Patent Application
20250177546
The present application relates to antibodies that specifically recognize ROR1, and preparation methods and uses thereof. In addition, the present application also relates to antibody-drug conjugates (ADCs) targeting ROR1 and compositions containing the molecule. In addition, the present invention also relates to the therapeutic and diagnostic uses of these antibodies, antibody fragments and antibody-drug conjugates.
Antibody-Drug Conjugates (ADCs) are a class of drugs derived from traditional antibody therapies. They consist of monoclonal antibodies targeting specific antigens conjugated to small-molecule cytotoxic agents through linkers. This combination provides the targeted specificity of antibody drugs and the cytotoxic effects of traditional small molecules, making ADCs particularly suited for the treatment of malignant tumors. Due to their excellent tumor-killing effects. ADCs have quickly become a focal point in anticancer drug development. However, ADCs often have higher toxicity, necessitating high specificity for target antigens. Ideally, the targets for ADC development are antigens that are highly expressed in tumors but low or not expressed in healthy tissues. The target antigens should be surface receptors upregulated in tumor cells that promote tumor growth or survival, and they should possess internalization properties.
Both Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1, also known as Receptor-related Neurotrophic Tyrosine Kinase 1, NTRKR1) and Receptor Tyrosine Kinase-like Orphan Receptor 2 (ROR2) are single-pass transmembrane proteins, belonging to the family of receptor tyrosine kinase (RTK), extracellular parr thereof consists of an immunoglobulin-like domain (Ig) and two cysteine-rich domains (FZD domain and KRD domain), and the intracellular part thereof consists of a tyrosine kinase domain, two serine- or threonine-rich domains and a proline-rich domain. ROR1 and ROR2 participate in the non-canonical Wnt signaling pathway by binding to the ligand Wnt5a through the FZD domain (Oishi I, Suzuki H, Onishi N, Takada R, Kani S, Ohkawara B, Koshida I, Suzuki K, Yamada G, Schwabe G C et al (2003). Genes Cells 8:645-654; Fukuda T, Chen L, Endo T. Tang L, Lu D, Castro J E, Widhopf G F I I, Rassenti L Z, Cantwell M J, Prussak C E et al (2008). Proc Natl Acad Sci USA 105:3047-3052; Paganoni S, Bernstein J, Ferreira A (2010). Neuroscience 165:1261-1274). ROR1 can inhibit apoptosis, enhance EGFR signaling, and induce epithelial-mesenchymal transition (EMT) (Fukuda T. Chen L, Endo T, Tang L, Lu D, Castro J E, Widhopf G F I I, Rassenti L Z, Cantwell M J, Prussak C E et al (2008). Proc Natl Acad Sci USA 105:3047-3052; Yamaguchi T, Yanagisawa K, Sugiyama R. Hosono Y, Shimada Y, ArimaC, KatoS, TomidaS, Suzuki M, OsadaHet al (2012). Cancer Cell 21:348-361; Cui B. Zhang S, Chen L, Yu J, Widhopf G F, Fecteau J F, Rassenti L Z, Kipps T J (2013). Cancer Res 73:3649-3660).
ROR1 is a conserved embryonic protein, the expression of which gradually decreases with embryonic development and is almost absent or lowly expressed in most adult tissues. However, it has been found in more and more literatures that ROR1 is expressed in a variety of cancer cells, such as B-cell chronic lymphocytic leukemia (CLI) and other hematological malignancies, renal cell cancer, colon cancer and certain other cancer cell lines of breast cancer. Furthermore, ROR1 plays an important role in the progression of many hematological and solid malignancies. Therefore, as a cancer marker, ROR1 becomes an ideal drug target for cancer treatment.
Although several antibody drugs against ROR1 have been disclosed in the prior art, there is still an urgent need to develop anti-ROR1 antibodies with high-quality due to ROR1 as tumor markers for pan-cancers. Such antibodies can be used as the basis for developing antibody-based targeted therapies for cancers expressing ROR1, and can also be used as a diagnostic tool to detect ROR1 expression in ROR1-related diseases. In addition, in view of the good prospects shown by ADCs in the field of tumor treatment, there is still an urgent need for ADCs containing ROR1 with effective therapeutic effects, and the present invention meets these needs.
In a first aspect, the present invention provides an antibody targeting ROR1, which has the following advantages:
In one embodiment, the present invention provides an anti-ROR1 antibody specifically binding to ROR1 and antigen-binding fragment thereof, comprising:
In one embodiment, the present invention provides an anti-ROR1 antibody specifically binding to ROR1 and antigen-binding fragment thereof, comprising:
In one embodiment, the present invention provides an anti-ROR1 antibody specifically binding to ROR1 and antigen-binding fragment thereof, comprising a heavy chain variable region, wherein:
In one embodiment, the present invention provides an anti-ROR1 antibody specifically binding to ROR1 and antigen-binding fragment thereof, comprising a light chain variable region, wherein:
In another embodiment, the present invention provides an anti-ROR1 antibody specifically binding to ROR1 and antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein:
In some embodiments, the above mentioned antibody or antigen-binding fragment thereof further comprises a heavy chain and/or light chain constant region sequence derived from human antibody germline consensus sequence. The light chain constant region is preferably a human kappa or lambda chain constant region. The heavy chain constant region can be γ, ρ, α, δ, or ε chain. In some embodiments, the heavy chain constant region is preferably derived from the constant region sequence of human IgG1, IgG2, IgG3, or IgG4. In one embodiment, the light chain constant region comprises the sequence shown in SEQ ID NO: 53, or consists of the sequence. In another embodiment, the heavy chain constant region comprises the sequence shown in SEQ ID NO: 52.
It will be appreciated that sequence variants of these constant region domains may also be used, for example comprising one or more amino acid modifications, wherein amino acid positions are identified according to the EU Index System of Kabat et al. (1991).
In a specific embodiment, the present invention provides an anti-ROR1 antibody specifically binding to ROR1 and antigen-binding fragment thereof, comprising a heavy chain and a light chain, wherein:
In certain embodiments of any of the antibodies described above, the antibody is monoclonal.
In certain embodiments of any of the antibodies described above, the antibody is a full length antibody.
In one embodiment, the anti-ROR1 antibody of the invention is an intact antibody, such as an IgG1, IgG2, IgG3, IgG4 antibody. In another embodiment, the anti-ROR1 antibody of the invention encompass only the antigen binding portion thereof, such as: Fab, Fab′-SH, Fv, scFv or (Fab′)2 fragment.
In a second aspect, the present invention provides an antibody-drug conjugate targeting human ROR1, the antibody-drug conjugate having the following advantages:
In one embodiment, the present application provides a conjugate comprising the anti-ROR1 antibody of the first aspect. In a specific embodiment, the molecules that can be conjugated to the anti-ROR1 antibody are, for example, cytotoxic agents, immunomodulators, imaging agents, fluorescent proteins, molecular markers, therapeutic proteins, biopolymers, and oligonucleotides, etc.
In one embodiment, the present invention provides an antibody-drug conjugate (ADC), comprising the anti-ROR1 antibody or antigen-binding fragment thereof according to the first aspect and at least one therapeutically active substance or pharmaceutically active ingredient, having the structure of Ab-(L-D)n, wherein: Ab is the antibody binding to ROR1 or antigen-binding fragment thereof as described in the first aspect of the present invention; L is a linker; D is a therapeutically active substance or a pharmaceutically active ingredient, and n represents an integer from 1 to 20, such as 1, 2, 3, 4, 5, etc.
In one embodiment, the antibody-drug conjugate comprises a plurality of D components, which may be a combination of different therapeutically active substances or pharmaceutically active ingredients, or a combination of the same therapeutically active substances or pharmaceutically active ingredients. In one embodiment, the antibody-drug conjugate has a drug/antibody ratio (DAR) from 1 to 20, such as a DAR value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In one embodiment, the DAR is an average DAR. In one embodiment, the average DAR ranges from 1 to 15, e.g., from 1 to 10, from 2 to 8, from 2 to 6 or from 3 to 5. In a specific embodiment, the average DAR is 3.7.
In a specific embodiment, the therapeutically active substance or pharmaceutically active ingredient is a cytotoxin, a plant toxin, a small molecule toxin, a radioactive isotope, maytansine alkaloids, or the like. In a specific embodiment, the cytotoxin is dolastatin and auristatin derivatives thereof, such as 0101(2-methylpropionyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxy-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)-ethyl]amino}propyl]pyrrolidine-1-yl}-5-methyl-1-oxyheptane-4-yl]-N-methyl-L-valinamide), 8261 (2-methylpropionyl-N-[(3R,4S,5S)-1-{(2S)-2-1(1R,2R)-3-{[(S)-1-carboxyl-2-phenethyl]amino}-1-methoxy-2-methyl-3-oxypropyl]pyrrolidine-1-yl}-3-methoxy-5-methyl-1-oxyheptane-4-yl]-N-methyl-L-valinamide), Dolastatin 10, Dolastatin 15, auristatin E, auristatin PE, monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), auristatin F hydroxypropylamide (AFHPA). In another embodiment, the dolastatin and auristatin derivatives thereof are auristatin, dolastatin, MMAE, MMAF, auristatin F hydroxypropylamide or auristatin F phenylenediamine.
In one embodiment, the cytotoxin is covalently linked to the anti-ROR1 antibody or antigen-binding fragment thereof in a non-site-specific or in site-specific manner via a linker.
In one embodiment, the linker is selected from the group consisting of maleimido-hexanoyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), amino PEG6-propionyl, maleimidocaproyl (mc), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB). N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl 3-(pyridin-2-yldithio)-propionate (SPDP).
In one embodiment, the antibody-drug conjugate comprises the anti-ROR1 antibody or antigen-binding fragment thereof described in the first aspect and tubulin inhibitor (MMAE). In a further embodiment, MMAE is conjugated to the thiol group of cysteine on the anti-ROR1 antibody via a MC-VC-PAB linker, having the structure of anti-ROR1 antibody-MC-VC-PAB-MMAE. IgG1 antibodies have 16 pairs of cysteine residues, which are present in the form of 12 intrachain and 4 interchain disulfide bonds. The interchain disulfide bonds are accessible solvent and can be reduced by reducing agents to form eight sulfhydryl groups, which then become conjugation targets (McCombs J, Owen S. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J. 2015, 17:339-51).
