Anti-ROR1 antibodies and anti-ROR1 antibody-drug conjugates and their medical uses

Abstract

Anti-ROR1 antibodies and anti-ROR1 antibody-drug conjugates and their medical uses.

Description

[Technical field]

[0001] This application claims priority to a Chinese patent application filed on December 28, 2021 (application number CN202111623091.0) and a Chinese patent application filed on March 10, 2022 (application number CN202210230033.X).

[0002] The present disclosure relates to anti-ROR1 antibodies, anti-ROR1 immunoconjugates, methods for their preparation, pharmaceutical compositions containing them, and their use in the preparation of medicaments for treating ROR1-mediated diseases or conditions, in particular in the preparation of anti-cancer drugs. [Background technology]

[0003] The statements herein are not necessarily intended to constitute prior art, but merely to provide background information relevant to the present disclosure.

[0004] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) belongs to the receptor tyrosine kinase (RTK) family. ROR1 is a typical type I single transmembrane protein on the cell membrane and contains three distinct domains on the extracellular side: immunoglobulin-like (Ig-like) domain, cysteine-rich frizzled domain (FZD), and kringle domain (KD). Intracellularly, it is composed of a tyrosine kinase domain and is mainly involved in downstream RTK signaling. As an RTK protein, the cytoplasmic activation of ROR1 requires two steps: 1. an increase in intrinsic catalytic activity, and 2. the formation of binding sites for downstream signaling pathway proteins. These two steps are achieved by inducing autophosphorylation of intracellular tyrosine residues after ligand-mediated oligomerization. Current studies have shown that ROR1 can be expressed in the skeletal system, respiratory and cardiac systems, and synaptic cells of central neurons at different stages of embryonic development, but its expression is obviously downregulated after birth. In tumor cells, ROR1 can bind to WNT5a protein to activate the non-classical WNT pathway and maintain the stem cell properties of tumor cells, including proliferation, migration and differentiation, and is one of the surface markers of tumor stem cells (CSCs). The correlation of ROR1 expression status with tumors was first identified in B-cell chronic lymphoma leukemia (CLL). Studies have found that ROR1 is highly expressed in various hematological tumors, including mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), etc. Further research into ROR1 expression profiles has been extended to solid tumors. Studies have shown that a series of solid tumors, such as triple-negative breast cancer (TNBC), various subtypes of lung cancer, various subtypes of ovarian cancer, and pancreatic cancer, all showed different degrees of ROR1 expression.

[0005] No expression of ROR1 was detected in most normal tissues, including important human organs such as the heart, spleen, lung, and liver, but some expression was detected only in parathyroid, pancreatic islet, and gastrointestinal tissues. Considering that ROR1 expression is highly different in normal tissues and tumor tissues, and has different degrees of expression in various tumors (including blood tumors and solid tumors), it can be a desirable target for the development of ADC molecules. Summary of the Invention

[0006] The present disclosure relates to an isolated anti-ROR1 antibody, HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 2; and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:3.

[0007] In some embodiments, the anti-ROR1 antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 4, 16 or 22, HCDR2 shown in SEQ ID NO: 5, 17 or 23, and HCDR3 shown in SEQ ID NO: 6, 18 or 24, the light chain variable region comprises an LCDR1 represented by SEQ ID NO: 7, 19 or 25, an LCDR2 represented by SEQ ID NO: 8, 20 or 26, and an LCDR3 represented by SEQ ID NO: 9, 21 or 27, The above CDR regions are determined according to the Kabat, IMGT or Chothia numbering conventions.

[0008] In some embodiments, the anti-ROR1 antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; The above CDR regions are determined by the Kabat numbering convention.

[0009] In some embodiments, the anti-ROR1 antibody according to any one of the above, wherein the anti-ROR1 antibody is a full-length human antibody or an antigen-binding fragment thereof.

[0010] In some embodiments, the anti-ROR1 antibody of any one of the preceding claims, which comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region has an amino acid sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO:2; and / or The light chain variable region has an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:3.

[0011] In some embodiments, the anti-ROR1 antibody of any one of the preceding claims, which comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO:2; and The amino acid sequence of the light chain variable region is shown in SEQ ID NO:3.

[0012] In some embodiments, the anti-ROR1 antibody according to any one of the above, wherein the antibody further comprises an antibody heavy chain constant region and a light chain constant region. Preferably, the heavy chain constant region is selected from human IgG1, IgG2, IgG3 and IgG4 constant regions, and the light chain constant region is selected from human antibody κ and λ chain constant regions. More preferably, the antibody comprises the heavy chain constant region shown in SEQ ID NO: 10 and the light chain constant region shown in SEQ ID NO: 11.

[0013] In some embodiments, the anti-ROR1 antibody of any one of the above is a heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 12, and / or and a light chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:13.

[0014] In some embodiments, the anti-ROR1 antibody of any one of the above is It comprises a heavy chain as shown in SEQ ID NO:12 and a light chain as shown in SEQ ID NO:13.

[0015] In some embodiments, the anti-ROR1 antibody according to any one of the above, wherein the anti-ROR1 antibody is at a concentration of 1×10 -8 The antibody binds to human ROR1 or an epitope thereof with a KD of less than M, and the KD is measured by surface plasmon resonance.

[0016] In some embodiments, the disclosure further provides an isolated anti-ROR1 antibody that binds to human ROR1 competitively with any one of the above anti-ROR1 antibodies.

[0017] In some embodiments, the disclosure further provides an isolated nucleic acid molecule encoding any one of the above anti-ROR1 antibodies.

[0018] In some embodiments, the disclosure further provides a host cell comprising any one of the above described nucleic acid molecules.

[0019] In some embodiments, the present disclosure further provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the anti-ROR1 antibodies described above, or the nucleic acid molecule described above, and one or more pharma- ceutically acceptable vectors, diluents, or excipients.

[0020] In some embodiments, the disclosure further provides a method for immunologically detecting or measuring ROR1, the method comprising contacting a subject or a sample derived from a subject with an anti-ROR1 antibody described in any one of the above.

[0021] In some embodiments, the present disclosure further provides an immunoconjugate or a pharma- ceutically acceptable salt thereof, comprising any one of the above-described anti-ROR1 antibodies and an effector molecule, wherein the effector molecule is coupled to the anti-ROR1 antibody. Preferably, the effector molecule is selected from a radioisotope, an antitumor agent, an immunomodulator, a biological response modifier, a lectin, a cytotoxic drug, a chromophore, a fluorophore, a chemiluminescent compound, an enzyme, a metal ion, and any combination thereof.

[0022] In some embodiments, the immunoconjugate or a pharma- ceutically acceptable salt thereof is provided, wherein the structure of the immunoconjugate is [ka] Among them, n is an integer or decimal number from 1 to 10, and n is preferably an integer or decimal number from 1 to 8; Pc is any one of the anti-ROR1 antibodies described above, and L is a linker unit.

[0023] In some embodiments, the immunoconjugate or a pharma- ceutically acceptable salt thereof is provided, wherein the structure of the immunoconjugate is [ka] Among them, n is an integer or decimal number from 1 to 10, and n is preferably an integer or decimal number from 1 to 8; Pc is any one of the anti-ROR1 antibodies described above, and L is a linker unit.

[0024] In some embodiments, the immunoconjugate or pharma- ceutically acceptable salt thereof according to any one of the above embodiments, wherein n is an integer or decimal number of 3 to 5, and preferably 4.

[0025] In some embodiments, the immunoconjugate or pharma- ceutically acceptable salt thereof according to any one of the above, wherein n is an integer or decimal number of 6 to 8, for example, but not limited to, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, or a range between any two of the above values.

[0026] In some specific embodiments, the immunoconjugate or pharma- ceutically acceptable salt thereof according to any one of the above, wherein n is 8.

[0027] In some specific embodiments, n is expressed in the form of a DAR value, as will be understood by those skilled in the art. Since the DAR value is a measurement, when a specific n value (or DAR value) is mentioned in this disclosure, it should be understood to include an appropriate error range, and the size of the error range can be determined by those skilled in the art depending on the measurement system.

[0028] In some specific embodiments, the immunoconjugate or pharma- ceutically acceptable salt thereof according to any one of the above, wherein n is 7.39±0.05.

[0029] In some embodiments, the immunoconjugate or pharma- ceutically acceptable salt thereof according to any one of the above, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 - and L 1 -(succinimid-3-yl-N)-WC(O)-, -CH 2 -C(O)-NR 1 -WC(O)- or -C(O)-WC(O)-, where W is C 1-8 Alkyl group or C 1-6alkyl-cycloalkyl groups, L 2 -NR 2 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 2 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 )p 1 C(O)- or a chemical bond, of which p 1 is an integer from 1 to 20, preferably a chemical bond; L 3 is a peptide residue consisting of 2 to 7 amino acid residues, among which the amino acid residues are selected from the amino acid residues consisting of the amino acids phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E) and aspartic acid (D); L 4 -NR 3 (CR 4 R 5 ) t -, -C(O)NR 3 -, -C(O)NR 3 (CH 2 ) t - or a PAB group, in which t is an integer from 1 to 6; R 1 , R 2 and R 3 are the same or different and each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group; R 4 and R 5 are the same or different and each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

[0030] In some embodiments, the immunoconjugate or a pharma- ceutically acceptable salt thereof according to any one of the above, wherein L 3 is a dipeptide residue, preferably valine-citrulline (VC).

[0031] In some embodiments, the immunoconjugate or a pharma- ceutically acceptable salt thereof according to any one of the above, wherein L 3 is a tetrapeptide residue, preferably GGFG.

[0032] In some embodiments, the immunoconjugate or a pharma- ceutically acceptable salt thereof according to any one of the above, wherein L 4 -NR 3 (CR 4 R 5 )t-, of which R 3 , R 4 or R 5 are the same or different and each independently represent a hydrogen atom or C 1-6 is an alkyl group, and t is 1 or 2.

[0033] In some embodiments, the immunoconjugate or a pharma- ceutically acceptable salt thereof according to any one of the above, wherein L 4 is a PAB group.

