackson Immuno Research) diluted 1/15,000 in binding buffer for 1 hour at 37 ℃, washed again, developed with TMB and with 1m h2so4. The absorbance at 450nm-620nm was measured.

Representative binding curves for these antibodies are shown in FIG. 3, indicating that ROR1-14 binds to human ROR2 and ROR1-12 does not bind to human ROR 2.

Example 8 preparation of antibody drug conjugates ROR1-4-BL20MMAE, ROR1-12-BL20MMAE

The ROR 1-targeting antibodies ROR1-4 and ROR1-12 stock solutions were replaced with 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate (NaH ₂PO₄-Na₂HPO₄)/150 mM sodium chloride (NaCl)/2 mM ethylenediamine tetraacetic acid (EDTA), and the concentration was adjusted to 10mg/mL in a reaction buffer having pH 7.4, and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was added in a 10-fold molar excess, and the reaction solution was stirred at 28℃for 4 hours. The reaction solution was cooled to 20℃and a proper amount of Diethylacetamide (DMA) was added thereto, followed by addition of compound Ic-4 (10 mg/ml, previously dissolved in DMA) in a 5-fold molar ratio, so that the volume ratio of DMA in the reaction system was not more than 10%, and the reaction system was stirred at 25℃for 1.0 hour for coupling. The coupling reaction mixture was gel-filtered using a desalting column with sodium dihydrogen phosphate-disodium hydrogen phosphate/sucrose buffer at pH 7.4 and peak samples were collected according to UV280 UV absorbance. Then sterilized by a 0.22 micron pore size filter device and stored at-80℃and the resulting antibody conjugates were designated ROR1-4-BL20MMAE, ROR1-12-BL20MMAE.

The results are shown in FIGS. 4-9, wherein the mass spectrum desugared pattern of the humanized antibody ROR1-4 (FIG. 4) and the HIC and mass spectrum desugared pattern of the antibody conjugate ROR1-4-BL20MMAE (ROR 1-4-BL 20E) respectively show that the antibody ROR1-4 forms the antibody conjugate ROR1-4-BL20MMAE after coupling reaction, the molecular weight of the conjugate accords with the expected value, and DAR is about 4.0. The mass spectrum desugared spectrum of the humanized antibody ROR1-12 (figure 7) and the HIC and mass spectrum desugared spectrum of the antibody conjugate ROR1-12-BL20MMAE (figures 8 and 9) show that the antibody ROR1-12 forms an antibody conjugate ROR1-12-BL20MMAE (abbreviated as ROR1-12-BL 20E) after coupling reaction, and the molecular weight of the conjugate accords with an expected value, and DAR is about 4.0.

Example 9 preparation of ROR1-4-GGFG-Dxd, ROR1-12-GGFG-Dxd

The ROR1-12 stock solution of the ROR 1-targeting antibody was replaced with 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate (NaH ₂PO₄-Na₂HPO₄)/150 mM sodium chloride (NaCl)/2 mM ethylenediamine tetraacetic acid (EDTA), and the concentration was adjusted to 10mg/mL in a reaction buffer of pH7.0, and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was added in a molar ratio of 10-fold excess, and the reaction solution was stirred at 28℃for 4 hours. The reaction solution was cooled to room temperature, a proper amount of Diethylacetamide (DMA) was added, and then a 12-fold excess molar ratio of compound GGFG-DXd (purchased from Shanghai Hao Yuan chemical, 10mg/ml was previously dissolved in DMA) was added to ensure that the volume ratio of DMA in the reaction system was not more than 10%, and the reaction system was stirred at 25 ℃ for 1.0 hour for coupling. The coupling reaction mixture was gel-filtered using a desalting column with 4-morpholinoethanesulfonic acid (MES) -Tris/sucrose buffer at pH 6.6 and peak samples were collected according to UV280 UV absorbance. Then sterilized by a 0.22 micron pore size filter device and stored at-80℃and the resulting antibody conjugate was designated ROR1-12-GGFG-Dxd.

The results are shown in FIGS. 10, 11 and 12, wherein the mass spectrum desugared pattern of the humanized antibody ROR1-12 (FIG. 10) and the HIC and mass spectrum desugared pattern of the antibody conjugate ROR1-12-GGFG-Dxd (abbreviated as ROR 1-12-DX) respectively show that the antibody ROR1-12 forms the antibody conjugate ROR1-12-DX after the coupling reaction, the molecular weight of the conjugate is consistent with the expected value, and DAR is about 8.0.