In a specific embodiment, the antibody-drug conjugate comprises or consists of anti-ROR1 monoclonal antibody B62-H3L3 and MC-VC-PAB-MMAE. In a further embodiment, MMAE is conjugated to the thiol group of cysteine on B62-H3L3 via a MC-VC-PAB linker.
In another specific embodiment, the antibody-drug conjugate comprises or consists of anti-ROR1 monoclonal antibody B31-H3L3 and MC-VC-PAB-MMAE. In a further embodiment, MMAE is conjugated to the thiol group of cysteine on B31-H3L3 via a MC-VC-PAB linker.
In a third aspect, the present invention provides a pharmaceutical composition comprising (I) the antibody or antigen-binding fragment thereof of the first aspect or the antibody-drug conjugate of the second aspect, and (2) a pharmaceutically acceptable carrier.
In a fourth aspect, the present invention provides an isolated polynucleotide molecule encoding any one of the antibodies or antigen-binding fragments thereof described in the first aspect.
In a fifth aspect, the present invention provides a vector comprising the nucleic acid molecule of the fourth aspect. In one embodiment, the vector is an expression vector.
In a sixth aspect, the present invention provides a host cell comprising the vector of the fifth aspect or the nucleic acid molecule of the fourth invention. In some embodiments, the host cell is prokaryotic, such as E. coli. In other embodiments, the host cell is eukaryotic, such as HEK293 cell, CHO cell, yeast cell, or plant cell.
In a seventh aspect, the present invention provides a method for preventing or treating a disease associated with abnormal expression of ROR1 in a subject in need thereof, comprising administering to the subject a preventively or therapeutically effective amount of the antibody or antigen-binding fragment thereof of the present invention, or a preventively or therapeutically effective amount of the antibody-drug conjugate of the present invention, or a preventively or therapeutically effective amount of the pharmaceutical composition of the present invention.
In one embodiment, the disease associated with abnormal expression of ROR1 is a cancer that highly expresses ROR1, such as chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma, renal cell carcinoma, colon cancer, gastric cancer, breast cancer, neuroblastoma, lung cancer, head and neck cancer, and melanoma. In a specific embodiment, the lung cancer is non-small cell lung cancer. In a specific embodiment, the breast cancer is triple-negative breast cancer.
In some embodiments, the ADC molecules or pharmaceutical compositions of the present invention can also be administered in combination with one or more other therapies, such as treatment modalities and/or other therapeutic agents, for the uses described herein, such as for preventing and/or treating the relevant diseases or conditions mentioned herein.
In a specific embodiment, the present invention provides a method for killing cells expressing ROR1 or inhibiting the growth of cells expressing ROR1, comprising contacting the cells with an effective amount of the antibody or antigen-binding fragment thereof of the present invention, or an effective amount of the antibody-drug conjugate of the present invention, or an effective amount of the pharmaceutical composition of the present invention.
In an eighth aspect, the present invention provides use of an anti-ROR1 antibody or antigen-binding fragment thereof in the preparation of an antibody-drug conjugate for preventing or treating cancer.
The present invention also provides use of an antibody-drug conjugate comprising an anti-ROR1 antibody or antigen-binding fragment thereof in the preparation of a drug for preventing or treating cancer.
Before describing the present invention in detail, it is to be understood that this invention is not limited to the particular methods and experimental conditions described herein as such methods and conditions may vary. Additionally, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. For purposes of the present invention, the following terms are defined below.
The term “about” when used in connection with a numerical value is meant to encompass numerical values within the range between the lower limit of 10% less than the specified numerical value and the upper limit of 10% greater than the specified numerical value.
The term “and/or”, when used in conjunction with two or more alternatives, should be understood to mean any one of the alterative or any two or more of the alterative.
As used herein, the term “comprising”, “comprise”, “including” or “include” means to include the stated elements, integers or steps, but does not exclude any other elements, integers or steps. When the term “comprising” or “including” is used herein, unless otherwise specified, it also encompasses the situation consisting of the stated elements, integers or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to encompass an antibody variable region consisting of the specific sequence.
The term “ROR1” refers to any recombinant or naturally occurring form of Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1), variant or homolog thereof, which maintains, for example, at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the activity of ROR1. The variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the naturally occurring ROR1 protein in the entire sequence or a partial sequence (e.g., 50, 100, 150 or 200 consecutive amino acids). In one embodiment, the ROR1 protein includes the amino acid sequence of Uniprot ID: Q01973.
ROR1 is highly expressed in the embryo, and then its expression level decreases significantly in the adult stage. However, it was found that the expression of ROR1 was significantly increased in various hematologic malignancies and solid tumors. Hematologic malignancies that highly express ROR1 include B-cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL) and myeloid hematologic malignancy. Among solid tumors, cancers that express ROR1 include colon cancer, breast cancer, intestinal cancer, lung cancer, pancreatic cancer, ovarian cancer, etc.
The term “antibody” is used in the broadest sense herein and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they show desired antigen-binding activity. An intact antibody generally contains at least two full-length heavy chains and two full-length light chains, but may include fewer chains in some cases, for example, antibodies naturally occurring in camels may contain only heavy chains.
The term “anti-ROR1 antibody” refers to an antibody molecule that specifically binds to ROR1 and is capable of inhibiting ROR1 activity. An anti-ROR1 antibody can inhibit ROR1 activity relative to the absence of the ROR1 antibody, e.g., by at least partially or completely blocking the stimulation of ROR1, reducing, preventing or delaying the activation of ROR1, or inactivating, desensitizing or down-regulating the signaling, activity or amount of ROR1. In some embodiments, the antibody can inhibit ROR1 activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control.
The term “antibody fragment” refers to a molecule other than an intact antibody, such molecule comprises a portion of the intact antibody and binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; bivalent or bispecific antibodies or fragments thereof; camelid antibodies (heavy chain antibodies); and multispecific antibodies (e.g., bispecific antibodies) composed of antibody fragments.
The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in antibody binding to an antigen. The heavy and light chain variable domains of naturally occurring antibodies generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three complementarity determining regions (see, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co. page 91 (2007)). A single VH or VL domain is sufficient to confer antigen binding specificity.
The term “Complementarity-determining regions” or “CDR regions” or “CDRs” are the regions in antibody variable domains, which are highly variable in sequence and form structurally defined loop (“hypervariable loop”), and/or include antigen-contacting residues (“antigen-contacting points”). The CDRs are mainly responsible for binding to antigenic epitopes. The CDRs in the variable domain are commonly designated as CDR1, CDR2, and CDR3, sequentially numbered from the N-terminus. The precise amino acid sequence boundaries of each CDR in a given variable region can be determined by using any one or a combination of various established CDR assignment scheme, including, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948(1997)), Kabat based on the variability of antibody sequences (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (http://imgt.cines.fr/), and North CDR definition based on affinity propagation clustering by using a large number of crystal structures.
Unless otherwise indicated, in the present invention, the term “CDRs” or “CDR sequences” encompass CDR sequences determined in any of the ways described above.
The CDRs can also be determined based on a reference CDR sequence (for example, any of the exemplary CDRs of the present invention) having the same AbM numbering position. In one embodiment, the CDRs of the antibody of the invention are positioned according to the AbM numbering scheme.
Unless otherwise indicated, in the present invention, when referring to residue positions in antibody variable regions and CDRs (including heavy chain variable region residues), we refer to those according to the AbM numbering system.
“Humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all CDRs (e.g., CDRs) in a humanized antibody correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of antibody constant region derived from human antibody. The “humanized form” of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.
As used herein, the term “binding” or “specific binding” mean that the binding is selective for an antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen binding site to bind a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or biofilm thin layer interferometry or MSD assay or surface plasmon resonance (SPR).
The term “half effective concentration (EC50)” refers to the concentration of a drug, antibody or toxic agent that induces a response that is 50% between baseline and maximum after a specified exposure time.
The term “therapeutic agent” as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).
The term “antibody-drug conjugate” or “ADC” refers to an antibody or antibody fragment covalently coupled to a therapeutically active substance or active pharmaceutical ingredient, such that the therapeutically active substance or active pharmaceutical ingredient is targeted to the binding target of the antibody to exhibit its pharmacological function. The therapeutically active substance or active pharmaceutical ingredient may be a cytotoxin capable of killing cells, preferably cancer cells, targeted by the ADC. The covalent attachment of therapeutically active substances, active pharmaceutical ingredients or cytotoxins can be performed in a non-site-specific manner using linkers, or in a site-specific manner.
The term “site-specific conjugation” refers to a method of specifically linking a therapeutically active substance or active pharmaceutical ingredient to a specific site of an antibody. In one embodiment, the conjugation is accomplished via a linker.
The term “cytotoxic agent” and “cytotoxin” are used interchangeably and refer to a substance that inhibits or disrupts cellular function and/or causes cell death or destruction in the present invention. In one embodiment, the cytotoxic agent may include, but is not limited to, bacterial toxins (e.g, diphtheria toxin), plant toxins (e.g., ricin), small molecule toxins, radioactive isotopes, maytansine alkaloids, and the like, specifically, such as anthracycline, camptothecin, combretastatin, dolastatin and their auristatin derivatives, duocarmycin, enediyne, geldanamycin, indolino-benzodiazepine dimer, maytansine, puromycin, pyrrolobenzodiazepine dimer, taxane. vinca alkaloid, tubulysin, hemiasterlin, spliceostatin, pladienolide and calichearmicin.