[0034] In some embodiments, the immunoconjugate or a pharma- ceutically acceptable salt thereof according to any one of the above, wherein the structure of the immunoconjugate is [ka] and [ka] Selected from among these, n and Pc are defined as above.

[0035] In some embodiments, the immunoconjugate or pharma- ceutically acceptable salt thereof of any one of the above, wherein the structure is [ka] Among them, n is an integer or decimal number from 1 to 10, and n is preferably an integer or decimal number from 1 to 8; Pc is an anti-ROR1 antibody comprising a heavy chain shown in SEQ ID NO:12 and a light chain shown in SEQ ID NO:13.

[0036] In some embodiments, the immunoconjugate or pharma- ceutically acceptable salt thereof of any one of the above, wherein the structure is [ka] Among them, n is an integer or decimal number from 1 to 10, and n is preferably an integer or decimal number from 1 to 8; Pc is an anti-ROR1 antibody comprising a heavy chain shown in SEQ ID NO:12 and a light chain shown in SEQ ID NO:13.

[0037] In some embodiments, in any one of the above-mentioned immunoconjugates or pharma- ceutically acceptable salts thereof, n is an integer of 3 to 5 or a decimal number.

[0038] In some embodiments, n is selected from an average value of 1 to 10, or 1 to 8, or 2 to 8, or 2 to 7, or 2 to 4, or 3 to 8, or 3 to 7, or 3 to 6, or 4 to 7, or 4 to 6, or 4 to 5. In some embodiments, n is an average value calculated from one or more of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, n is preferably 6 to 8, with an average value of 8 being more preferred.

[0039] In some embodiments, the present disclosure further provides a pharmaceutical composition comprising any one of the anti-ROR1 antibodies described above, or any one of the nucleic acid molecules described above, or any one of the immunoconjugates or pharma- ceutically acceptable salts thereof, and one or more pharma- ceutically acceptable excipients, diluents, or vectors.

[0040] In some embodiments, the present disclosure further provides a use of any one of the anti-ROR1 antibodies described above, or any one of the nucleic acid molecules described above, or any one of the immunoconjugates or pharma- ceutically acceptable salts thereof described above, or any one of the pharmaceutical compositions described above, in the preparation of a medicament for treating a ROR1-mediated disease or condition.

[0041] In another aspect, the present disclosure provides a use of any one of the immunoconjugates or pharma- ceutically acceptable salts thereof, or a pharmaceutical composition comprising the same, in the preparation of a medicament for treating a ROR1-mediated disease or condition. In some embodiments, the ROR1-mediated disease or condition is a ROR1 high-, moderate-, or low-expressing cancer.

[0042] In some embodiments, the present disclosure further provides the use of any one of the anti-ROR1 antibodies described above, or any one of the nucleic acid molecules described above, or any one of the immunoconjugates or pharma- ceutically acceptable salts thereof described above, or any one of the pharmaceutical compositions described above, in the preparation of a medicament for treating a tumor or cancer, wherein the tumor and cancer are selected from breast cancer, pancreatic cancer, lung cancer, esophageal cancer, non-small cell lung cancer, laryngeal tumor, sarcoma, pharyngeal tumor, oral tumor, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, colorectal cancer, lymphoma and leukemia.

[0043] In some embodiments, the present disclosure further provides a reagent kit comprising any one of the anti-ROR1 antibodies described above, or any one of the nucleic acid molecules described above, or any one of the immunoconjugates or pharma- ceutically acceptable salts thereof described above, or any one of the pharmaceutical compositions described above.

[0044] In another aspect, the present disclosure provides a pharmaceutical composition comprising any one of the above-described anti-ROR1 antibodies, immunoconjugates, or pharma- ceutically acceptable salts thereof, and one or more pharma- ceutically acceptable excipients, diluents, or vectors. In some embodiments, the unit dose of the pharmaceutical composition comprises 0.1-3000 mg or 1-1000 mg of the anti-ROR1 antibody or the immunoconjugate.

[0045] In another aspect, the present disclosure provides the use of any one of the above described immunoconjugates or pharma- ceutically acceptable salts thereof, or a pharmaceutical composition comprising same, as a medicament.

[0046] In some embodiments, the disclosure further provides a method of preventing or treating a disease or condition, the method comprising administering to a subject a therapeutically effective amount of any one of the anti-ROR1 antibodies described above, or any one of the nucleic acid molecules described above, or any one of the immunoconjugates or pharma- ceutically acceptable salts thereof, or any one of the pharmaceutical compositions described above. In some embodiments, the disease or condition is a disease or condition associated with ROR1.

[0047] In another aspect, the present disclosure further relates to a method for treating a tumor, the method comprising administering to a subject in need thereof a therapeutically effective amount of any one of the above described immunoconjugates or pharma- ceutically acceptable salts thereof, or a pharmaceutical composition comprising same, wherein in some embodiments, the tumor is a cancer associated with high, moderate or low ROR1 expression.

[0048] In another aspect, the present disclosure further relates to a method for treating a tumor or cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of any one of the above described immunoconjugates or pharma- ceutically acceptable salts thereof, or a pharmaceutical composition comprising same, wherein in some embodiments, the tumor or cancer is selected from breast cancer, pancreatic cancer, lung cancer, esophageal cancer, non-small cell lung cancer, laryngeal tumor, sarcoma, pharyngeal tumor, oral cavity tumor, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, colorectal cancer, lymphoma and leukemia.

[0049] In another aspect, the present disclosure further provides any one of the above-described anti-ROR1 antibodies or immunoconjugates thereof as a medicament. In some embodiments, the medicament for treating cancer or tumor is preferably a medicament for treating ROR1-mediated cancer.

[0050] The active compound (e.g., an anti-ROR1 antibody or immunoconjugate thereof according to the present disclosure) can be prepared in a form suitable for administration by any suitable route. The unit dose presentation of the compound or composition according to the present disclosure can be a tablet, capsule, cachet, bottled drug solution, drug powder, granule, topical tablet, suppository, reconstituted powder or liquid formulation.

[0051] The dose of the compound or composition used in the therapeutic method according to the present disclosure will generally vary depending on the severity of the disease, the weight of the subject, and the relative potency of the compound. However, as a general guide, a suitable unit dose may be 0.1 to 1000 mg.

[0052] The pharmaceutical composition according to the present disclosure may contain one or more additives in addition to the active compound, and the additives are selected from components such as fillers, diluents, binders, wetting agents, disintegrants, or excipients. Depending on the method of administration, the composition may contain 0.1-99% by weight of the active compound.

[0053] The anti-ROR1 antibody provided in the present disclosure has good affinity for cell surface antigens and good cell endocytosis efficiency. The anti-ROR1 antibody immunoconjugate provided in the present disclosure has strong tumor inhibition efficiency, as well as better efficacy and lower toxicity and side effects in the animal body.

[0054] Detailed Description of the Invention term Unless otherwise limited, all technical and scientific terms used herein are consistent with those commonly understood by those skilled in the art. Although any methods and materials similar or equivalent to those described herein may be used to practice or test the present disclosure, the preferred methods and materials are described herein. In describing and claiming the present disclosure, the following terms are used in accordance with the following definitions.

[0055] When trade names are used in this disclosure, they are intended to include formulations of that trade name product, drugs and active drug portions of that trade name product.

[0056] Unless specifically stated to the contrary, terms used in the specification and claims have the following meanings.

[0057] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0058] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those amino acids that are coded for by codons, and those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an α-carbon bonded to a hydrogen, a carboxy group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of amino acids, but act in a manner similar to naturally occurring amino acids.

[0059] The term "antibody" herein is used in the broadest sense and covers a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), provided they exhibit the desired antigen-binding activity.

[0060] "Native antibody" refers to a naturally occurring immunoglobulin molecule. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has one variable region (VH), also called the variable heavy domain or the heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has one variable region (VL), also called the variable light domain or the light chain variable domain, followed by one constant light domain (also called the light chain constant domain, CL).

[0061] The terms "full length antibody," "complete antibody," and "whole antibody" may be used interchangeably herein and refer to an antibody that comprises a structure essentially similar to that of a native antibody or whose heavy chain comprises an Fc region as defined herein.

[0062] An "isolated antibody" is an antibody that has been separated from a component of its natural environment. In some embodiments of the present disclosure, the antibody can be purified to greater than 90% or 99% purity. In some embodiments, the antibody is purified and measured by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[0063] The term "variable region" or "variable domain" refers to the domains involved in antibody binding antigen in the antibody heavy and / or light chains. Natural IgG antibody VH and VL each contain four conserved framework regions (FR) and three complementarity determining regions (CDR). Among them, the term "complementarity determining region" or "CDR" refers to the region that mainly promotes antigen binding in the variable domain, and "framework" or "FR" refers to the variable domain residues excluding the CDR residues in the variable domain. VH contains three CDR regions, HCDR1, HCDR2, and HCDR3, and VL contains three CDR regions, LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL is sufficient to confer antigen binding specificity.

[0064] The boundaries of the amino acid sequence of the CDRs can be determined by various known methods, such as the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention, the "ABM" numbering convention, the "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001), and the ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003), Front Immunol. 2018 Oct 16; 9: 2278), and the correspondence between various numbering systems is well known to those skilled in the art. Exemplary numbers are shown in Table 1 below. [Table 1]

[0065] The term "light chain" comprises a variable region domain, VL, and a constant region domain, CL. The VL is at the amino-terminus of the light chain. Light chains include kappa chains and lambda chains.

[0066] The term "heavy chain" comprises a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. VH is at the amino-terminus of the heavy chain and the CH domain is at the carboxy-terminus, of which CH3 is closest to the carboxy-terminus of the polypeptide. The heavy chain may belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0067] The term "antibody fragment" refers to a molecule that is distinct from a complete antibody and includes a portion of the complete antibody that binds to the antigen that the complete antibody binds to. Antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab') 2 These include, but are not limited to, single domain antibodies, single chain Fab (scFab), diabodies, linear antibodies, single chain antibody molecules (e.g., scFv), and multispecific antibodies consisting of antibody fragments.