Example 10 in vivo anti-tumor Activity of ROR1 antibody-conjugated drugs (ROR 1-ADCs) against human mantle cell lymphoma JeKo-1

In vitro antitumor activity of ROR1 antibody-conjugated drugs (ROR 1-ADCs) against human mantle cell lymphoma JeKo-1, triple negative breast cancer cells MDA-MB-231, non-small cell lung cancer cells NCI-H1975 (all purchased from the national academy of sciences cell bank), i.e. allograft or xenograft of cancer cells in rodents, was determined by in vivo experiments and tumors were treated with the combination. The mice tested were treated with drugs or controls and monitored for weeks or more to measure time to tumor doubling, log cell killing, and tumor inhibition.

The curative effect of ROR1-12-BL20E, ROR-12-DX on human mantle cell lymphoma JeKo-1 mouse subcutaneous transplantation tumor.

Each NOD-Scid mouse (Shanghai Ling Biotechnology Co., ltd.) was inoculated with JeKo-1 cells subcutaneously, and when tumors grew to about 100mm ³, the (IV) drugs were intravenously injected 1 time (Q4D) every 4 days, 2 times (D0, D4) in total, with an injection volume of 10mL/Kg; the dosage and regimen are shown in Table 4. Tumor diameter was measured twice weekly with vernier calipers and tumor volume (V) was calculated as: v=1/2×a×b ² (where a and b represent length and width, respectively).

TABLE 4 Table 4

T/C (%) = (T-T0)/(C-C0) ×100 where T, C is tumor volume at the end of the experiment; t0 and C0 are tumor volumes at the beginning of the experiment. Tumor growth inhibition rate (TGI%) =100-T/C (%).

Tumor growth inhibition% (TGI%) =100- (T-T0)/t0×100 when tumor regression occurs

Comparisons between two groups of tumor volumes or tumor weights were tested using the two-tailed Student's t, P <0.05 defined as statistically significant differences, unless otherwise specified.

ROR1-12-BL20E (5 mg/kg, IV, D0, 4) has obvious inhibition effect on the growth of human mantle cell lymphoma JeKo-1 mouse subcutaneous transplantation tumor, the tumor inhibition rate is 92%, and 1/6 tumor part is resolved; the tumor inhibition rate of ROR1-12-DX (5 mg/kg, IV, D0, 4) on JeKo-1 mice was 81%; the tumor-bearing mice can better tolerate the drugs, and no obvious symptoms such as weight reduction occur. In comparison, the efficacy of ROR1-12-BL20E on JeKo-1 subcutaneous grafts was significantly greater than that of ROR1-12-DX (P <0.05, equivalent dose group comparison), and the results are shown in FIG. 13 (the effect of ROR1-12-BL20E, ROR1-12-DX on human mantle cell lymphoma JeKo-1 subcutaneous graft growth).

EXAMPLE 11 efficacy of ROR1 antibody-coupled drugs (ROR 1-ADCs) ROR1-12-BL20E, ROR1-4-BL20E, VLS101-MMAE on subcutaneous transplantation tumor in human breast cancer MDA-MB-231 nude mice

ROR1-12-BL20E, ROR-4-BL 20E, VLS-MMAE (5 mg/kg, IV, QW. Times.3) inhibited growth of human breast cancer MDA-MB-231 mice subcutaneous transplantation tumor at rates of 49%, 39% and 24%, respectively (FIG. 14 influence of ROR1-12-BL20E, ROR1-4-BL20E, VLS101-MMAE on growth of human breast cancer MDA-MB-231 subcutaneous transplantation tumor); the tumor-bearing mice can better tolerate the drugs, and no obvious weight reduction and other symptoms occur (the influence of ROR1-12-BL20E, ROR1-4-BL20E, VLS101-MMAE on the weight of the tumor-bearing mice in FIG. 15). In comparison, ROR1-12-BL20E has relatively better drug effect on MDA-MB-231 mice subcutaneous transplantation tumor.

EXAMPLE 12 efficacy of ROR1 antibody-coupled drugs (ROR 1-ADCs) ROR1-12-BL20E, ROR1-4-BL20E, VLS-MMAE on human lung cancer H1975 mouse subcutaneous transplantation tumor

Each BALB/c nude mouse (Beijing Warcon Biotechnology Co., ltd.) was inoculated subcutaneously with 5X 10 ⁶ H1975 cells and when the tumor had grown to 100-150 mm ³, the drugs were intravenously Injected (IV) in groups according to the tumor volume 3 times in total, with an injection volume of 10mL/Kg. Tumor diameters were measured twice weekly with vernier calipers.