Any antibody-drug conjugate of the present invention can be prepared b % conjugating the antibody to dolastatin and its auristatin derivatives. Dolastatin and its auristatin derivatives are important cytotoxins used in antibody drug conjugates (ADCs). They interfere with microtubule dynamics, cell division, etc., and have anti-tumor and anti-fungal activities. The modification of its skeleton has been widely reported in the literatures, mainly the modification of the terminal subunits: modification of PI (N-terminus) and P5 (C-terminus). The modification of the central peptide subunit also results in effective in vitro cytotoxicity of this type of substance. In an aspect, dolastatin and auristatin derivatives thereof can be, e.g., 0101(2-methylpropionyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxy-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)-ethyl]amino}propyl]pyrrolidine-1-yl}-5-methyl-1-oxyheptane-4-yl]-N-methyl-L-valinamide), 826](2-methylpropionyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxyl-2-phenethyl]amino}-1-methoxy-2-methyl-3-oxypropyl]pyrrolidine-1-yl}-3-methoxy-5-methyl-1-oxyheptane-4-yl]-N-methy 1-L-valinamide), Dolastatin 10, Dolastatin 15, auristatin E, auristatin PE, monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), auristatin F hydroxypropylamide (AF AFHPA), and other auristatins, e.g., auristatins described in US publication No. 20130129753.
Monomethyl auristatin (MMAE), also known as demethyl-auristatin E, is a well-known member of the auristatin compound family. It has the following structural formula:
MMAE plays an effective inhibitory role in mitosis by inhibiting tubulin polymerization. It cannot be used as a drug due to its cytotoxicity, but it has been widely used to prepare antibody conjugates. MMAE was conjugated to monoclonal antibody (MAB) via a linker to form MMAE-MAB. Generally, MMAE-MAB targets tumor cells through antibodies, and the linker is cleaved after MMAE-MAB enters the tumor cells, thereby releasing MMAE, allowing it to exert its cytotoxic effect and kill tumor cells.
The terms “linker” and “connector” are used interchangeably in this application to refer to a chemical moiety that covalently links an antibody to a therapeutically active substance or active pharmaceutical ingredient in an ADC. In one embodiment, the linker may comprise amino acid residues that connect the antibody to the payload. The amino acid residues may form dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide units. Amino acid residues include those occurring naturally as well as non-naturally occurring amino acid analogs, such as citrulline or β-amino acids, such as β-alanine, or ω-amino acids such as 4-amino-butyric acid.
According to the property, the linkers suitable for the present invention can be classified as linkers degradable by cathepsin, such as valine-citrulline (val-cit) linkers, cBu-Cit linkers and CX linkers; non-cleavable linkers such as SMCC linkers or MD linkers; acid-sensitive linkers, silicone-structured linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared-sensitive linkers, ultraviolet-sensitive linkers such as PC4AP.
The linker of the present invention may also be a combination of one or more linkers. For example, a linker degradable by cathepsin may be combined with other types of linkers to form a new linker. Therefore, the “linker” described in the present invention encompasses a single type of linker, or a combination of different types of linkers, as long as it is capable of coupling the antibody of the present invention to a drug.
In a specific embodiment, the linker includes but is not limited to maleimido-hexanoyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), amino PEG6-propionyl, maleimidocaproyl (mc), maleimidopropionyl (MP), valine-citrulline (val-cit). alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (STAB), N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl 3-(pyridin-2-yldithio)-propionate (SPDP).
The term “load” or “drug loading” or “payload” refers to the average number of effective payloads per antibody within an ADC molecule (“payload” is used interchangeably with “therapeutically active substance or active pharmaceutical ingredient” herein). Drug loading can range from 1 to 20 therapeutically active substances or active pharmaceutical ingredients per antibody. The term “drug/antibody ratio” or “DAR” refers to the ratio of therapeutically active substance or active pharmaceutical ingredient (D) conjugated to an antibody to the antibody. The ADCs described herein typically have a DAR from 1 to 20, and in certain embodiments have a DAR from 1 to 8, from 2 to 8, from 2 to 6, from 2 to 5, from 2 to 18, from 4 to 16, from 5 to 12, from 6 to 10, from 3 to 8, from 4 to 6, from 6 to 10, and from 2 to 4. Representative DAR value is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, usually expressed as a combination of the letter D and a number, where the number represents the numerical value of the DAR, for example, D2 represents the drug/antibody ratio, i.e., DAR value is 2. In some embodiments, the DAR is an average DAR, i.e., characterized by a detection method (e.g., by conventional methods such as UV/visible spectroscopy, mass spectrometry. ELISA assay, and HPLC). Quantitative DAR values can also be determined.
DAR may be limited by the number of binding sites on the antibody. For example, where the binding site is cysteine thiol, the antibody may have only one or a few cysteine thiol groups or may have only one or a few sufficiently reactive thiol groups through which the linker unit may be attached. In some embodiments, the average DAR value of the conjugate of the invention is from 1 to 20, such as from 2 to 18, from 4 to 16, from 5 to 12, from 6 to 10, from 2 to 8, from 3 to 8, from 2 to 6, from 4 to 6, from 6 to 10, such as from 1.0 to 8.0, from 2.0 to 6.0, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0, and ranges with two of these values as endpoints.
The term “treatment” refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing disease appearance or recurrence, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or to slow the progression of a disease.
The term “prevention” includes the inhibition of the occurrence or progression of a disease or disorder or symptoms of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.
The term “effective amount” refers to an amount or dose of an antibody or conjugate or composition of the invention to produce the desired effect in a patient in need of treatment or prevention after being administered to a patient in single or multiple doses. An effective amount can be readily determined by the attending physician skilled in the art, by taking into account various factors such as the species; body weight; age and general health condition of the mammals; the specific disease involved; the degree or severity of the disease; the response of the individual patient; the specific antibody to be administered; the mode of administration; the bioavailability profile of the preparation to be administered; the selected dosing regimen; and the use of any concomitant therapy.
The term “therapeutically effective amount” refers to an amount effective to achieve the desired therapeutic result, at necessary dosages and for periods of time necessary. A therapeutically effective amount of the antibody or antibody fragment or conjugate or composition thereof may vary according to factors such as the disease state, age, sex, and body weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or conjugate or composition thereof are outweighed by the therapeutically beneficial effects. The “therapeutically effective amount” preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60% or 70%, and still more preferably at least about 80% or 90% relative to untreated subjects. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system in which the efficacy in human tumors is predictive.
The term “preventively effective amount” refers to an amount effective to achieve the desired preventive result, at necessary dosages and for periods of time necessary. Typically, since a preventive dose is used in subjects prior to or at an earlier stage of disease, the preventively effective amount will be less than the therapeutically effective amount.
The term “pharmaceutical composition” refers to a composition that allows active ingredients contained therein are in a form of maintaining their biological activity and does not contain additional ingredients unacceptably toxic to a subject who would be administered with the composition.
In some embodiments, the present invention provides a composition comprising any anti-ROR1 antibody described herein, or an ADC molecule thereof, preferably a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutically acceptable adjuvant, such as a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, including a buffer, known in the art. In one embodiment, the composition (e.g., the pharmaceutical composition) comprises an anti-ROR1 antibody of the invention, or an ADC molecule thereof, in combination with one or more other therapeutic agents.
As used herein, “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersion media, isotonic agents and absorption delaying agents, and the like.
For the use of pharmaceutically acceptable adjuvant, see also “Handbook of Pharmaceutical Excipients”, Eighth Edition, R. C. Rowe, P. J. Sheskey and S. C. Owen, Pharmaceutical Press. London, Chicago.
The compositions of the present invention may be present in a variety of forms. These forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.
The administration route of the composition of the invention is based on the known methods, for example, orally, by intravenous injection, intraperitoneally, intracerebrally (intraparenchymal), intracerebroventricularly, intramuscularly, intraocularly, intraarterially, intraorally or intralesionally; by sustained release systems or by implanted devices. In certain embodiments, the compositions may be administered by bolus injection or by continuous infusion or by an implant device.
The subject can be a mammal, e.g., a primate, preferably, a higher order primate. e.g., a human (e.g., an individual having or at risk of having a disease described herein). In one embodiment, the subject has or is at risk of having a disease described herein (e.g., cancer). In certain embodiments, the subject is receiving or has received other treatments, such as chemotherapy treatment and/or radiation therapy. In some embodiments, the subject has previously received or is currently receiving immunotherapy.
A medicament comprising the antibody described herein can be prepared by mixing the anti-ROR1 antibody of the present invention, or ADC molecule thereof, having a desired purity with one or more optional pharmaceutically acceptable adjuvants, preferably in the form of a lyophilized preparation or an aqueous solution.
The pharmaceutical composition or formulation of the present invention may also contain more than one active ingredient as required for the particular indication to be treated, preferably those with complementary activities that do not adversely affect each other. For example, it is also desirable to provide other therapeutic agents, including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists), and the like. The active ingredients are suitably present in combination in amounts effective for the intended purpose.
Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, and the matrices are in the form of shaped articles, e.g. films, or microcapsules.
In one embodiment, the present invention provides a method for preparing an anti-ROR1 antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an anti-ROR1 antibody or an expression vector comprising the nucleic acid under conditions suitable for expression of the nucleic acid encoding the anti-ROR1 antibody, and optionally isolating the anti-ROR1 antibody. In certain embodiment, the method further comprises recovering the anti-ROR1 antibody from the host cell (or host cell culture medium).
For recombinant production of the anti-ROR1 antibody of the present invention, a nucleic acid encoding the anti-ROR1 antibody of the present invention is first isolated and inserted into a vector for further cloning and/or expression in a host cell. Such nucleic acid is readily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that are capable of binding specifically to the nucleic acid encoding the anti-ROR1 antibody of the invention.
The present anti-ROR1 antibodies prepared as described herein can be purified by techniques known in the prior art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also be dependent on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art. The purity of the anti-ROR1 antibody of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
Various methods for conjugating cytotoxic or other therapeutic agents to antibodies have been described in the prior art. For example, conjugation can occur through the amino group of lysine side chains and the amino group at the N-terminus of the antibody, the carboxyl group of aspartic acid, glutamic acid and the C-terminus, or the activated cysteine sulfydryl group in the antibody.