[0068] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, and includes native sequence Fc regions and modified Fc regions. In some embodiments, the Fc region of a human IgG heavy chain is defined to extend from the amino acid residue at position Cys226, or from Pro230 to its carboxy terminus. The boundaries of the Fc region of an antibody heavy chain may be altered, for example, by deletion of the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region, or by deletion of the C-terminal glycine and lysine (residues 446 and 447 according to the EU numbering system) of the Fc region. Thus, in some embodiments, a composition of whole antibodies may include antibodies in which any K447 and / or G446+K447 residues have been removed. In some embodiments, a composition of whole antibodies may include antibodies in which the K447 and / or G446+K447 residues have not been removed. In some embodiments, the composition of whole antibodies comprises a mixture of antibodies with and without the K447 residue and / or the G446+K447 residue. Suitable native sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described, for example, in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0069] The term "human antibody" refers to an antibody in which the variable and constant regions are human sequences. The term covers antibodies derived from human genes but with altered sequences, e.g., reduced potential immunogenicity, increased affinity, removal of cysteines or glycosylation sites that may result in undesired folding, etc. The term covers such antibodies that are recombinantly produced in non-human cells (which may confer glycosylation not characteristic of human cells). The term also covers antibodies produced in transgenic mice that contain human immunoglobulin heavy and light chain loci. The definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0070] The term "affinity matured" antibody refers to an antibody that has one or more modifications in one or more CDRs of the antibody that improves the affinity of the antibody for its antigen compared to the parent antibody. In some embodiments, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies can be generated by methods known in the art. Marks et al., Bio / Technology 10:779-783 (1992) describes affinity maturation by shuffling VH and VL domains. The following publications describe random mutagenesis of CDR and / or framework residues: Barbas et al., PNAS, 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0071] The term "monoclonal antibody" refers to a population of essentially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules in the population are the same, except for minor natural mutations that may exist. In contrast, a polyclonal antibody preparation usually contains a plurality of different antibodies having different amino acid sequences in their variable domains, which are generally specific for different epitopes. "Monoclonal" describes the character of the antibody as being obtained from an essentially 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 from the hybridoma method described in Kohler et al., (1975) Nature 256:495, or by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Alternatively, the "monoclonal antibodies" can be isolated from phage antibody libraries by the techniques described in Clackson et al., (1991) Nature 352:624-628 and Marks et al., (1991) J. Mol. Biol. 222:581-597. "Monoclonal antibodies" can also be prepared by methods see Presta (2005) J. Allergy Clin. Immunol. 116:731, or using transgenic animals containing all or part of the human immunoglobulin loci.

[0072] The term "antigen" refers to a molecule or portion of a molecule capable of being selectively bound by an antibody. An antigen may have one or more epitopes capable of interacting with different antibodies.

[0073] The term "epitope" refers to an area or region on an antigen capable of specific binding to an antibody. An epitope may be formed by a contiguous string of amino acids (linear epitope) or may comprise non-contiguous amino acids (conformational epitope) that are spatially proximate, for example, due to folding of the antigen (i.e., tertiary folding of the antigen). Conformational and linear epitopes differ in that the binding of an antibody to a conformational epitope is not detectable in the presence of denaturing solvents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.

[0074] Screening for antibodies that bind to a specific epitope (i.e., antibodies that bind the same epitope) can be performed using methods well known in the art, including, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide truncation analysis, epitope cutting, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0075] An "antibody that binds to the same epitope" as a reference antibody or an "antibody that binds competitively" to a reference antibody refers to an antibody that inhibits the binding of the reference antibody to an antigen by 50% or more in a competitive assay, or an antibody whose binding to an antigen is inhibited by the reference antibody by 50% or more. Also, for example, to determine whether the antibody to be measured binds to the same epitope as the reference antibody, the reference antibody is allowed to bind to the antigen under saturating conditions. After removing the excess reference antibody, the ability of the antibody to be measured to bind to the antigen is evaluated. To confirm whether the antibody to be measured binds to the same epitope or whether the binding is hindered only by spatial reasons, conventional experiments (e.g., peptide mutations and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art) can be used. Such a measurement method should be performed in two settings, i.e., both antibodies are saturating antibodies. If in both sets only the first (saturating) antibody is able to bind to the antigen, it can be concluded that the antibody to be measured binds to the antigen competitively with the reference antibody.

[0076] In some embodiments, two antibodies are considered to bind to the same or overlapping epitope if a 1-fold, 5-fold, 10-fold, 20-fold or 100-fold excess of one antibody inhibits binding of the other antibody by at least 50%, at least 75%, at least 90%, or even 99% or more, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

[0077] In some embodiments, with respect to amino acid mutations in an antigen that can reduce or eliminate binding of one antibody, two antibodies are considered to bind the "same epitope" if essentially all of them are capable of reducing or eliminating binding of another antibody, and two antibodies are considered to have "overlapping epitopes" if only some of them are capable of reducing or eliminating binding of the other antibody.

[0078] The term "affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity and reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can usually be expressed as an equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.

[0079] As used herein, the term "kassoc" or "ka" refers to the association rate of a particular antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a particular antibody-antigen interaction. The term "KD" refers to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed as a molar concentration (M). The KD value of an antibody can be measured by methods known in the art. For example, the affinity in solution is measured by a biosensing system such as a surface plasmon resonance system, or by solution equilibrium titration (SET).

[0080] The terms "specifically bind," "specific binding," or "binding" refer to an antibody that binds to an antigen or epitope thereof with higher affinity than it has for other antigens or epitopes. Typically, antibodies bind to an antigen or epitope with a higher affinity than it has for other antigens or epitopes with a higher affinity than it has for other antigens or epitopes with a higher affinity than it has for other antigens or epitopes. -7 M or less (e.g., about 1 × 10 -8 M or less, about 1×10 -9 In some embodiments, the antibody binds to an antigen or epitope thereof with an equilibrium dissociation constant (KD) of 100 M or less. In some embodiments, the antibody binds to an antigen with a KD that is 10% or less (e.g., 1%) of the antibody's KD for binding to a non-specific antigen (e.g., BSA, casein). KD can be measured by methods known in the art, for example, by BIACORE. (登録商標)It is measured by surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or its epitopes may have cross-reactivity to other related antigens, for example, to corresponding antigens from other species (homologs) (e.g., humans or monkeys such as Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp) and Callithrix jacchus (common marmoset, marmoset)).

[0081] The terms "anti-ROR1 antibody" and "antibody that binds to ROR1" refer to an antibody that can bind to ROR1 with sufficient affinity. In some embodiments, an antibody that binds to ROR1 has an affinity of < about 1 μM, < about 100 nM, < about 10 nM, < about 1 nM, < about 0.1 nM, < about 0.01 nM, or < about 0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 ~10 -9 M, for example 10 -9 M or less). In some embodiments, the anti-ROR1 antibody binds to a conserved ROR1 epitope in ROR1 from different species.

[0082] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcR) on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophage cells) to specifically bind these cytotoxic effector cells to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "arm" the cytotoxic cells and are essential for such killing. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch & Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, for example, as described in No. 5500362 or 5821337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, the ADCC activity of a target antibody may be assessed in vivo, for example, in an animal model (for example, as disclosed in Clynes et al. (USA) 95:652-656 (1998)).

[0083] The term "antibody-dependent cellular phagocytosis" ("ADCP") refers to the mechanism by which antibody-coated target cells are removed by internalization by phagocytes (eg, macrophages or dendritic cells).

[0084] The term "complement dependent cytotoxicity" or "CDC" refers to a mechanism for inducing cell death in which the Fc effector domain of a target-binding antibody binds and activates complement component C1q, which in turn activates the complement cascade, resulting in target cell death. Complement activation can also deposit complement components on the surface of target cells that promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0085] The terms "nucleic acid" and "polynucleotide" may be used interchangeably herein and refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The terms cover nucleic acids containing known nucleotide analogs or modified backbone residues or linkers, synthetic, naturally occurring, and non-naturally occurring, that have similar binding properties as the reference nucleic acid and are metabolized in a similar manner as the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell, as described below, which generally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. A "nucleic acid encoding an anti-ROR1 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody.

[0086] Unless otherwise stated, a particular nucleic acid sequence also implicitly covers its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence specified. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0087] The terms "polypeptide" and "protein" may be used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, and apply to naturally occurring and non-naturally occurring amino acid polymers.

[0088] The term "naked antibody" refers to an antibody that is not conjugated to a heterologous module (e.g., a cytotoxic module) or a radioactive marker. Naked antibodies can be present in pharmaceutical formulations.

[0089] The term "identity", "sequence identity" or "amino acid sequence identity" refers to the degree (percentage) to which the amino acids / nucleic acids of two sequences are identical at equivalent positions when the two sequences are optimally aligned (introducing gaps as necessary to obtain the maximum sequence identity percentage, and any conservative substitutions are not considered part of the sequence identity). To measure the percentage of sequence identity, alignment can be achieved by various methods known in the art, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR). Those skilled in the art can determine the parameters to be applied for the measured alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences to be compared.

[0090] The term "conservatively modified variant" or "conservative substitution" refers to the substitution of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophilicity / hydrophobicity, backbone configuration and rigidity, etc.) such that such changes can usually be made without altering the biological activity of the protein. In general, it is known to those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not essentially alter the biological activity (see, for example, Watson et al., (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). The term "conservatively modified variant" when applied to a nucleic acid sequence refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, to the essentially the same sequence. Due to the degeneracy of genetic codons, any given protein can be encoded by multiple nucleic acids having the same function. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at each position of a codon that specifies alanine, the codon can be changed to any corresponding codon without changing the encoded polypeptide. Such a nucleic acid variation is a "silent variation", which is a type of conservative modification variation. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variation of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG--generally the only codon for methionine and TGG--generally the only codon for tryptophan) may all be modified to produce a molecule with the same function. Thus, in each of the above sequences, each silent variation of the nucleic acid that encodes a polypeptide is implicitly included.

[0091] The term "expression vector" or "expression construct" refers to a vector that is applied for transformation of a host cell and contains nucleic acid sequences that direct and / or control the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and RNA splicing of the coding region operably linked thereto, if any, introns.