ROR1-12-BL20E, ROR-4-BL 20E, VLS101-MMAE (5 mg/kg, IV, 1 times per week, 3 times total) inhibited growth of human lung cancer H1975 mice subcutaneous transplantable tumors at tumor growth inhibition rates (TGI%) of 69%, 43% and 19%, respectively (FIG. 16 effect of ROR1-12-BL20E, ROR1-4-BL20E, VLS-MMAE on growth of human lung cancer H1975 subcutaneous transplantable tumors); the tumor-bearing mice can better tolerate the drugs, and no obvious weight reduction and other symptoms occur (the influence of ROR1-12-BL20E, ROR1-4-BL20E, VLS101-MMAE on the weight of the tumor-bearing mice in FIG. 17). In comparison, ROR1-12-BL20E showed the best effect on H1975 mice with subcutaneous transplants (P >0.05, comparison in the equivalent dose group).

The present application has been described in terms of several embodiments, but the description is illustrative and not restrictive, and it will be apparent to those of ordinary skill in the art that many more embodiments and implementations are possible within the scope of the described embodiments. Although many possible combinations of features are shown herein and discussed in the detailed description, many other combinations of the disclosed features are possible. Any feature or element of any embodiment may be used in combination with or in place of any other feature or element of any other embodiment, unless expressly limited otherwise.

Claims

1. An antibody or antigen-binding fragment thereof that targets ROR1, comprising:

And

2. The antibody or antigen-binding fragment thereof of claim 1, comprising:

b) A complementarity determining region VH CDR2 comprising the amino acid sequence shown in SEQ ID No. 3 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and

b) A complementarity determining region VH CDR2 comprising the amino acid sequence shown in SEQ ID No. 19 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and

b) A complementarity determining region VH CDR2 comprising the amino acid sequence shown in SEQ ID No. 35 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and

3. The antibody or antigen-binding fragment thereof of claim 2, comprising:

4. The antibody or antigen binding fragment thereof of claim 3, comprising the amino acid sequence set forth in SEQ ID NOs 15-16, 31-32 or 47-48 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, selected from the group consisting of a monoclonal antibody, a chimeric antibody, a single chain antibody, an Fv, a single chain Fv (scFv), fd, fab, fab ', and F (ab') 2.

6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, which binds human ROR1 with a KD of 2.0 x 10 ^-9 M or less.

7. The antibody or antigen binding fragment thereof of any one of claims 1-6, which does not cross-react with mouse ROR 1.

8. A nucleic acid sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-7.

9. A plasmid or vector comprising the nucleic acid sequence of claim 8.

10. A host cell comprising and expressing the nucleic acid sequence of claim 8 or the plasmid or vector of claim 9.

11. Antibody coupling drug with structure shown in formula I

Ab- (L-D) _n (formula I),

Wherein Ab is the antibody or antigen-binding fragment thereof of any one of claims 1-7;

L is a linker;

D is a cytotoxic drug; and is also provided with

12. The antibody conjugated drug of claim 11, wherein L is selected from the group consisting of Maleimide Caproyl (MC), maleimide (MAL), succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate) (SMCC) linkers attached to the antibody moiety, disubstituted maleimides, and comprising one or more linkers of valine-citrulline (VC), valine-alanine (VA), glycine-phenylalanine-glycine (GGFG), alanine-alanine (AAA), p-aminobenzyloxycarbonyl (PAB), polyethylene glycol (PEG).

13. The antibody conjugated drug of claim 11, wherein D is selected from one or more of the group consisting of:

(iii) Topoisomerase inhibitors, camptothecins, SN38, irinotecan, dxd.

14. A kit for diagnosing a disease, the kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-7, and instructions for use.

15. A composition comprising the antibody-conjugated drug of claim 11.

16. Use of an antibody or antigen binding fragment thereof according to any one of claims 1-7 in the manufacture of a kit for diagnosing a disease.

17. Use of an antibody-conjugated drug according to claim 11 or a composition according to claim 15 in the manufacture of a medicament for the treatment of cancer.

18. A method of treating a disease comprising administering to a subject in need thereof an antibody-conjugated drug according to claim 11 or a composition according to claim 15.

19. A method for diagnosing a disease comprising using the antibody or antigen-binding fragment thereof according to any one of claims 1-7 or the kit of claim 14.