The present invention is further illustrated according to the following examples, however, it should be understood that the examples are described in an illustrative rather than a limiting manner, and various modifications can be made by those skilled in the art.
The present invention will be implemented by conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology in the art, unless clearly indicated to the contrary.
Preparation of ROR1 control antibody; in the application, the anti-ROR1 antibody Cirmtuzumab is used as a positive control antibody. The gene for the target fragment was synthesized by General Biotech Co., Ltd. according to the sequence disclosed in WO/2019/173843. The gene was then cloned into the eukaryotic expression vector pcDNA3.4 (Invitrogen) via homologous recombination. The recombinant protein expression vector was transformed into E. coli DH5a, cultured overnight at 37° C., and plasmid DNA was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-O1). The obtained expression vector, which expresses Cirmtuzumab, was designated with clone number 99961.1, and the expressed Cirmtuzumab will be referred to as 99961.1. The expression vector was transfected into 293 cells using ExpiFectamine™ CHO Transfection Reagent (Thermo Fisher. A29129). After 7 days, the cell culture supernatant was collected, centrifuged at 15,000 g for 10 minutes, and filtered through a 0.22 μm membrane. The antibody in the supernatant was purified using a Protein A/G affinity chromatography column. The target antibody was eluted with 100 mM glycine (pH 3.0) and then exchanged into PBS buffer using an ultrafiltration tube (Millipore, UFC901096).
Assessment of ROR1 control antibody; The activity of the prepared positive control antibody 99961.1 (IgG1) was detected with the purchased huROR1-His antigen protein (Kaixia Biotechnology, ROR-HM401). The specific method was as follows: a 96-well ELISA plate was coated with huROR1-His (2 μg/mL, 30 μL/well) at 4° C. overnight; after washing three times, the plate was blocked with 5% skim milk prepared in PBS at room temperature for 1 hour; after washing three times, the plate was added the control antibody 99%1.1 gradient diluted in PBS and incubated at room temperature for 1 hour; after washing, the plate was added the secondary antibody Anti-human-IgG-Kappa+Lambda-HRP (Millipore, AP502P+AP506P) diluted with PBS (1:6000) and incubated at room temperature for 1 hour, the plate was then washed six times and added TMB for color development for 5-20 min. The data was read at OD450 with the microplate reader after the color development was terminated, and the data was processed and plotted using Graphpad prism. The results showed that the expressed control antibody 99961.1 could bind to the ROR1 protein and had normal anti-ROR1 activity.
Preparation of antigen protein: Through genetic manipulation at the coding gene level, His tag or human Fc (SEQ ID NO: 51) tag was added to the C-terminus of the sequences of human ROR1 protein huROR1 ECD AA30-406 (Uniprot ID: Q01973), mouse ROR1 protein MusROR1 ECD AA30-406 (Uniprot ID: Q9Z139), and human ROR2 protein huROR2 ECD AA34-403 (Uniprot ID: Q01974). The obtained nucleic acid sequences were constructed into pcDNA3.4 vector, which was then transformed into Escherichia coli DH5a, cultured at 37° C. overnight, and then the plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01). The resulting plasmid was transiently transfected into HEK293 cells (ATCC® CRL-1573™) using the ExpiFectamine™ 293 Transfection Kit (Gibco™, A14524). After 7 days of expression, the cell culture supernatant was collected and the protein containing Fc-tagged was subjected to affinity purification using COLUMN XK16/20 (Cytiva). After purification, the target protein was eluted with 100 mM glycine (pH=3.0), concentrated, and subjected to buffer exchange, finally resulting in the antigen protein (huROR1-huFc). The His-tagged protein was subjected to affinity purification using Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then the target protein was eluted using imidazole gradient. The eluted proteins were respectively placed into PBS buffer through ultrafiltration concentration tubes (Millipore, UFC901096), and finally resulting in the antigen proteins (huROR1-His, MusROR1-His, huROR2-His).
Antigen assessment: The prepared antigens (huROR1-His, huROR1-huFc) were detected using the antibody 99961.1 (IgG1) obtained in Example 1.1, which passed quality inspection. The specific method was as follows: ELISA plate was coated with 2 μg/mL huROR1-His and huROR1-huFc respectively at 4° C. overnight. The purchased antigen proteins huROR1-His and huROR1-huFc (Kaixia Biology, ROR-HM201) were used as positive controls. After washing three times, the plate was blocked with 5% skim milk prepared in PBS at room temperature for 1 hour; after washing three times, the plate was added antibody 99961.1 gradient diluted in PBS and incubated at room temperature for 1 hour; after washing, the plate was added the secondary antibody Anti-human-IgG-Kappa+Lambda-HRP (Millipore, AP502P+AP506P) diluted with PBS (1:6000) and incubated at room temperature for 1 hour, the plate was then washed six times and added TMB for color development for 5-20 min. The data was read at OD450 with the microplate reader after the color development was terminated, and the data was processed and plotted using Graphpad prism. The results show that antibody 99961.1 can bind to the antigens huROR1-His and huROR1-huFc expressed in house with an affinity comparable to that of the purchased ROR1 antigen protein.
Construction of HEK293 cell line overexpressing human ROR1 (hereafter referred to as huROR1-HEK293): The nucleic acid sequence encoding full-length human ROR1 (Uniprot ID: Q01973) was constructed into pLVX-puro plasmid (Clontech, Cat #632164). Then, the resulting plasmid was electroporated into HEK293 cells (ATCC® CRL-1573™) using an electroporator (Invitrogen, Neon™ Transfection System, MP922947). After electroporation, the obtained cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) by volume and without antibiotics, and then transferred to 10×10 cm cell culture dishes for culture for 48 hours. The cells were then distributed into 96-well cell culture plate at an average density of 104 cells/well, and puromycin was added at a final concentration of 2 μg/mL for screening pressure. After about 2 weeks, the cell lines that formed clones were picked up for identification.
Flow cytometric assay of huROR1-HEK293 cells; The above obtained cell lines in the logarithmic growth phase were digested and plated into 96-well plates. After washing with FACS buffer (1× PBS buffer containing 2% FBS by volume), the primary antibody (99961.1) gradient diluted in PBS was added and incubated at 4° C. for 30 min. After washing, the prepared fluorescent secondary antibody anti-human IgG Fc (abcam, 985%) was added and incubated at 4° C. for 30 min. Finally, the cells were detected by flow cytometer (Beckman, CytoFLEXAOO-1-1102). The results show that huROR1-HEK293 cell line with high expression of human ROR1 on the surface was obtained.
Three Balb/C mice (Shanghai Lingchang Biotechnology Co., Ltd.) were cross-immunized with huROR1-huFc and huROR1-His antigens by subcutaneous injection and intraperitoneal injection, once every two weeks, for a total of four immunizations. One week after the fourth immunization, the blood was collected from mice for immune titer assay, and finally, huROR1-huFc was used for booster immunization once.
ELISA plate was coated with 2 μg/mL huROR1-His and huROR1-huFc respectively at 4° C. overnight (30 μL/well). After washing three times, the plate was blocked with 5% skim milk prepared in PBS at room temperature for 1 hour; after washing three times, the plate was added mouse serum gradient diluted in PBS, and added antibody 99961.1 as positive control, and incubated at room temperature for 1 hour; after washing, the plate was added the secondary antibody Goat-anti-mouse-lgG (1+2a+2b+3)-HRP (Jackson, 115-035-164) or Goat-anti-human-Kappa+Lambda-HRP (Millipore, AP502P+AP506P) diluted in PBS and incubated at room temperature for 1 hour, the plate was then washed six times and added TMB for color development for 5-20 min. The data was read at OD450 with the microplate reader after the color development was terminated, and the data was processed and plotted using Graphpad prism. The results showed that the serum titers of all three mice met the standards.
After the immunization was completed, the spleens were taken from mice, and the splenocytes were collected after grinding and filtering. 1 mL of TRIzol™ Reagent (Thermo Fisher, 15596026) was added to lyse the splenocytes, and the total RNAs were extracted by the phenol-chloroform method. The extracted RNAs were reverse transcribed into cDNAs using a reverse transcription kit (TaKaRa, 6210A). The cDNAs were then used as PCR templates and specific primers for the mouse antibody sequences were used to amplify the antibody light and heavy chain variable region genes respectively. The PCR product was digested with two enzymes NcoI and NotI to obtain the antibody gene fragments, which were inserted into the phage display vector and ligated with T4 ligase. The ligation products were recovered using a DNA recovery kit (Omega, D6492-02) and finally transformed into competent Escherichia coli SS320 (Lucigen, MC1061F) using Electroporator (Bio-Rad, MicroPulser). The electroporated bacteria were spread on a 2-YT (C W/K+ 2-YT) solid plate containing ampicillin and tetracycline, and the SS320 bacteria that were correctly transformed with the antibody plasmid were amplified and packaged using VSCM13 helper phage (purchased from Stratagene) to obtain a phage display library containing Fab sequences.
hROR1-HEK293T cells were cultured in T25 flasks. When the cell growth density was close to 90%, the culture supernatant was removed and the cells were washed once with PBS (Yuanpei, B310KJ), then 2 mL of 4% paraformaldehyde (Shenggong, E672002-0500) was added for fixation for 0.5 h, and finally washed twice with PBS and used as the screening raw material. During screening, the phage display library was incubated with fixed hROR1-HEK293T cells at room temperature for 1 hour, washed three times with 1×PBS, and then 2 mL of glycine-HCl (pH=2.0) was added and gently mixed for 10 minutes to elute the phage that specifically binds to human ROR1. The eluted supernatant then infected SS320 bacteria at logarithmic phase (Lucigen, 60512-1), allowed to stand for 30 minutes, and then cultured at 37° C., 220 rpm for 1 hour. VSCM13 helper phage was added, allowed to stand for 30 minutes, and continued to be cultured at 37° C., 220 rpm for 1 hour. After centrifugation, the cells were transfers to C+/K+ 2-YT medium. The final phage was used for the second round of screening. The screening was repeated several times, and 10 clones were randomly selected for sequence analysis in each round to evaluate the library. After three rounds of screening, the sequences in the library were significantly enriched.