[0092] As used herein, "operably linked" refers to a relationship that permits the components to which the term is applied to carry out their inherent functions under appropriate conditions. For example, a control sequence "operably linked" to a protein coding sequence in an expression vector is ligated thereto such that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.

[0093] The terms "host cell," "host cell line," and "host cell culture," which may be used interchangeably, refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the original transformed cell and its derived progeny, without regard to the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, and may contain mutations. The term includes mutant progeny that have the same function or biological activity as the cell screened or selected from the original transformed cell. Host cells include prokaryotic and eukaryotic host cells, of which eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Exemplary host cells are Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells, Pichia pastoris, Pichia finlandica, Candida albicans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei.

[0094] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably and include the progeny of such cells. Thus, the terms "transformant" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of passages. It is to be understood, further, that due to deliberate or unintentional mutations, not all progeny will have exactly the same DNA content. Mutant progeny that have the same function or biological activity as the initially transformed cell from which they were screened are included.

[0095] Methods for producing and purifying antibodies and antigen-binding fragments well known in the art are described, for example, in chapters 5-8 and 15 of the Cold Spring Harbor Antibody Laboratory Techniques Manual. The antibodies or antigen-binding fragments described in the disclosure have one or more human FR regions added to the non-human CDR regions by genetic engineering methods. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) homepage by aligning the IMGT human antibody variable region germline gene database with MOE software, or from the Immunoglobulin journal, 2001 ISBN 012441351.

[0096] The engineered antibodies or antigen-binding fragments according to the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector. The recombinant immunoglobulin expression vector can be stably transfected into host cells. In one of the more preferred prior art, a mammalian expression system will cause glycosylation of the antibody, especially at the N-terminal site of the Fc region. Stable clones are obtained by expressing an antibody that specifically binds to human ROR1. Positive clones are expanded in a bioreactor medium to produce the antibody. The culture medium from which the antibody is secreted can be purified by conventional techniques. For example, purification is performed on an A or G Sepharose FF column. Non-specifically bound components are washed away. Furthermore, the bound antibody is eluted by a pH gradient method, and the antibody fragment is detected and collected by SDS-PAGE. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers may be removed by conventional methods such as molecular sieving, ion exchange, etc. The resulting product must be immediately frozen, such as at -70°C, or lyophilized.

[0097] "Isolated" means removed from its naturally occurring state, and in this case the designated molecule is essentially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other materials such as cellular debris, growth medium, etc. In general, the term "isolated" is not intended to imply the total absence of these materials, or the absence of water, buffers, or salts, unless, for example, they are present in amounts that would significantly interfere with the experimental or therapeutic uses of the compounds described herein.

[0098] The term "drug" refers to chemical substances that can modify the physiological functions and pathological conditions of the body and can be applied to the prevention and treatment of diseases. Drugs include cytotoxic drugs. There is no strict boundary between drugs and poisons. A poison refers to a chemical substance that has a toxic effect on the body and damages human health in a relatively small dose, while any drug can cause a toxic reaction when the dose is too large.

[0099] A cytotoxic drug is a substance that inhibits or prevents the function of cells and / or causes the death or destruction of cells. In principle, cytotoxic drugs can kill tumor cells if they are present in high enough concentrations, but due to lack of specificity, they may also cause apoptosis of normal cells while killing tumor cells, resulting in severe side effects. Cytotoxic drugs include, for example, toxins such as small molecule toxins and enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioisotopes of Lu), chemotherapy drugs, antibiotics and nucleolytic enzymes.

[0100] An "antibody drug conjugate (ADC) or immunoconjugate" is an antibody linked to a biologically active or cell-killing drug via a linking unit.

[0101] The antibody or antibody fragment described in this disclosure may be coupled to an effector molecule in any manner. For example, the antibody or antibody fragment may be chemically or recombinantly attached to a cytotoxic drug. Chemical methods for preparing fusions or conjugates are known in the art and can be used to prepare immunoconjugates. The method for coupling the antibody or antibody fragment to a drug must be capable of linking the antibody to the drug without interfering with the ability of the antibody or antibody fragment to bind to the target molecule.

[0102] In one embodiment, both the antibody and the cytotoxic drug are proteins and can be coupled by techniques well known in the art. Hundreds of crosslinkers disclosed in the art can couple the two proteins. The crosslinker is generally selected according to the reactive functional groups available or insertable on the antibody or the cytotoxic drug. Also, photoreactive crosslinkers can be used if there are no reactive groups. An intron may have to be provided between the antibody and the cytotoxic drug. Crosslinkers known in the art include homobifunctional agents such as glutaraldehyde, dimethyl adipimidate, and bis(diazobenzidine), and heterobifunctional agents such as m-maleimidobenzoyl-N-hydroxysuccinimide and sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide.

[0103] Crosslinkers available for coupling effector molecules to antibody fragments include, for example, TPCH (S-(2-thiopyridyl)-L-cysteine ​​hydrazide) and TPMPH (S-(2-thiopyridinyl)mercapto-propionyl hydrazide). TPCH and TPMPH have previously reacted partially with carbohydrates of glycoproteins that have been oxidized by moderate periodate treatment, resulting in the formation of hydrazone bonds between the hydrazide moiety of the crosslinker and the periodate-derived aldehyde. The heterobifunctional crosslinkers GMBS (N-(γ-maleimidobutyryloxy)-succinimide) and SMCC (succinimidyl 4-(N-maleimido-methyl)cyclohexane) react with primary amines, thereby introducing a maleimide group into the moiety. This maleimide group can then react with a crosslinker-introducible sulfhydryl group on another moiety, resulting in the formation of a stable thioether bond between the moieties. In cases where steric hindrance between the components would interfere with the activity of any one of the components, a long spacer arm can be introduced between the components by a cross-linker such as 3-(2-pyridyldithio)propionic acid n-succinimidyl ester (SPDP).Therefore, there are many suitable cross-linkers available, each selected for its effect on optimal immunoconjugate yield.

[0104] The "drug loading amount" is also called the drug-to-antibody ratio (DAR), that is, the average number of drugs conjugated to each antibody in the ADC. It may be, for example, in the range of about 1 to about 10 drugs coupled to each antibody. In some embodiments, it is in the range of about 1 to about 8 drugs coupled to each antibody, preferably selected from the ranges of 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 4, 3 to 5, 5 to 6, 5 to 7, 5 to 8, and 6 to 8. Exemplarily, the drug loading amount may be one or more calculated average values ​​among 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The general formula of the ADC according to the present disclosure includes a collection of immune complexes within the above certain range. In an embodiment of the present disclosure, the drug loading amount may be represented by n, which is a decimal or an integer. The drug loading amount can be measured by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA test, and HPLC.

[0105] The term "linker unit" or "linking fragment" or "linking unit" refers to a chemical structural fragment or bond that is linked at one end to an antibody or antigen-binding fragment thereof and at the other end to a drug, and may be linked to another linker and then to a drug.

[0106] The term linker may include one or more linker elements. Exemplary linker elements include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("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 include an extender, a spacer, and an amino acid unit, and can be synthesized by methods known in the art, for example, the methods described in US2005-0238649A1. The linker may be a "cleavable linker" that facilitates release of the drug in cells. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992), U.S. Patent No. 5,208,020) may be used.

[0107] Linker elements include, but are not limited to, the following: MC=6-maleimidocaproyl, having the following structure: [ka] Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in a protease cleavable linker); Citrulline = 2-amino-5-ureidopentanoic acid, The PAB group is p-aminobenzyloxycarbonyl (an example of a "self-immolative" linker moiety) having the following structure: [ka] Me-Val-Cit = N-methyl-valine-citrulline (in which the linker peptide bond was modified to prevent cleavage by cathepsin B); MC(PEG)6-OH = maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteines); SPP = N-succinimidyl 4-(2-pyridylthio)pentanoate; SPDP = N-succinimidyl 3-(2-pyridyldithio)propionate, SMCC = succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate; IT=iminothiolane.

[0108] In one embodiment of the disclosure, the cytotoxic drug is coupled to the antibody by a linking unit.

[0109] The drug loading of the complex is (1) controlling the molar ratio of the drug linker fragment to the monoclonal antibody; (2) controlling reaction time and temperature; (3) selecting different reaction reagents; The amount of oxygen can be controlled by a number of methods, including but not limited to:

[0110] The term “alkyl group” refers to a saturated, straight or branched chain aliphatic hydrocarbon group, which has 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms (i.e., C 1-20 The alkyl group is an alkyl group having 1 to 12 carbon atoms (i.e., C 1-12 alkyl group) is preferred, and alkyl groups having 1 to 6 carbon atoms (i.e., C 1-6Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-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, 6-methylhexyl, 7-methylhexyl, 8-methylhexyl, 9-methylhexyl, 10-methylhexyl, 11-methylhexyl, 12-methylhexyl, 13-methylhexyl, 14-methylhexyl, 15-methylhexyl, 16-methylhexyl, 17-methylhexyl, 18-methylhexyl, 19-methylhexyl, 22-methylhexyl, 23-methylhexyl, 24-methylhexyl, 25-methylhexyl, 26-methylhexyl, 27-methylhexyl, 28-methylhexyl, 29-methylhexyl, 30-methylhexyl, 31-methylhexyl, 32-methylhexyl, 33-methylhexyl, 34-methylhexyl, 35-methylhexyl, 36-methylhexyl, 37-methylhexyl, 38-methylhexyl, 39-methylhexyl, 40-methylhexyl, 41-methylhexyl, 42-methylhexyl, 43-methylhexyl, 44-methylhexyl, 45-methylhex Examples of the branched chain isomers include 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-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, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched chain isomers thereof. The alkyl group may be substituted or unsubstituted and, when substituted, it may be substituted at any available attachment point, and the substituents are preferably one or more selected from D atoms, halogens, alkoxy groups, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclyloxy groups, hydroxy groups, hydroxyalkyl groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups and heteroaryl groups.

[0111] The term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic all-carbocyclic ring (i.e., a monocyclic cycloalkyl group) or polycyclic ring system (i.e., a polycyclic cycloalkyl group) having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 3- to 20-membered cycloalkyl group). The cycloalkyl group is preferably a cycloalkyl group having 3 to 12 ring atoms (i.e., a 3- to 12-membered cycloalkyl group), more preferably a cycloalkyl group having 3 to 8 ring atoms (i.e., a 3- to 8-membered cycloalkyl group), and most preferably a cycloalkyl group having 3 to 6 ring atoms (i.e., a 3- to 6-membered cycloalkyl group) or a cycloalkyl group having 5 ring atoms (i.e., a 5-membered cycloalkyl group).

[0112] The monocyclic cycloalkyl groups include, by way of non-limiting example, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, and cyclooctyl groups.

[0113] The term "haloalkyl group" refers to the replacement of a hydrogen on an alkyl group with one or more halogens, where the alkyl group is as defined above.

[0114] The term "deuterated alkyl group" refers to a group in which a hydrogen on an alkyl group is replaced with one or more deuterium atoms, wherein the alkyl group is as defined above.

[0115] The term "haloalkoxy" refers to an alkoxy group in which a hydrogen is replaced with one or more halogens, wherein the alkoxy group is as defined above.

[0116] The term "hydroxyalkyl group" refers to an alkyl group substituted with one or more hydroxy groups, wherein the alkyl group is as defined above.

[0117] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0118] The term "hydroxy group" refers to --OH.

[0119] The term "amino group" means -NH 2 Refers to...

[0120] The term "cyano" refers to -CN.

[0121] "Optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not. For example, "C optionally substituted with a halogen or cyano group" 1-6 By "alkyl group" is meant that a halogen or cyano group may or may not be present, and this description includes cases where the alkyl group is substituted with a halogen or cyano group and cases where the alkyl group is not substituted with a halogen or cyano group.

[0122] "Substituted" refers to one or more hydrogen atoms, preferably 1 to 6, more preferably 1 to 3 hydrogen atoms in a group being independently replaced with a corresponding number of substituents. A person skilled in the art can determine (experimentally or theoretically) possible or impossible substitutions without much effort. For example, an amino group or a hydroxy group having a free hydrogen may be unstable if it is attached to a carbon atom having an unsaturated (e.g., olefinic) bond.

[0123] A "pharmaceutical salt" refers to a salt of a compound according to the present disclosure, which may be selected from inorganic salts or organic salts. Such salts have the desired biological activity while being safe and effective when used in a mammalian body. The salts may be prepared separately during the final isolation and purification process of the compound, or by reacting a suitable group with a suitable base or acid. In general, bases for forming pharmaceutical acceptable salts include inorganic bases such as sodium hydroxide and potassium hydroxide, and organic bases such as ammonium. In general, acids for forming pharmaceutical acceptable salts include inorganic acids and organic acids.

[0124] The concentration of the immune complex in this disclosure is measured in terms of the protein concentration, i.e., the concentration of the antibody moiety in the immune complex.

[0125] With respect to a drug or pharmacologically active agent, the term "therapeutically effective amount" refers to a sufficient dose of the drug or agent to produce a desired effect while being non-toxic. The effective amount is determined by the person and depends on the age and general condition of the subject, and also on the specific active agent, and the appropriate effective amount for an individual can be determined by one skilled in the art through routine testing.

[0126] The compounds and intermediates of the present disclosure may exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can be interconverted by a low energy barrier. For example, proton tautomers (also called protolytic tautomers) include interconversions by protolysis, such as keto-enol and imine-enamine, lactam-lactim isomerization. An example of lactam-lactim equilibrium is between A and B as shown below.

[0127] [ka]

[0128] All of the compounds in this disclosure can be depicted in Form A or Form B. All tautomeric forms are within the scope of this disclosure. The naming of a compound does not exclude any tautomeric forms.

[0129] A "prodrug" is a substance that can be transformed in vivo to yield the active drug compound under physiological conditions, for example, by hydrolysis in blood.

[0130] As used herein, the term "pharmacologically acceptable" means that these compounds, materials, compositions and / or dosage forms can be applied in contact with the tissues of a subject without undue toxicity, irritation, allergic response or other problem or complication, within the scope of reasonable medical judgment, and are effective for the desired use, with a reasonable benefit / risk ratio.

[0131] The preparation of conventional pharmaceutical compositions is set out in the Chinese Pharmacopoeia.

[0132] As used herein, the singular forms "a," "an," and "the" include plural references and vice versa unless the context clearly indicates otherwise.

[0133] "About" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which is determined in part by how the value is measured or measured, i.e., limited by the measurement system. In the context of a particular measurement, result, or embodiment, unless expressly stated otherwise in the examples or elsewhere in the specification, "about" means within one standard deviation, as is customary in the art.

[0134] The term "pharmacologically acceptable vector" or "medicable vector" refers to an ingredient in a pharmaceutical formulation that is not toxic to a subject, unlike the active ingredient. Pharmaceutically acceptable vectors include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0135] The term "package insert" is used to refer to instructions typically included in the commercial packaging of a therapeutic product that contain information on indications, usage, dosage, administration, concomitant therapy, contraindications and / or warnings associated with the use of such therapeutic product.

[0136] The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals), non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise indicated, the terms "patient" or "subject" may be used interchangeably herein. As used herein, the term "cyno" or "cynomolgus" refers to cynomolgus monkeys (Macaca fascicularis). In some embodiments, the individual or subject is a human.

[0137] The term "excipient" refers to an additive other than an active ingredient in a pharmaceutical formulation, and may be called an additive. For example, any of the following may be called an excipient: binders, fillers, disintegrants, and lubricants in tablets, base parts in ointments and creams that are semisolid preparations, preservatives, antioxidants, flavorings, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and coloring agents in liquid preparations.

[0138] The term "diluent" is also called filler, and its main use is to increase the weight and volume of tablets. The addition of diluent not only ensures a certain volume size, but also reduces the dose deviation of the main ingredient, improves the compression moldability of the drug, etc. When the tablet drug contains oily ingredients, it is necessary to add an absorbent to absorb the oily substance in order to maintain a "dry" state and contribute to the preparation into tablets. For example, starch, lactose, inorganic salts of calcium, microcrystalline cellulose, etc.

[0139] The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein or their physiological / medicinal salts or prodrugs and other chemical components, together with other components such as physiological / medicinal vectors and excipients, to facilitate administration to the body and contribute to the absorption of the active ingredients to further exert biological activity.

[0140] The pharmaceutical compositions may be in the form of a sterile injectable aqueous solution. Acceptable solvents and vehicles used may include water, Ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerin and processed to form a microemulsion. The injectable solution or microemulsion may be injected into the subject's bloodstream by localized bolus injection. Alternatively, the solutions and microemulsions are preferably administered by a method capable of maintaining a constant cyclic concentration of the compounds of the present disclosure. In order to maintain such a constant concentration, a continuous intravenous administration device may be used. An example of such a device is the Deltec CADD-PLUS.TM.5400 type intravenous pump.

[0141] The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension for use in intramuscular and subcutaneous administration. Such suspensions can be prepared according to known techniques using suitable dispersing or wetting agents and suspending agents as described above. The sterile injectable preparation may be a sterile injectable solution or suspension prepared in a non-toxic, gastrointestinal-tolerable diluent or solvent, for example, a solution prepared in 1,3-butanediol. Sterile fixed oils are also conveniently used as a solvent or suspending medium. For this purpose, any suitable fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acids, can also be used to prepare injectables.

[0142] "Administration" or "giving," as applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous agent, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid.

[0143] The term "sample" refers to a collection of fluids, cells or tissues isolated from a subject, as well as fluids, cells or tissues within a subject. Exemplary samples are biological fluids, such as blood, serum and serous fluid, plasma, lymphatic fluid, urine, saliva, cyst fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleural, pericardial, peritoneal, abdominal and other body cavities fluids, fluids collected from bronchial washings, synovial fluid, liquid solutions that have come into contact with a subject or biological source, such as cell and organ media (including cell or organ conditioned media), lavage fluids, tissue biopsies, fine needle aspirates, surgically excised tissues, organ cultures or cell cultures.

[0144] The term "pharmaceutically acceptable salt" or "medicinal salt" refers to a salt of an immunoconjugate according to the present disclosure, or a salt of a compound described in the present disclosure, which is safe and effective when used in a mammalian body and has the desired biological activity, and the antibody-drug conjugate according to the present disclosure contains at least one amino group and is therefore capable of forming a salt with an acid.

[0145] "Treatment" (and grammatical variations thereof) refers to a clinical intervention that seeks to alter the natural course of the individual being treated, and may be performed prophylactically or during clinical pathological processes. The desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction / reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, amelioration or alleviation of the disease state, and regression or improvement of prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the formation of disease or to slow the progression of disease.

[0146] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or underlying causes, prevent the appearance of symptoms and / or their underlying causes, and / or improve or ameliorate damage caused by or associated with a disease state (e.g., pulmonary disease). In some embodiments, the effective amount is a therapeutically or prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptom, particularly a condition or symptom associated with the disease state, or to prevent, inhibit, delay or reverse the progression of the disease state or any other undesirable symptoms associated in any way with the disease state in any other manner. A "prophylactically effective amount" is an amount that, when administered to a subject, confers a desired prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. Complete treatment or prevention does not necessarily occur after administration of a single dose, but may occur after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or multiple doses. A "therapeutically effective amount" and a "prophylactically effective amount" can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, an improvement in the subject's health status.

[0147] The above specification provides details of one or more embodiments of the present disclosure. Although the present disclosure can be practiced or tested by any methods and materials similar or equivalent to those described herein, the following describes the preferred methods and materials. Other features, objects, and advantages of the present disclosure will become apparent from the specification and claims. In the specification and claims, the singular forms include plural referents unless otherwise indicated by the context. Unless otherwise defined, all technical and scientific terms used herein have the common meanings understood by those skilled in the art. All patents and publications referenced in the specification are incorporated by reference. The following examples are presented to more fully illustrate preferred embodiments of the present disclosure. These examples should not be construed in any manner as limiting the scope of the present disclosure, which is limited by the scope of the claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0148] The present invention will be further described below in conjunction with examples, but these examples are not intended to limit the scope of the present invention.