The immunotube and magnetic bead were used to enrich specific antibodies against antigens, and the two methods complemented and verified each other.
Immunotube-based screening is a panning process, consisting of coating the antigen protein huROR1-His or huROR1-huFc on the surface of an immunotube with high adsorption capacity, adding the phage display antibody library to the immunotube to incubate with the antigen protein adsorbed on the surface of the immunotube, washing and eluting. After 2-4 rounds of panning, the specific monoclonal antibody Fab against the antigen is finally enriched. In this example, monoclonal antibody Fab against human ROR1 was enriched after three rounds of panning. For the specific method, refer to Example 2.4.2 in patent CN112250763B.
Magnetic bead-based screening is a panning process, consisting of labeling the antigen protein huROR1-His with biotin, binding the labeled antigen protein with magnetic beads coupled with streptavidin, incubating the antigen-bound magnetic beads with the antibody gene phage display library, washing and eluting. Usually specific monoclonal antibody against the antigen can be enriched in large quantities by 3-4 rounds of panning. In this example, biotin-labeled huROR1-His was used for phage display library screening, and monoclonal antibody Fab against human ROR1 was primarily screened after three rounds of panning. For the specific method, refer to Example 2.4.1 in patent CN112250763B.
The phage pool eluted in each round was tested by ELISA to evaluate the enrichment effect, and 10 clones were randomly selected from the phage pool in each round of screening for sequence analysis. The enrichment effect and the ratio of the sequencing repeatability were comprehensively analyzed to select the appropriate round for single clone selection.
The antigen protein huROR1-His was used for the ELISA monoclonal primary screening, and the antibody Fab binding to huROR1-His obtained from the primary screening was prepared as Fab lysate, and then the huROR1-overexpression HEK293 cells prepared in Example 2.1 were used for detection and verification by flow cytometry (FACS). A total of 11 antibody Fab molecules that specifically bind to human ROR1 were screened, and the obtained 11 mouse antibody Fabs were named according to the corresponding clone numbers (B362, B31, B32, B74, B34, B39, C38. C77, C42, M71 and M78). The specific FACS results are shown in
The VH coding sequence in the Fab sequence of the screened monoclonal antibodies B62, B31, B32, B74, B34, B39, C38, C77, C42, M71 and M78 was connected to the coding sequence of the human IgG1 heavy chain constant region (SEQ ID NO: 52) to obtain the heavy chain coding sequence of the chimeric antibody, and the VL coding sequence in the Fab sequence was connected to the coding sequence of the kappa type (SEQ ID NO: 53) of the human light chain constant region (CL) to obtain the light chain coding sequence of the chimeric antibody. The antibody heavy and light chain coding sequence was respectively inserted into the eukaryotic expression vector plasmid pcDNA3.4 (Invitrogen), transformed into Escherichia coli DH5a, and cultured at 37° C. overnight. The plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free antibody plasmid for eukaryotic expression.
The full-length sequence of the antibody obtained above was expressed by the Expi CHO transient expression system (Thermo Fisher, A29133). The specific method was as follows: on the day of transfection, the CHO cell density was about 7×106 to 1×107 viable cells/mL, and the cell survival rate was >98%. At this time, the cells were adjusted to a final concentration of 6×106 cells/mL using fresh ExpiCHO expression medium pre-warmed at 37° C. The target plasmid was diluted with OptiPRO™ SFM pre-cooled at 4° C. (1 μg plasmid was added to 1 mL of the culture medium), and ExpiFectamine™ CHO reagent was diluted with OptiPRO™ SFM. The two was mixed in equal volumes and gently mixed by pipetting, resulting in ExpiFectamine™ CHO/plasmid DNA mixture, which was incubated at room temperature for 1-5 min, slowly added to the prepared cell suspension with gently shaking, and finally placed in a cell culture shaker and cultured at 37° C., 8% CO2.
18-22 hours after transfection, ExpiCHO™ Enhancer reagent and ExpiCHO™ Feed reagent were added to the culture medium, and the flask was placed on a shaker at 32° C. with 5% CO2 to continue culturing. On day 5 after transfection, the same volume of ExpiCHO™ Feed reagent was added slowly, along with gently mixing the cell suspension. Seven days after transfection, the cell culture supernatant expressing the target protein was collected and centrifuged at 15,000 g for 10 min. The resulting supernatant was subjected to affinity purification using MabSelect SuRe LX (GE, 17547403), and then the target protein was eluted with 100 mM sodium acetate (pH 3.0), followed by neutralization with 1 M Tris-HCl, and finally the resulting protein was placed into PBS buffer using ultrafiltration concentration tubes (Millipore, UFC901096).
In this example, the relative molecular weight and purity of the antibodies obtained above were detected by SDS-PAGE and SEC-HPLC.
Preparation of non-reducing solution: 1 μg of each obtained antibody and quality control product IPI (Ipilimumab) were separately added to 5× SDS loading buffer and 40 mM iodoacetamide, heated in a 75° C. dry bath for 10 min, cooled to room temperature, and centrifuged at 12,000 rpm for 5 min to obtain the supernatant.
Preparation of reducing solution: 2 μg of each obtained antibody and quality control product IPI were separately added to 5×SDS loading buffer and 5 mM DTT, heated in a 100° C. dry bath for 10 min, cooled to room temperature, and centrifuged at 12000 rpm for 5 min to obtain the supernatant. The supernatant was added to Bis-tris 4-15% gradient gel (GenScript) for gel electrophoresis and the protein bands were visualized by Coomassie brilliant blue staining.
The protein gel with chromogenic protein bands was scanned using EPSON V550 color scanner (decolorization was performed until the gel background was transparent), and the purity of the reduced and non-reduced bands was calculated using ImageJ according to the peak area normalization method.
The results show that the bands of each antibody in non-reducing gel were around 150 kD, and the bands in reducing gel were around 55 kD and 25 kD, which were in line with the expected size. The purity of all antibodies obtained in this application detected by reducing gel was greater than 95% (Table 2).
Preparation of materials: 1. Mobile phase: 150 mmol/L phosphate buffer, pH 7.4; 2. Sample preparation: Each antibody and quality control product IPI were diluted to 0.5 mg/mL with mobile phase solution, respectively. Agilent HPLC 1100 or Shimadzu LC2030C PLUS liquid chromatograph was used, column was XBridge BEH (SEC 3.5 μm, 7.8 mm I.D.×30 cm). Waters flow rate was set to 0.8 mL/min, injection volume was 20 μL, VWD detector wavelengths were 280 nm and 214 nm. Blank solution, IPI quality control solution and antibody sample solution were injected sequentially. The percentages of high molecular weight polymers, antibody monomers and low molecular weight substances in the samples were calculated according to the area normalization method.
The results are shown in
In this example, the binding profiles of 11 antibodies (B62, B31, B32, B34, B39, B74, C38, C42, C77, M71 and M78) to human ROR1 antigen protein huROR1-His were detected by the ELISA assay, and the binding abilities of the antibodies (B62, B31, B32, B34, B39, B74, C38, C42, C77, M71 and M78) to huROR1-HEK293 cells over-expressing human ROR1 and A549 tumor cells were also detected by the FACS assay. A549 tumor cells are a human non-small cell lung cancer cell line that over-expresses human ROR1.
96-well ELISA plate was coated with 2 μg/mL huROR1-His (30 μL/well) at 4° C. overnight. The next day, the plate was washed three times with PBST and then blocked with 5% skim milk for 2 h. After washing with PBST for three times, the gradient dilution solution (3.00000, 0.33333, 0.11111, 0.03704, 0.01235, 0.00412, 0.00046, 0.00005 μg/mL) of each antibody and the positive control antibody 99961.1 were added and incubated for 1 h. After washing with PBST for three times, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 h. After incubation, the plate was washed with PBST for 6 times and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2 M HCl was added to terminate the reaction, and the data were read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).
The results are shown in
In this example, the binding activity of the antibody was evaluated using two types of cells, huROR1-HEK293 cells over-expressing human ROR1 and A549 tumor cells.
The specific method was as follows: huROR1-HEK293 cells or A549 cells in the logarithmic growth phase were prepared to a single-cell suspension, the density was adjusted to 1×106 cells/mL, then 100 μL/well cells were added to a 96-well round-bottom plate, centrifuged at 4° C., 300 g, and the supernatant was removed. The gradient dilution solution of antibodies obtained in this application and the positive control antibody 99961.1 were added to the corresponding wells, thoroughly mixed and incubated at 4° C. for 30 min. After the incubation, the cell mixture was washed three times and then 100 μL of 1:300 diluted secondary antibody Goat F(ab′)2 Anti-Human IgG-Fc (abcam, ab98596) was added. The mixture was incubated at 4° C. in the dark for 30 min. After washing three times, the cells were detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).
The results are shown in the figure. On A549 cells (
In this example, the cross-reactivity of each of the antibodies obtained in the present application was tested between species and homologous groups. The cross-activity of the antibodies obtained in the present application between species and homologous groups was identified with the mouse ROR1 antigen protein MusROR1-His and the human ROR2 antigen protein huROR2-His prepared in Example 1.2.
96-well ELISA plate was coated with 2 μg/mL MusROR1-His (30 μL/well) at 4° C. overnight. The next day, the plate was washed three times with PBST and then blocked with 5% skim milk for 2 h. After washing with PBST for three times, the gradient dilution solution (1.00000, 0.11111, 0.03704, 0.01235, 0.00412, 0.00137, 0.00015, 0.00002 μg/mL) of each antibody and the positive control antibody 99961.1 were added and incubated for 1 h. After washing with PBST for three times, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 h. After incubation, the plate was washed with PBST for 6 times and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2 M HCl was added to terminate the reaction, and the data were read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).