[0149] Experimental methods for which specific conditions are not specified in the Examples or Test Examples of this disclosure are generally in accordance with common conditions or conditions recommended by the raw material or product manufacturers. See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, and Modern Methods in Molecular Biology, Ausubel et al., Greene Publishing Company, Wiley Interscience, NY. Reagents for which specific sources are not specified are conventional commercially available reagents.

[0150] I. Antibody Examples Example 1: Construction of ROR1-highly expressing cell line The pBABE-ROR1 lentivirus expression vector plasmid and pVSV-G, pGag-pol lentivirus packaging vectors were transfected into virus packaging cells 293T (Chinese Academy of Sciences Cell Bank, SCSP-502) with Lipofectamine 3000 (ThermoFisher, L3000001) transfection reagent, the culture supernatant containing the virus was collected, filtered, centrifuged at ultra-high speed, and the concentrated virus was infected into Chinese hamster ovary cells CHO-K1 (Chinese Academy of Sciences Cell Bank, SCSP-507) and screened with puromycin for 2 to 3 weeks, and then FACS single cell sorting was performed.

[0151] We detected ROR1 expression on the surface of lentivirus-infected CHO-K1 cells by FACS, and selected a monoclonal cell line with high ROR1 expression, which we named ROR1-CHO-K1. The selected monoclonal cell line was expanded and frozen in a refrigerator for further experiments.

[0152] Human ROR1 amino acid sequence (UniProtKB-Q01973) TIFF2025503493000011.tif154155

[0153] Example 2: Screening of anti-human ROR1 monoclonal antibodies Using a fully human semi-synthetic phage antibody library and antigen biotinylated human ROR1 (Kactus Biosystems (Shanghai) Co., Ltd., product number: ROR-HM401B), three rounds of panning were performed, and phage detection was performed by ELISA to obtain positive clones. The positive clones were sequenced to obtain the sequences, and then the positive clone sequences were inserted into the protein expression vector Phr-IgG and expressed in HEK293 and Expi-CHO-S. After purification, FACS and endocytosis activity confirmation experiments were performed to obtain the ROR1 fully human antibody molecule 347.

[0154] Fully human ROR1 antibody molecule 347 heavy chain variable region: TIFF2025503493000012.tif32155

[0155] Fully human ROR1 antibody molecule 347 light chain variable region: TIFF2025503493000013.tif27155

[0156] Below are the CDR regions determined according to the Kabat numbering convention for anti-ROR1 antibody 347 of the present disclosure:

[0157] [Table 2]

[0158] The CDR regions of the anti-ROR1 antibody 347 according to the present disclosure are numbered and determined according to the Chothia and IMGT rules: [Table 3]

[0159] The anti-ROR1 antibody may further comprise an antibody heavy chain constant region and a light chain constant region, the heavy chain constant region may be selected from human IgG1, IgG2, IgG3 and IgG4 constant regions, and the light chain constant region may be selected from human antibody kappa and lambda chain constant regions. In the present disclosure, the antibody comprises a heavy chain constant region whose sequence is set forth in SEQ ID NO: 10 and a light chain constant region whose sequence is set forth in SEQ ID NO: 11:

[0160] Human IgG1 heavy chain constant region: TIFF2025503493000016.tif64155

[0161] Human kappa light chain constant region: TIFF2025503493000017.tif27155

[0162] Anti-ROR1 antibody 347 sequence according to the present disclosure: Fully human ROR1 antibody 347 heavy chain: TIFF2025503493000018.tif80155

[0163] Fully human ROR1 antibody 347 light chain: TIFF2025503493000019.tif43155

[0164] In the present disclosure, the anti-ROR1 antibody Ab1 in WO2018237335 was used as a positive control, and its sequences are as follows: Ab1 heavy chain: TIFF2025503493000020.tif80155

[0165] Ab1 light chain: TIFF2025503493000021.tif43155

[0166] II. ADC Example Measuring the DAR value of an ADC The method for calculating the DAR value of the ADC according to the present disclosure employs RP-HPLC (reverse-phase high performance liquid chromatography) and is specifically as follows.

[0167] 1, Method of measurement: The naked antibody and the ADC sample (concentration 1 mg / mL) to be tested were reduced by adding 4 μL of DDT (sigma), and then the sample was placed in a water bath at 37 ° C for 1 hour, and then removed and placed in an inner tube. Detection was performed with a high-performance liquid chromatograph Agilent 1200, and the column was selected as Agilent PLRP-S 1000A 8 μm 4.6 × 250 mm, with column temperature: 80 ° C, DAD detector wavelength: 280 nm, flow rate: 1 mL / min, and injection volume: 40 μL. Then, the positions of the light and heavy chains were distinguished by comparing the spectrum of the sample with that of the naked antibody, and the DAR value was calculated by integrating the spectrum of the detected sample.

[0168] 2. Preparation of Solutions 1) 0.25M DTT solution: Preparation example: 5.78 mg of DTT was taken and thoroughly dissolved in 150 μL of purified water, after which a 0.25 M DTT solution was prepared and stored at -20°C. 2) Mobile phase A (0.1% TFA in water): Preparation example: Measure out 1000mL of purified water using a measuring cylinder, add 1mL of TFA (Sigma), mix thoroughly and homogenously, then use. Store at 2-8℃ for 14 days. 3) Mobile phase B (0.1% TFA in acetonitrile): Preparation example: Measure out 1000 mL of acetonitrile using a measuring cylinder, add 1 mL of TFA, mix thoroughly and homogenously, then use and store at 2-8°C for 14 days.

[0169] 3. Data analysis By comparing the spectra of the sample and the naked antibody, the positions of the light and heavy chains were distinguished, and the DAR value was calculated by integrating the spectrum of the detected sample.

[0170] The calculation formula is as follows: TIFF2025503493000022.tif43155 LC peak total area = LC peak area + LC+1 peak area Total HC peak area = HC peak area + HC+1 peak area + HC+2 peak area + HC+3 peak area LC DAR = Σ(number of linked drugs × peak area percentage) / LC peak total area HC DAR = Σ(number of linked drugs × peak area percentage) / total HC peak area DAR=LC DAR+HC DAR

[0171] Example 3: Preparation of ADC-1 [ka] Under the condition of 37°C, the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) hydrochloride (10 mM, 55.2 μL, 552 nmol) was added to an aqueous PBS buffer solution of antibody 347 (0.05 M PBS buffer solution with pH=6.5, 10.0 mg / mL, 3.14 mL, 212 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to terminate the reaction. The reaction solution was cooled to 25°C in a water bath.

[0172] Compound D1 (Hanxiang Biotechnology, CAS: 646502-53-6, 2.92 mg, 2218 nmol) was dissolved in 140 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted for 3 hours while shaking at 25 ° C., and the reaction was terminated. The reaction solution was desalted and purified with a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA), and the title product ADC-1 in PBS buffer (2.25 mg / mL, 13.5 mL) was obtained and stored in a refrigerator at 4 ° C.

[0173] The mean drug loading was calculated by RP-HPLC: n=4.04.

[0174] Example 4: Preparation of ADC-2 [ka] Under the condition of 37°C, the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) hydrochloride (10 mM, 25.3 μL, 253 nmol) was added to an aqueous PBS buffer solution of antibody Ab1 (0.05 M PBS buffer solution with pH=6.5, 10.0 mg / mL, 1.44 mL, 97.3 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to terminate the reaction. The reaction solution was cooled to 25°C in a water bath.

[0175] Compound D1 (1.28 mg, 972 nmol) was dissolved in 65 μL of DMSO, added to the above reaction solution, placed on a water bath shaker, and reacted with shaking at 25 ° C. for 3 hours to terminate the reaction. The reaction solution was desalted and purified with a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA) to obtain the title product ADC-2 in PBS buffer (1.24 mg / mL, 11 mL), which was then refrigerated and stored at 4 ° C.

[0176] The mean drug loading was calculated by RP-HPLC: n=4.43.

[0177] Example 5: Preparation of ADC-3 [ka] Under the condition of 37°C, the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) hydrochloride (10 mM, 101.4 μL, 1014 nmol) was added to an aqueous PBS buffer solution of antibody Ab1 (0.05 M PBS buffer solution with pH=6.5, 10.0 mg / mL, 6 mL, 405 nmol), and the solution was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours to terminate the reaction. The reaction solution was cooled to 25°C in a water bath.

[0178] Compound D1 (5.34 mg, 4058 nmol) was dissolved in 300 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25 ° C. for 3 hours to terminate the reaction. The reaction solution was desalted and purified with a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA) to obtain the title product ADC-3 in PBS buffer (3.33 mg / mL, 18 mL), which was stored in a refrigerator at 4 ° C.

[0179] The mean drug loading was calculated by RP-HPLC: n=4.76.

[0180] Example 6: Preparation of ADC-4 [ka] Under the condition of 37°C, the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) hydrochloride (10 mM, 1.145 mL, 11.45 μmol) was added to an aqueous solution of antibody 347 in PBS buffer (0.05 M aqueous solution of PBS buffer with pH=6.5, 10.0 mg / mL, 30 mL, 2027 nmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3.5 hours to terminate the reaction. The reaction solution was cooled to 25°C in a water bath.

[0181] Compound 9A (prepared from compound 9-A in Example 9 with reference to WO2020063676, the entire contents of which are incorporated herein by reference) (32.7 mg, 30.45 μmol) was dissolved in 1.7 mL of dimethyl sulfoxide, added dropwise to the above reaction solution, placed on a water bath shaker, and reacted at 25 ° C. for 3 hours while shaking to terminate the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA), to obtain the title product ADC-4 in PBS buffer (2.6 mg / mL, 80 mL), which was stored in a refrigerator at 4 ° C.

[0182] The mean drug loading was calculated by RP-HPLC: n=7.39.

[0183] Example 7: Preparation of ADC-5 [ka] Under the condition of 37°C, the prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 169 μL, 1690 nmol) was added to the PBS buffer solution of antibody 347 (0.05 M PBS buffer solution with pH=6.5, 10.0 mg / mL, 10 mL, 675 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3.5 hours to terminate the reaction. The reaction solution was cooled to 25°C in a water bath.

[0184] Compound 9A (7.26 mg, 6.76 μmol) was dissolved in 500 μL of dimethyl sulfoxide, added dropwise to the above reaction solution, placed on a water bath shaker, and reacted with shaking at 25 ° C for 3 hours to terminate the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-5 in PBS buffer (1.92 mg / mL, 48 mL), which was stored in a refrigerator at 4 ° C.

[0185] The average loading amount was calculated by RP-HPLC: n=3.99.

[0186] Test Example Biological evaluation Test Example 1: Protein-level affinity and kinetics experiments of antibodies Anti-ROR1 antibodies were analyzed by Biacore T200 (Cytiva) to characterize the affinity and binding kinetics. IgG antibodies were affinity captured on a Protein A biosensor chip (Cat.#29127556, Cytiva), and then a gradient series of human ROR1 His (Cat.#19771-H08H, Yiqiao Shenzhou) antigen diluted in HBS-EP buffer (pH 7.4, Cat.#BR-1006-69, Cytiva) was flowed over the chip surface. The antigen-antibody binding kinetics was tracked for 3 min, and the dissociation kinetics was tracked for 10 min, and the reaction signals were detected in real time by the Biacore T200 instrument to obtain binding-dissociation curves. After the dissociation of each experimental cycle was completed, the biosensor chip was regenerated by washing with 10 mM Gly-HCl pH 1.5 (Cat.#BR-1003-54, Cytiva). The data obtained was analyzed using Cytiva's BIAevaluation software with a 1:1 (Langmuir) binding model, and the ka (kon), kd (koff) and KD values ​​determined by this method are shown in the table below.

[0187] [Table 4]

[0188] Test Example 2: In vitro cell binding experiment of antibodies ROR1-CHO-K1 cells (Example 1), Jeko-1 cells (ATCC, CRL-3006), or MDA-MB-231 cells (ATCC, CRM-HTB-26) were each cultured at 1 × 10 6 Cell suspensions were prepared at 100 μL / well and added to 96-well round-bottom plates. After centrifugation to remove the supernatant, 50 μL / well of the antibody to be tested at various concentrations diluted in FACS buffer was added and incubated in the dark for 1 h in a 4°C refrigerator. After washing with FACS buffer by centrifugation three times at 500 g, working concentrations of fluorescent secondary antibody were added and incubated in the dark for 40 min in a 4°C refrigerator. After washing with FACS buffer by centrifugation three times at 500 g, the binding EC of the antibody to ROR1-expressing cells was detected using a BD FACSCantoII flow cytometer using geometric mean fluorescence intensity. 50 The value was calculated.

[0189] [Table 5]

[0190] Test Example 3: Endocytosis experiment of DT3C antibody This experiment was conducted to indirectly reflect the endocytosis status of ROR1 antibody by injuring cells with activated diphtheria toxin (DT) after DT3C protein enters cells. 50 The in vitro endocytosis activity of anti-ROR1 antibodies was evaluated according to the maximum killing value.

[0191] DT3C is a recombinantly expressed fusion protein that is a fusion of Fragment A of diphtheria toxin (toxin portion only) and 3C fragment of group G streptococcus (IgG binding portion). This protein has high affinity with the IgG portion of antibodies, and when endocytosis occurs in antibodies, it enters cells together and releases toxic DT under the action of intracellular furin. DT inhibits the activity of EF2-ADP ribosylation, blocks the protein translation process, and ultimately causes cell death. DT3C that has not entered cells does not have the activity of killing cells. The endocytosis activity of antibodies was evaluated according to the cell killing situation.

[0192] Prepare ROR1-CHOK1 cell suspension in fresh cell culture medium containing 20% ​​low IgG FBS at a cell density of 2 × 10 4 cells / mL, and 50 μL / well was added to the cell culture plate and incubated at 37°C with 5% carbon dioxide for 16 hours. DT3C was diluted to 1.6 μM in serum-free medium, and anti-ROR1 antibody was diluted to 266.4 nM in serum-free medium. 80 μL of DT3C and 80 μL of anti-ROR1 antibody were mixed uniformly in a 1:1 volume and incubated at room temperature for 30 minutes. The molar concentration of DT3C is 6 times that of anti-ROR1 antibody.

[0193] The mixture of DT3C and anti-ROR1 antibody was diluted 4-fold in serum-free medium, totaling 8 gradients, with the 9th and 10th points being pure medium. 50μL of the diluted mixture was added to 50μL of cells and incubated in an incubator for 3 days. 50μL of CTG was added to each well and incubated at room temperature in the dark for 10 minutes, and the white bottom membrane was attached to the bottom of the cell culture plate and placed in a plate reader Victor3 to read the chemiluminescence value.

[0194] [Table 6]

[0195] Test Example 4: Cross-linking ability to rat / mouse ROR1 antigen Anti-ROR1 antibodies were analyzed by Biacore T200 (Cytiva) to characterize the affinity and binding kinetics. IgG antibodies were affinity captured with a Protein A biosensor chip (Cat.#29127556, Cytiva), and then a series of gradient concentrations of mouse ROR1-His (Cat.#RO1-M522, Acro) antigen and rat ROR1-His (Cat.#RO1-R5221, Acro) antigen diluted in HBS-EP buffer (pH 7.4, Cat.#BR-1006-69, Cytiva) were flowed over the chip surface. The antigen-antibody binding kinetics was followed for 3 min, and the dissociation kinetics was followed for 15 min, and the reaction signals were detected in real time by a Biacore T200 instrument to obtain binding-dissociation curves. After the dissociation of each experimental cycle was completed, the biosensor chip was washed and regenerated with 10 mM Gly-HCl pH 1.5 (Cat.#BR-1003-54, Cytiva). The obtained data was analyzed using the 1:1 (Langmuir) binding model by Cytiva's BIAevaluation software, and the ka (kon), kd (koff) and KD values ​​measured by this method are shown in the table below.

[0196] [Table 7]

[0197] Conclusion: The 347 antibody has strong cross-linking to rat / mouse ROR1.

[0198] Test Example 5: In vivo efficacy evaluation of ADC molecule high expression CDX model JEKO-1 cells (human mantle cell lymphoma, MCL, 5 × 10 6 The mice were inoculated with 100 μL of 100% Matrigel (100 μL / mouse + 50% Matrigel / mouse / 200 μL) and divided into groups of 8 mice after 8 days. The average tumor volume of each group on the day of grouping was 223.09 mm. 3The ADC compound was administered by intraperitoneal injection at a dose of 5 mpk once a week for a total of one dose, and the mice were observed for 21 days.

[0199] Tumor volume and body weight were measured twice a week and the data were recorded. Mean values ​​were calculated using avg, SD values ​​using STDEV, SEM values ​​using STDEV / SQRT, and P values ​​for differences between groups using TTEST using Excel 2003 statistical software.

[0200] Tumor volume (V) calculation formula: V = 1 / 2 × L 長 ×L 短 2 Tumor growth rate T / C%=(T-T0) / (C-C0)×100% Tumor inhibition rate TGI(%)=1-T / C(%).

[0201] Among them, T and C are the tumor volumes of the treatment group and the control group at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment.

[0202] [Table 8]

[0203] Conclusion: In this experiment, the tumor inhibition rate of the test ADC-1 (5mpk) reached 93.4% (P<0.0001 vs blank control), and the tumor inhibition rate of the positive control ADC-2 (5mpk) was 85.6% (p<0.0001 vs blank control). In terms of tumor inhibition rate, the tumor inhibition rate of ADC-1 (5mpk) is superior to that of the positive control ADC-2 (5mpk).

[0204] Test Example 6: Evaluation of cellular activity of ADC molecules The purpose of this experiment was to detect the cell killing effect of ROR1-ADC samples, ADC-4 and ADC-5, and to measure the IC 50 and to evaluate the in vitro activity of ROR1-ADC by maximum killing value.

[0205] HCC827 / ROR1 (ROR1 overexpressing cell line), Jeko-1, MDA-MB-231 and CHO-K1 cells were digested with pancreatin, neutralized with fresh medium, centrifuged at 1000 rpm, resuspended in culture medium, counted, and the cell suspension was adjusted to the appropriate density and added to a 96-well cell culture plate at 135 μL per well, with 150 μL of medium alone added to the remaining peripheral wells. The culture plate was incubated in a 5% carbon dioxide, 37°C incubator for 24 hours.

[0206] ADC samples were prepared at various concentrations in PBS and diluted 5-fold with PBS for a total of 8 concentrations. Cell culture plates were removed and 15 μL of 10× solution was added to each well. The plates were incubated at 37°C with 5% carbon dioxide for 6 days.

[0207] 70μL of CTG was added to each well, incubated at room temperature in the dark for 10 minutes, and the white bottom membrane was attached to the bottom of the cell culture plate and placed on Victor 3 to read the chemiluminescence. The data from this experiment was processed using the data processing software GraphPad prism 5.0.

[0208] [Table 9]

[0209] Conclusion: The inhibitory activity of ADC-4 and ADC-5 against different cell lines was positively correlated with the antigen expression level, i.e., the higher the antigen expression, the stronger the inhibitory activity of the drug.

[0210] Test Example 7: Evaluation of in vivo activity of ADC molecules in the CDX model 1. Human mantle cell lymphoma (MCL) subcutaneous tumor model in mice JEKO-1 cells (5 × 10 6 The mice were inoculated with 100 μL of 50% Matrigel per mouse (100 μL per mouse) and divided into groups of 8 mice per group 7 days later. The tumor volume of each group on the day of grouping was 266.7 mm on average. 3The ADC compound was administered intraperitoneally at a dose of 2.5mpk / 5mpk, twice in total on the 1st and 14th days of grouping, respectively, and observed for 21 days.

[0211] Tumor volume and body weight were measured twice a week and the data were recorded. Mean values ​​were calculated using avg, SD values ​​using STDEV, SEM values ​​using STDEV / SQRT, and P values ​​for differences between groups using TTEST using Excel 2003 statistical software.