The results are shown in
96-well ELISA plate was coated with 2 μg/mL huROR2-His (30 μL/well) at 4° C. overnight. The next day, the plate was washed three times with PBST and then blocked with 5% skim milk for 2 h. After washing with PBST for three times, the gradient dilution solution of the antibody and positive control antibody 99961.1 were added and incubated for 1 h. After washing with PBST for three times, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 h. After incubation, the plate was washed with PBST for 6 times and TMB (SurModics, TMBS-1000-0) was added for color development. According to the color development results, 2 M HCl was added to terminate the reaction, and the data were read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).
The results are shown in
In this example, the internalization rate of the antibodies obtained in this application was detected by using two detection methods, FACS and Fab-Zap. The FACS-based internalization detection is to detect the antibody internalization rate in a short period of time by using supersaturated antibody-binding cells; and the Fab-Zap method reflects the long-term cumulative effect of internalization rate by binding cells with different concentrations of Fab-Zap toxin-conjugated antibodies, and killing the target cells by the toxins entering the cells through internalization.
Antibody dilution: The test antibody was diluted with DMEM complete medium to a final concentration of 10.0000 μg/mL.
Cell treatment: huROR1-HEK293 cells were digested and added into complete DMEM medium. After thoroughly mixing, the cells was counted and determined for their viability, 106 cells were taken and added to 1.5 mL centrifuge tubes, centrifuged at 300 g for 5 minutes, the supernatant was discarded, 1 mL pre-cooled DMEM medium was used for re-suspension, centrifuged at 300 g for 5 minutes, and the supernatant was discarded.
Primary antibody incubation: 1000 μL of the pre-cooled diluted antibody was taken and added to the centrifuge tube containing cells to prepare the antibody-cell suspension, which was then added to a 96-well plate and incubated at 4° C.
Extracellular secondary antibody incubation: The suspension in the 96-well plate was quickly transferred to a second 96-well plate. 180 μL of pre-cooled FACS buffer was added to each well of the second 96-well plate and washed twice. Then, 100 μL of diluted secondary antibody FITC-labeled anti-huFc or RPE-labeled anti-huFc (the secondary antibody was diluted 1:150 using FACS buffer) was added to each well; incubated at 4° C. for 30 min.
Cell fixation: After incubation, the supernatant was discarded by centrifugation, 180 μL of pre-cooled FACS buffer was added to each well, and the cells were washed twice. The supernatant was removed by centrifugation, and the cells were fixed with 100 μL of 4% paraformaldehyde in each well for 30 min at room temperature.
Cell membrane disruption: After fixation, 180 μL FACS buffer was added and washed twice. 100 μL of pre-warmed 0.5% Triton X-100 was added to each well and permeabilization for 5 min at room temperature.
Intracellular secondary antibody incubation: After washing the 96-well plate, 180 μL of pre-warmed Permeabilization Buffer (Invitrogen™ eBioscience™, 00-8333-56) was added to each well and washed twice. 100 μL of diluted secondary antibody RPE-labeled anti-huFc (diluted 1:150 using Perm buffer) was added to each well, and to be incubated at room temperature for 60 min.
Fluorescence Detection: After washing the 96-well plate. 100 μL FACS buffer was used for re-suspension, and detection was performed by flow cytometry.
Two groups of different fluorescence settings were used in the example to stain each sample. The first group was set as FITC+PE, that is, FITC secondary antibody was first used for staining the extracellular primary antibody, and then the PE secondary antibody was used for staining the intracellular primary antibody after the membrane was permeabilized. In this group, PE was the intracellular signal and FITC was the extracellular signal. The second group was set as PE+PE, that is, PE secondary antibody was first used for staining the extracellular primary antibody, and then the PE secondary antibody was used for staining the intracellular primary antibody after the membrane was permeabilized. In this group, PE was the sum of intracellular and extracellular signals. At the same time, both groups of samples were detected using FITC and PE channels, and the values of internalization rate were calculated as detection values for the PE channel. The specific formula is:
Internalization rate=group 1(FITC+PE)PE channel/group 2(PE+PE)PE channel×100%.
The results are shown in
The internalization activity of antibodies was detected in this experiment by using the cytotoxicity of antibody-mediated Fab-ZAP internalization. Fab-ZAP is a Fab fragment linked to saporin, a ribosomal inhibitor that can inhibit protein synthesis and cause cell death. The Fab-ZAP used in this experiment is a Fab fragment that can bind to the human Fc of the chimeric antibody. After incubation with the chimeric antibody, Fab-ZAP makes the chimeric antibody carry the toxin. When the chimeric antibody is internalized, the toxin enters the cell along with the chimeric antibody, resulting in cell death. Then, the activity of the cell is detected by MTS (Promega, G3580) to determine whether the antibody is internalized.
The detail experimental procedures were as follows: first, Fab-Zap was diluted to 0.4 nM with DMEM complete medium, and then each of the antibodies obtained in the application and the positive control antibody were gradiently diluted (0.020000, 0.006667, 0.002222, 0.000741, 0.000247, 0.000082, 0.000027, 0.000009 μg/mL) with the 0.4 nM Fab-Zap to prepare antibody dilution solutions. The huROR1-HEK293 cells in the logarithmic growth phase were made into a single cell suspension, the density was adjusted to 6×106 cells/mL, and 50 μL was inoculated into each well of a 96-well plate. Then, the above antibody dilution was taken and added into the cell culture plate, 50 μL per well, and the suspension was mixed thoroughly by pipetting. The cell culture plate was placed in a 37° C. cell culture incubator and incubated for 48 hours. After the incubation, 7.5 μL of TritonX-100 solution was added to each well, gently mixed by tapping, and the cell culture plate was placed in a 37° C. cell culture incubator for 0.5 h. Then, 20 μL of MTS was added to each well and incubated at 37° C. for 1-4 hours. Finally, the cell culture plate was centrifuged at 1000 rpm for 5 min, and the data was read by a microplate reader at a detection wavelength of A492.
The results are shown in
In this example, the affinity of the antibodies obtained in the present application and the positive control antibody 99961.1 to the antigen protein huROR1-His was detected based on the Gator device.
First, Q buffer was prepared with PBS (10 mM pH7.4) (IgG-free, purchased from Jackson ImmunoResearch Lab)+0.02% Tween 20 (purchased from thermo)+0.2% BSA (purchased from source culture), and the stock solution of the test antibody was diluted with the prepared Q buffer to a working solution with a final concentration of 30 nM, the stock solution of the antigen protein huROR1-His was prepared with Q buffer into a working solution with multiple dilutions (480, 240, 120, 60, 30, 15, 7.5 nM), and detection and analysis was then performed with Gator instrument and its accompanying software, selecting the Advanced Kinetics experimental mode is used. The results are shown in Table 4.
The results show that except for C42 antibody, which has comparable affinity to the control antibody 99961.1, the other antibodies all have affinity superior to that of the control antibody 99961.1 by one order of magnitude or more. Among them, B62 antibody has affinity of 9.84E-10, superior to that of the control antibody 99961.1 by about two orders of magnitude.
In this example, the affinity of the antibodies obtained in the present application and the positive control antibody 99961.1 to the antigen protein huROR1-His was detected based on the Biacore device.
Protein coupling: The huROR1-His protein produced in Example 1.2 was diluted to 5.6 μg/mL with NaAc buffer, pH 5.0, the flow rate was set to 10 μL/min, the chip activation time was set to the default value of 420 s with a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the antigen protein huROR1-His was fixed to a level of about 75 RU using a coupling mode with a preset coupling amount, and the activated groups that were not bound to the test sample were blocked with ethanolamine.
Sample test conditions: PBS buffer (pH 7.4) containing 0.05% Tween-20 was used as the running buffer, and the running buffer was used as the control test sample. A series of antibody concentrations (4 nM, 20 nM) were set. During sample analysis, the flow rate was set to 30 μL/min, the binding time was 120 s, and the dissociation time was 360 s. After the dissociation was completed, 10 mM Gly-HCl (pH 2.0) was used for regeneration for 20 s to completely remove the antibody bound to the ligand.
Parameter fitting: The experiment was run in multiple cycles, with the response signal taking the analysis time as the horizontal axis and the response value as the vertical axis. After double reference subtraction, the obtained data were fitted by BiAcore T200 analysis software. The fitting model adopted was 1:1 Langmuir binding model, and Affinity metrics such as the binding dissociation constant were determined.
The results are shown in Table 5. Antibody B62, with a KD of 6.39E-11, has affinity about 2 orders of magnitude better than that of control antibody 99961.1, the later has KD of 1.98E-9: in addition, the affinity of antibody B31 is comparable to that of control antibody 99961.1.
The antibodies obtained in this application and the positive control antibody 99961.1 were divided into various groups based on the epitope in this example by using the affinity kinetics method.
The detail experimental procedures were as follows: First, prepare Q buffer by mixing PBS (10 mM, pH 7.4) (IgG-free, purchased from Jackson ImmunoResearch Lab) with 0.02% Tween 20 (purchased from Thermo) and 0.2% BSA (purchased from Source culture). Dilute the antibody storage solution to a final concentration of 100 nM in the prepared Q buffer. Similarly, dilute the antigen huROR1-His storage solution to a 50 nM working solution in Q buffer. Detection and analysis were then conducted using the Gator instrument and its accompanying software, employing the Tandem settings based on the Epitope Binning experimental mode.
The specific detection and analysis steps were as follows:
The results of the epitope grouping of each antibody obtained are shown in Table 6. The results show that the antibodies of the present application and the control antibody 99961.1 are divided into 2 epitope-based groups, in which only C42 and the control antibody share the same binding epitope, and the remaining antibodies have epitope different from that of the control antibody.
The VH and VL sequences of the murine antibodies B31 and B62 screened in Example 4 were separately compared with known human antibody databases to find the human germline gene VH and VL sequences with the highest homology to the mouse VH and VL sequences respectively. The framework regions of the corresponding human germline gene VH and VL sequences (CDR and framework regions were defined by using AbM) were determined, and the complementarity determining region (CDR) sequences of the germline genes were replaced with the corresponding CDR sequences in the murine antibodies B31 and B62 of the present application. Then, with the help of computer prediction simulation, the murine amino acids in the framework regions of the murine antibodies B31 and B62 that have an important effect on antigen binding were retained by back mutation. The amino acid sequences of the CDR regions of the humanized antibodies resulting from humanization of the murine antibodies B31 and B62 are shown in Table 7. Various antibodies resulting from humanization of B31 and B62 were constructed, expressed and purified by the method of Example 5, and the various antibody proteins resulting from humanization of B31 and B62 were identified by SDS-PAGE and SEC-HPLC. The results are shown in Table 8. The results show that each antibody exhibits band at around 150 kD in non-reducing gel, and bands at around 55 kD and 25 kD in reducing gel, which are consistent with the expected size. The purity of all antibodies obtained in this application is greater than 95% by reducing gel detection, the SEC monomer purity of all antibodies is greater than 96%.
In this example, the affinity of the humanized antibody to the human ROR1 antigen protein huROR1-His was detected based on the ELISA method, and the affinity of the humanized antibody to A549 tumor cells was also detected based on the FACS assay. Specific testing method was referred to Example 7.
The ELISA results are shown in
The FACS results are shown in
In this example, the humanized antibodies B31-H3L3 and B62-H3L3 in Example 13 were subjected to Fab-Zap-based internalization detection. The specific method was referred to Example 9. The results are shown in
In this example, antibodies B62-H3L3, B31-H3L3 and control antibody 99961.1 were conjugated with toxin MMAE (a tubulin inhibitor with anticancer activity) to construct antibody-drug conjugates. MMAE was connected to the linker MC-VC-PAB to form MC-VC-PAB-MMAE, and the linker was covalently linked to the sulfhydryl group on the cysteine of the antibody, thereby conjugating the antibody to MMAE to obtain an antibody-drug conjugate. IgG1 antibodies have 16 pairs of cysteine residues, which are present in the form of 12 intrachain and 4 interchain disulfide bonds. The interchain disulfide bonds are solvent accessible and can be reduced by reducing agents to form eight sulfhydryl groups, which then become conjugation targets (McCombs J, Owen S. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J. 2015, 17:339-51).
The specific preparation method was as follows:
Antibodies B62-H3L3, B31-H3L3 and control antibody 99961.1 were taken out of the −80° C. refrigerator, thawed and transferred to 15 mL 30 KD ultrafiltration centrifuge tubes separately, and coupling buffer (components per 1 L: Na2HPO4·2H2O 6.86 g, NaH2PO4·H2O 1.58 g, purified water to a total of 1000 g, pH7.4) was added to a total of 15 mL. Then, the solutions were centrifuged at 4500 rpm for about 30 min, concentrated to 2-3 mL, and replenished with dialysate (components per 1 L: histidine 0.73 g, histidine hydrochloride monohydrate 1.12 g, purified water to a total of 1000 g, pH6.0) to a final volume of 15 mL. The dialysis was repeated 8-10 times to obtain the antibody stock solution, and the antibody concentration was measure after dialysis.
The thiol group on the cysteine of the antibody was reduced by a reduction reaction system, which was formed by adding the antibody stock solution, 10 mM disulfide bond reducing agent TCEP stock solution (tris(2-carboxyethyl)phosphine hydrochloride stock solution, components per 1 L: Na2HPO4·2H2O 6.86 g, NaH2PO4·H2O 1.58 g, purified water to a total of 1000 g), 10 mM DTPA stock solution (diethylenetriaminepentaacetic acid stock solution, contents per 1 L: DTPA 3.90 g, NaOH 1.20 g, purified water to a total of 1000 g) and coupling buffer in order. The amount of each component added to the reduction reaction system is shown in Table 9, so that the antibody concentration in the reduction reaction system is 5 mg/mL, the final concentration of DTPA is 1 mM, the molar ratio of TCEP to B62-H3L3 or B31-H3L3 is 2, and the molar ratio of TCEP to 9996.1 is 2.2. After thoroughly mixing, the reduction reaction system was placed in a 25° C. constant temperature shaker, with a rotation speed of 400 rpm, for 2 h of reduction reaction.
MC-VC-PAB-MMAE was weighted and dissolved in DMSO to prepare a 5 mM MC-VC-PAB-MMAE stock solution. After the reduction reaction was completed, the MC-VC-PAB-MMAE stock solution was added to the reduction reaction system in an ice-water bath in order, and the added amount was as shown in Table 10, to prepare a coupling reaction system. After thoroughly mixing, the coupling reaction system was placed in a 25° C. constant temperature shaker at 400 rpm and coupled for 1 h, to obtain a solution containing ADC.
After the coupling reaction was completed, the solution containing ADC was centrifuged and filtered to obtain the ADC sample, which was transferred into a 15 mL 30 KD ultrafiltration centrifuge tube. Dialysate was added to 15 mL, centrifuged at 4500 rpm for 20 min to concentrate to 2-3 mL, and then dialysate was replenished to 15 mL again. The dialysis was repeated 8-10 times. The ADC samples after dialysis were subjected to SEC-HPLC detection. HIC-HPLC detection, concentration detection, free drug detection, etc. The results are shown in Table 11. The results show that the ADCs with a purity of more than 99% were obtained.
In this example, the ability of the prepared ADCs (B62-H3L3-MMAE and B31-H3L3-MMAE) binding to human ROR1 on tumor cells A549 and HT-29 (human colon cancer cells) was detected based on the FACS assay.
The specific method was as follows: A549 cells or HT-29 cells in the logarithmic growth phase were prepared to a single-cell suspension, the density was adjusted to 1×106 cells/mL, 100 L/well was added to a 96-well round-bottom plate, then centrifuged at 4° C., 300 g, and the supernatant was removed. The gradient dilution solutions (1.0000, 0.3333, 0.1111, 0.0370, 0.0123, 0.0041, 0.0014, 0.0001 μg/mL) of the antibodies of the present application (B62-H3L3 and B31-H3L3), ADCs of the present application (B62-H3L3-MMAE and B31-H3L3-MMAE), 99961.1, 99961.1-MMAE and negative control were added to the corresponding wells, respectively, mixed thoroughly and incubated at 4° C. for 30 min. After the incubation, the cell mixture was washed three times and then 100 μL of 1:300 diluted secondary antibody Goat F(ab′)2 Anti-Human IgG-Fc (abcam, ab98596) was added. The mixture was incubated at 4° C. in the dark for 30 min. After washing three times, the cells were detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).
The results are shown in
In this example, A549 and HT-29 cells were used to detect the killing effect of the ADCs of the present application and the control ADC.
The specific method was as follows: A549 cells or HT-29 cells in the logarithmic growth phase were prepared to a single-cell suspension, the density of A549 was adjusted to 1×104 cells/mL, and the density of HT-29 was adjusted to 1.5×104 cells/mL, and 100 μL/well was added to a 96-well cell culture plate and cultured at 37° C., 5% CO2 for 12 hours. Then, gradient diluted ADC samples (2000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 1.953 nM) were added and cultured at 37° C., 5% CO2 for 72 h (A549 cells) or 96 h (HT-29 cells). Then, 40 μL of MTS (Promega, G3580) was added to each well, incubated at 37° C. for 1 h, and the data were read at OD492 using a microplate reader.
The results are shown in
In the example, Jeko-1 cells (human mantle cell lymphoma cells), MDA-MB-468 cells (human breast cancer cells) and NCI-H1944 cells (human lung cancer cells) were used to detect the killing effect of B31-H3L3-MMAE, B62-H3L3-MMAE and control ADC.
The specific method was as follows: Jeko-1 cells, MDA-MB-468 cells or NCI-H1944 cells in the logarithmic growth phase were prepared to a single-cell suspension, the density of Jeko-1 was adjusted to 1×105 cells/mL, the density of MDA-MB-468 was adjusted to 2×105 cells/mL and the density of NCI-H1944 was adjusted to 1.2×105 cells/mL, and 90 μL/well single-cell suspension was added to a 96-well cell culture plate and cultured at 37° C., 5% CO2 for 12 hours. Then, the gradiently diluted ADC samples (500, 158, 50, 15.8, 5, 1.58, 0.5, 0.158, and 0.05 nM) were added. The cells were cultured at 37° C. and 5% CO2 for 72 h. Then 10 μL of CCK8 (Bimake, B34304) was added to each well, incubated at 37° C. for 1 h, and the data were read at OD450 using a microplate reader.
The experimental results are shown in
In this example, huROR1-HEK293 cells were used to detect the antigen-dependent killing effect of B31-H3L3-MMAE and control ADC.
The specific method was as follows: huROR1-HEK293 cells in the logarithmic growth phase were prepared to a single-cell suspension, the cell density was adjusted to 6×104 cells/mL, and 50 μL/well single-cell suspension was added to a 96-well cell culture plate. After culturing at 37° C., 5% CO2 for 24 h, 50 μL of 100 μg/mL antibody B31-H3L3 or positive control antibody 99961.1 were added to each well. After incubation at 37° C. for 2 h, the gradient dilution solution (42.6667, 21.3333, 10.6667, 5.3333) of the ADC B31-H3L3-MMAE or control ADC 99%1.1-MMAE were added correspondingly. After culturing at 37° C. 5% CO2 for 96 h, 30 μL of CCK8 (absin/Aibixin, abs50003) was added to each well and incubated at 37° C. for 1-4 h. Then, the plate was read at OD450 using a microplate reader. The system only adding ADC, but not adding antibody, was used as the control group.
The results are shown in
In this example, A549 and HT-29 tumor cells were used to detect the internalization efficiency of the ADC obtained in this application based on the FACS assay. For detail experimental methods, see Example 9.
The experimental results show that on A549 cells (
In this example, the anti-tumor effects of two candidate ADCs (B31-H3L3-MMAE and B62-H3L3-MMAE) in animals were verified, 99961.1-MMAE was used as a positive control, and the tumor cells used were colon cancer cells HT-29 (BNCC337732).