[0212] Tumor volume (V) calculation formula: V = 1 / 2 × L 長 ×L 短 2 Tumor growth rate T / C%=(T-T0) / (C-C0)×100% Tumor inhibition rate TGI(%)=1-T / C(%).

[0213] Among them, T and C are the tumor volumes of the treatment group and the control group at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment.

[0214] [Table 10]

[0215] Conclusion: In the Jeko-1 mouse subcutaneous tumor CDX model, the tumor-inhibiting activity of ADC-4 was superior to that of ADC-5, and the tumor-inhibiting rate of the tested ADC-4 (5 mpk) reached 100% (P<0.0001 vs blank control).

[0216] 2. Triple-negative breast cancer (TNBC) subcutaneous tumor model in mice Sixty NOD-SCID mice were subcutaneously injected with MDA-MB-231 cells (3 × 10 6 The mice were inoculated with 200 μL of cells per mouse, and 9 days later, they were divided into groups of 7 mice per group. The average tumor volume in each group on the day of grouping was 184.85 mm 3The ADC compounds were administered by intraperitoneal injection at a dose of 2.5mpk / 5mpk, administered only on the first day of grouping, and observed for 21 days.

[0217] Tumor volume and body weight were measured twice a week and the data were recorded. Mean values ​​were calculated using avg, SD values ​​using STDEV, SEM values ​​using STDEV / SQRT, and P values ​​for differences between groups using TTEST using Excel 2003 statistical software.

[0218] Tumor volume (V) calculation formula: V = 1 / 2 × L 長 ×L 短 2 Tumor growth rate T / C%=(T-T0) / (C-C0)×100% Tumor inhibition rate TGI(%)=1-T / C(%).

[0219] Among them, T and C are the tumor volumes of the treatment group and the control group at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment.

[0220] [Table 11]

[0221] Conclusion: In the MDA-MB-231 mouse subcutaneous tumor CDX model, after 20 days of observation, the tumor inhibition rate of the test ADC-4 (5mpk) reached 100% (P<0.0001 vs blank control), the tumor inhibition rate of the positive ADC-3 (5mpk) was 75.79% (p<0.0001 vs blank control), and the tumor inhibition rate of the test ADC-5 (5mpk) reached 92.21% (p<0.0001 vs blank control). In terms of tumor inhibition rate, the tumor inhibition rate of ADC-4 (5mpk) was superior to the positive ADC-3 (5mpk) and ADC-5 (5mpk), and in terms of body weight change, neither showed obvious toxicity.

[0222] Study Example 8: PK evaluation of ADC molecules The pharmacokinetics of a single dose of ROR1-ADC (ADC-4) was studied in adult SD rats (Zhejiang Weitong Lihua) and NDG C57 mice (Balbc). One molecule was distributed to one group of rats, and 10mpk of ROR1-ADC molecule was intravenously injected (n=4 / group), and one molecule was distributed to two groups of mice, and 10mpk of ROR1-ADC molecule was intravenously injected (n=3 / group). Serum was collected from rats at 0.083, 8, 24, 48, 96, 168, 240, 336, 504, and 672 hours after intravenous injection for biological analysis measurements, one group of mice was collected at 0.083, 8, 48, 168, 336, and 672 hours after intravenous injection for biological analysis measurements, and the other group was collected at 1, 24, 96, 240, and 504 hours after intravenous injection for biological analysis measurements. The rat / mouse serum samples were measured by HTRF method, and the contents of the samples waiting to be measured were quantitatively analyzed by the four-parameter model curve of the standard.

[0223] Pharmacokinetic parameters were analyzed by a noncompartmental model using WinNonlin software (6.4).

[0224] The results of the experiment are as follows:

[0225] [Table 12]

[0226] Conclusion: In both rats and mice, ADC-4 has good plasma stability.

[0227] Test Example 9: In vivo toxicity evaluation of ADC molecules in rats The ADC compound (prepared with saline) was injected into the tail vein of SD rats (male, Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.), 6 rats per group, once a week, twice in total, and each rat was injected at 5mL / kg according to body weight. The specific experimental design and experimental results are as follows:

[0228] [Table 13]

[0229] The results showed that the positive control ADC-3 caused rat deaths after a single dose, but ADC-4 did not cause any rat deaths after two doses of 35mpk and 70mpk, respectively. During the administration period, group C (ADC-3-35mpk) showed significant clinical side effects such as severe arching of the back, piloerection, emaciation, obvious hind limb weakness, slow movement, and hind limb paralysis after the first administration, and normal feeding and drinking were affected. Some of the animals died, and the remaining animals in group C were dissected early on the 6th day after drug administration to avoid censoring the data. No obvious abnormalities were observed in the animals in groups A and B after administration.

[0230] [Table 14]

[0231] Conclusion: ADC-4-35 / 70mpk: maximum tolerated dose (MTD) > 70mpk, toxicity was shown by slowed weight gain and thymic atrophy, etc. ADC-3-35mpk: MTD<35mpk (animals died) and toxicity was shown by weight loss, bone marrow suppression, significant atrophy of the thymus, and obvious effects on organ function such as the liver, lungs, and kidneys.

[0232] Experimental results suggest that ADC-4 has better in vivo safety, lower toxicity and side effects than the control molecule ADC-3.

Claims

1. 1. An isolated anti-ROR1 antibody, comprising: HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 2; LCDR1, LCDR2, and LCDR3 contained in the light chain variable region shown in SEQ ID NO: 3; An isolated anti-ROR1 antibody comprising:

2. 1. An isolated anti-ROR1 antibody, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; Isolated anti-ROR1 antibodies.

3. a heavy chain variable region and a light chain variable region, the heavy chain variable region has an amino acid sequence that is at least 90% identical to SEQ ID NO:2, and / or the light chain variable region has an amino acid sequence that is at least 90% identical to SEQ ID NO:3; Preferably, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 2; and The light chain variable region has an amino acid sequence set forth in SEQ ID NO:

3. The isolated anti-ROR1 antibody of claim 2.

4. a heavy chain having at least 85% sequence identity to SEQ ID NO: 12, and / or a light chain having at least 85% sequence identity to SEQ ID NO: 13; Preferably, comprising a heavy chain set forth in SEQ ID NO: 12 and a light chain set forth in SEQ ID NO: 13; The isolated anti-ROR1 antibody of claim 2.

5. 1 x 10 －８ 3. The isolated anti-ROR1 antibody of claim 2, which binds to human ROR1 or an epitope thereof with a KD of less than M, wherein the KD is measured by surface plasmon resonance.

6. 2. An isolated nucleic acid molecule encoding the isolated anti-ROR1 antibody of claim 1.

7. A host cell comprising the isolated nucleic acid molecule of claim 6.

8. A method for immunologically detecting or measuring ROR1, comprising contacting a subject or a sample derived from a subject with an isolated anti-ROR1 antibody according to any one of claims 1 to 5.

9. An immunoconjugate or a pharmaceutically acceptable salt thereof, 10. A method for the preparation of a medicament for the treatment of ROR1 comprising administering to a subject the isolated anti-ROR1 antibody of claim 1 and an effector molecule, wherein said effector molecule is coupled to said anti-ROR1 antibody; Preferably, the effector molecule is selected from a radioisotope, an anti-tumor agent, an immunomodulator, a biological response modifier, a lectin, a cytotoxic drug, a chromophore, a fluorophore, a chemiluminescent compound, an enzyme, a metal ion, and any combination thereof. An immunoconjugate or a pharmaceutically acceptable salt thereof.

10. The structure of the immune complex is 【Chemistry 1】 and 【Chemistry 2】 Selected from where: n is an integer or decimal number from 1 to 10, preferably an integer or decimal number from 1 to 8, more preferably 8; L is a linking unit, and Pc is the anti-ROR1 antibody of claim 2. The immunoconjugate according to claim 9 or a pharmaceutically acceptable salt thereof.

11. The linker unit -L- is -L １ -L ２ -L ３ -L ４ - and L １ -(succinimide-3-yl-N)-W-C(O)-, -CH ２ —C(O)—NR １ -WC(O)- or -C(O)-WC(O)-, where W is C １－８ Alkyl group or C １－６ alkyl-cycloalkyl groups, L ２ is -NR ２ (CH ２ CH ２ O) p １ CH ２ CH ２ C(O)-, -NR ２ (CH ２ CH ２ O) p １ CH ２ C(O)-, -S(CH ２ ) p １ C(O)— or a chemical bond, where p １ is an integer from 1 to 20, preferably a chemical bond; L ３ is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from the amino acid residues consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid; L ４ But, -NR ３ (CR ４ R ５ ) ｔ -, -C(O)NR ３ -, -C(O)NR ３ (CH ２ ) ｔ - or PAB groups, where t is an integer from 1 to 6; R １ , R ２ and R ３ are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group; R ４ and R ５ are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group; The immunoconjugate according to claim 10 or a pharmaceutically acceptable salt thereof.

12. The structure of the immune complex is 【Transformation 3】 and 【Chemistry 4】 are selected from, where n and Pc are as defined in claim 10; The immunoconjugate according to claim 10 or a pharmaceutically acceptable salt thereof.

13. 1. A pharmaceutical composition comprising: An isolated anti-ROR1 antibody according to any one of claims 1 to 5, or an isolated nucleic acid molecule according to claim 6, or an immunoconjugate or a pharmaceutically acceptable salt thereof according to any one of claims 9 to 12; one or more pharmaceutically acceptable excipients, diluents or vectors; A pharmaceutical composition comprising:

14. The pharmaceutical composition of claim 13 for treating a ROR1-mediated disease or condition.

15. The pharmaceutical composition of claim 13 for treating a tumor or cancer, comprising: Preferably, the tumor or cancer is selected from breast cancer, pancreatic cancer, lung cancer, esophageal cancer, non-small cell lung cancer, laryngeal tumor, sarcoma, pharyngeal tumor, oral tumor, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, colorectal cancer, lymphoma and leukemia; Pharmaceutical compositions.

Images