The specific method was as follows: 6-8 week-old female Balb/C nude mice weighing about 20 g (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used, and 1-106 HT-29 cells were injected subcutaneously unilaterally into each nude mouse. When the tumor volume reached about 100 mm3, the mice were randomly grouped into cages. Totally 9 groups were divided, 6 tumor-bearing nude mice in each group, including a PBS negative control group, 5 candidate ADC groups (B31-H3L3-MMAE 0.4 mg/kg (mpk), B31-H3L3-MMAE 2 mg/kg, B31-H3L3-MMAE 10 mg/kg, B62-H3L3-MMAE 2 mg/kg and B62-H3L3-MMAE 10 mg/kg) and 3 positive control ADC groups (99961.1-MMAE 0.4 mg/kg, 99961.1-MMAE 2 mg/kg and 99961.1-MMAE 10 mg/kg). The drugs were administered by tail vein injection, twice a week, and the tumor volume was measured twice, for a total of 8 administrations/4 weeks (BIW*4). Tumor volume (V) was calculated as follows: V=L×W2/2 (where L is the longest tumor diameter and W is the shortest tumor diameter). One week after the end of the administration, the mice were euthanized, and the tumors were removed and the tumor weight was measured. The data of the tumor volume, tumor weight and mouse body weight change were analyzed and the Tumor growth inhibition was calculated. The results are shown in
The experimental results show that there is no significant difference in the body weight of mice in each group, and there is no significant change in the body weight of mice in each group during the treatment, indicating that the mice have good tolerance to ADCs (
In this example, the anti-tumor effects of two candidate ADCs (B331-H3L3-MMAE and B62-H3L3-MMAE) in animals were detected. The tumor cells used were non-small cell lung cancer cells A549 (Shanghai Cell Bank. Chinese Academy of Sciences, C2107019), and 99961.1-MMAE was used as a positive control.
The specific method was as follows: 6-8 week-old female nude mice weighing about 18-20 g (Balb/C, Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used, and 1×106 A549 cells were injected subcutaneously unilaterally on the right dorsal side of each nude mouse. When the tumor volume reached about 100 mm3, the mice were randomly grouped into cages. Totally 4 groups were divided, 6 tumor-bearing nude mice in each group, including a PBS negative control group, 2 ADC groups (B31-H3L3-MMAE 10 mpk (mg/kg) and B62-H3L3-MMAE 10 mpk) and 1 positive control ADC group (99961.1-MMAE 10 mpk). The drugs were administered by tail vein injection, twice a week, and the tumor volume was measured twice, for a total of 6 administrations/3 weeks (BIW*3). Tumor volume (V) was calculated as follows: V=L×W2/2 (where L is the longest tumor diameter and W is the shortest tumor diameter). After a certain period of observation after the end of the drug administration, the mice were euthanized, the tumors were removed and the tumor weight was measured. The data of the tumor volume, tumor weight and mouse body weight change were analyzed, and the Tumor growth inhibition was calculated. The results are shown in
The experimental results show that there is no significant change in the body weight of mice in each group during the treatment, indicating that the mice have good tolerance to the ADCs (
In this example, the anti-tumor effect of a candidate ADC (B31-H3L3-MMAE) in animals was detected. The tumor cells used were gastric cancer cells NCI-N87 (Shanghai Cell Bank of the Chinese Academy of Sciences, C2009021, P3), and 99961.1-MMAE was used as a positive control.
The specific method was as follows: 6-8 week-old female nude mice weighing about 18-20 g (Balb/C, Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used, and 1×106 NCI-N87 cells were injected subcutaneously on the dorsal side of each nude mouse. When the tumor volume reached about 100 mm3, the mice were randomly grouped into cages. Totally 5 groups were divided, 10 tumor-bearing nude mice in each group including a PBS negative control group, 2 ADC groups (B31-H3L3-MMAE 5 mpk (mg/kg) and B31-H3L3-MMAE 10 mpk) and 2 positive control ADC groups (991.1-MMAE 5 mpk and 99961.1-MMAE 10 mpk) The drugs were administered by tail vein injection, once a week, and the tumor volume was measured twice, for a total of 3 administrations/3 weeks (BIW*3). Tumor volume (V) was calculated as follows: V=L×W/2 (where L is the longest tumor diameter and W is the shortest tumor diameter). The data of the tumor volume and mouse body weight change were analyzed, and the Tumor growth inhibition was calculated. The results are shown in
The experimental results show that there is no significant change in the body weight of mice in each group during the treatment, indicating that the mice have good tolerance to ADC (
In this example, the anti-tumor effect of a candidate ADC (B3I1-H3L3-MMAE) in animals was detected. The tumor cells used were triple-negative breast cancer cells MDA-MB-231 (Shanghai Cell Bank of the Chinese Academy of Sciences, C2006040), and 99961.1-MMAE was used as a positive control.
The specific method was as follows: 6-8 week-old female nude mice weighing about 20-22 g (NSG, Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used, and 1×106 MDA-MB-231 cells were injected subcutaneously on the dorsal side of each nude mouse. When the tumor volume reached about 1(0) mm3, the mice were randomly grouped into cages. Totally 5 groups were divided, 10 tumor-bearing nude mice in each group, including a PBS negative control group, 2 ADC groups (B31-H3L3-MMAE 5 mpk (mg/kg) and B31-H3L3-MMAE 10 mpk) and 2 positive control ADC groups (99961.1-MMAE 5 mpk and 99961.1-MMAE 10 mpk). The drugs were administered by tail vein injection, once a week, and the tumor volume was measured twice, for a total of 3 administrations/3 weeks (BIW*3). Tumor volume (V) was calculated as follows: V=L×W2/2 (where L is the longest tumor diameter and W is the shortest tumor diameter). The data of the tumor volume and mouse body weight change were analyzed, and the Tumor growth inhibition was calculated. The results are shown in
The results show that there was no significant difference in the early stage of drug administration among the mice in each group. The PBS group started to show body weight loss on day 35, which was expected to be due to tumor overloading. There is no significant change in the body weight of mice in the ADC group and the positive control group, indicating that the mice have good tolerance to ADCs (
In this example, the anti-tumor effect of a candidate ADC (B31-H3L3-MMAE) in animals was detected. The tumor cells used were triple-negative breast cancer cells MDA-MB-468 (Shanghai Cell Bank of the Chinese Academy of Sciences, TCHul36), and 99961.1-MMAE was used as a positive control.
The specific method was as follows: 6-8 week-old female nude mice weighing about 21-25 g (NOD SCID, Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used, and 1×107 MDA-MB-468 cells were injected subcutaneously on the dorsal side of each nude mouse. When the tumor volume reached about 200 mm3, the mice were randomly grouped into cages. Totally 5 groups were divided, 6 tumor-bearing nude mice in each group, including a PBS negative control group, 2 ADC groups (B31-H3L3-MMAE 5 mpk (mg/kg) (QW*3), B31-H3L3-MMAE 10 mpk (QW*3) and 2 positive control ADC groups (99961.1-MMAE 5 mpk (QW*3), 99961.1-MMAE 10 mpk (QW*3); totally 2 groups with 3 tumor-bearing nude mice in each group, including B31-H3L3-MMAE 10 mpk (single dose) and 99961.1-MMAE 10 mpk (single dose), and the administration method was tail vein injection, for a total of 3 administrations/3 weeks (QW*3) or single dose. Tumor volume (V) was calculated as follows: V=L×W2/2 (where L is the longest tumor diameter and W is the shortest tumor diameter). The data of the tumor volume and mouse body weight change were analyzed, and the Tumor growth inhibition was calculated. The results are shown in
The experimental results show that there is no significant change in the body weight of mice in each group during the treatment period, indicating that the mice have good tolerance to ADCs (
In this example, the anti-tumor effect of a candidate ADC (B331-H3L3-MMAE) in animals was detected. The tumor cells used were mantle cell lymphoma Jeko-1 cells (ATCC, CRL-3006), and 99961.1-MMAE was used as a positive control.
The specific method was as follows: 6-8 week-old female nude mice weighing about 21-25 g (BALB/c. Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used, and 1×107 Jeko-1 cells were injected subcutaneously on the dorsal side of each nude mouse. When the tumor volume reached about 100 mm3, the mice were randomly grouped into cages. Totally 9 groups were divided, 7 tumor-bearing nude mice per group, including a PBS negative control group, 4 ADC groups (B31-H3L3-MMAE 2.5 mpk (mg/kg) (QW*3), 1B31-H3L-MMAE 10 mpk (QW*3), B31-H3L3-MMAE 10 mpk (single dose) and B31-H3L3-MMAE 2.5+3.5 mpk (Q2W*2, i.e., 2.5 mpk on day 15 and 3.5 mpk on day 29, abbreviated as D15 2.5 mpk+D29 3.5 mpk)) and 4 positive control ADC groups (99961.1-MMAE 2.5 mpk (QW*3), 99961.1-MMAE 10 mpk (QW*3). 99961.1-MMAE 10 mpk (single dose) and 99961.-MMAE 2.5+3.5 mpk (Q2W*2, i.e. 3 2.5 mpk on day 15 and 3.5 mpk on day 29 abbreviated as D35 2.5 mpk+D29 3.5 mpk)) the administration method was tail vein injection, a total of 3 administrations/3 weeks (QW*3) or single dose (single dose) or 2 administrations/3 weeks (Q2W*2). Tumor volume (V) was calculated as follows: V=L×W2/2 (where L is the longest tumor diameter and W is the shortest tumor diameter). The data of the tumor volume and mouse body weight change were analyzed, and the Tumor growth inhibition was calculated. The results are shown in
The experimental results show that there is no significant change in the body weight of mice in each group during the treatment, indicating that the mice have good tolerance to ADC (
| Number | Date | Country | Kind |
|---|---|---|---|
| CN202210226048.9 | Mar 2022 | CN | national |
| CN202310099967.9 | Feb 2023 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2023/079562 | 3/3/2023 | WO |
