WO2025201493A1 - Molécule de liaison à dll3 et son utilisation - Google Patents

Molécule de liaison à dll3 et son utilisation

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Publication number
WO2025201493A1
WO2025201493A1 PCT/CN2025/085579 CN2025085579W WO2025201493A1 WO 2025201493 A1 WO2025201493 A1 WO 2025201493A1 CN 2025085579 W CN2025085579 W CN 2025085579W WO 2025201493 A1 WO2025201493 A1 WO 2025201493A1
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Prior art keywords
amino acid
seq
dll3
hcdr2
hcdr1
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PCT/CN2025/085579
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English (en)
Chinese (zh)
Inventor
黄冰
贾鸽子
王健
郑海林
杨媛媛
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Nona Biosciences Suzhou Co Ltd
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Nona Biosciences Suzhou Co Ltd
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Publication of WO2025201493A1 publication Critical patent/WO2025201493A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells

Definitions

  • DLL3 Delta-Like Ligand 3
  • SCLC small cell lung cancer
  • LNEC large cell neuroendocrine carcinoma
  • DLL3 is expressed in other tumor types of neuroendocrine origin, such as melanoma, glioblastoma multiforme, small cell bladder cancer, metastatic castration-resistant prostate cancer, and neuroendocrine lung tumors.
  • DLL3 In tumor cells, DLL3 is specifically expressed on the cell surface. In contrast, only a few normal cell types (such as neurons, pancreatic islet cells, and pituitary cells) express DLL3, and in normal cells, DLL3 expression is confined to the cytoplasm.
  • the DLL3 expression profile (high, homogeneous cell surface expression in tumors versus low, cytoplasmic expression in subsets of normal tissues) enables the development of therapies that specifically target tumor cells (e.g., SCLC cells) with DLL3.
  • therapies that specifically target tumor cells (e.g., SCLC cells) with DLL3.
  • tumor cells e.g., SCLC cells
  • DLL3-specific drugs against tumors (e.g., SCLC and other neuroendocrine tumors).
  • DLL3-targeting antibody drugs include Rova-T (Rovalpituzumab tesirine), an antibody-drug conjugate developed by AbbVie.
  • Rova-T TM is an antibody-drug conjugate that targets DLL3 in small cell lung cancer (SCLC) tumors, where it is internalized and releases the toxin pyrrolobenzodiazepine, which induces cell death.
  • TCE molecules such as Amgen's tarlatamab, Harpoon Therapeutics' HPN328, Boehringer Ingelheim's BI 764532, Qilu Pharmaceutical's QLS31904, Roche's RO7616789, and Phanes Therapeutics' PT217 are all in Phase I clinical trials.
  • Tarlatamab (AMG 757), developed by Amgen, has been optimized to extend the half-life of the BiTE molecule, activating T cells to target tumors expressing DLL3.
  • a Phase I clinical study of AMG 757 for SCLC patients (NCT03319940) is ongoing.
  • Chimeric antigen receptor T cell therapy genetically modifies the patient's autologous T cells to guide the patient's T cells to express chimeric receptors for tumor antigens, and then re-injects these cells into the patient's body to attack and kill target cells.
  • AMG119 is an adoptive cell therapy that modifies the patient's autologous T cells to express a transmembrane chimeric receptor (CAR) targeting DLL3 and attack DLL3-positive cells.
  • CAR transmembrane chimeric receptor
  • DLL3 As an inhibitory ligand for Notch signaling, DLL3 is closely associated with various tumors. In SCLC, in particular, extensive basic and clinical research evidence indicates that DLL3 is a promising therapeutic target. Therefore, exploring antibody drugs targeting DLL3 for cancer treatment is expected to provide more options for cancer treatment.
  • the present invention provides a DLL3 binding molecule comprising a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1 comprises the sequence shown in SEQ ID NO: 18 or 32 or an amino acid sequence that differs from the sequence shown in SEQ ID NO: 18 or 32 by no more than 2 amino acids in amino acid addition, deletion or substitution; and/or HCDR2 comprises the sequence shown in SEQ ID NO: 69, 74, 79, 80, 87 or 90 or an amino acid sequence that differs from the sequence shown in SEQ ID NO: 69, 74, 79, 80, 87 or 90 by no more than 2 amino acids in amino acid addition, deletion or substitution.
  • Sequence; and/or HCDR3 comprises the sequence shown in SEQ ID NO: 126, 127, 128, 129, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151 or 152, or an amino acid sequence that differs from the sequence shown in SEQ ID NO: 126, 127, 128, 129, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151 or 152 by amino acid addition, deletion or substitution of no more than 2 amino acids.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 60, 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 19, 60, 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 61, 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 20, 62, 127, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 18, 63, and 128, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 21, 64, and 129, respectively; or HCDR1, HCDR2, and HCDR3
  • R2 and HCDR3 contain the amino acid sequences shown in SEQ ID NO: 18, 64, 131, respectively; or HCDR1, HCDR2 and HCDR3 contain the amino acid sequences shown in SEQ ID NO: 19, 68, 126, respectively; or HCDR1, HCDR2 and HCDR3 contain the amino acid sequences shown in SEQ ID NO: 18, 68, 126, respectively; or HCDR1, HCDR2 and HCDR3 contain the amino acid sequences shown in SEQ ID NO: 20, 69, 132, respectively; or HCDR1, HCDR2 and HCDR3 contain the amino acid sequences shown in SEQ ID NO: 24, 66, 126, respectively; or HCDR1, HCDR2 and HCDR3 contain the amino acid sequences shown in SEQ ID NO: 24, 66, 126, respectively; or HCDR1, HCDR2 and HCDR3 contain the amino acid sequences shown in SEQ ID NO: 24, 66, 126, respectively; or CDR2 and HCDR3 comprise
  • the heavy chain variable region comprises 1) SEQ ID NO: 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 185, 186, 187, 188, 189, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 202, 213,
  • the invention provides a multispecific antigen-binding construct comprising a first antigen-binding moiety that binds DLL3, wherein the first antigen-binding moiety comprises the DLL3-binding molecule of the first aspect of the invention.
  • the multispecific antigen-binding construct comprises a first antigen-binding moiety that binds DLL3 and a second antigen-binding moiety that binds a second antigen, wherein the first antigen-binding moiety comprises the DLL3-binding molecule of the first aspect of the invention.
  • the present invention provides a chimeric antigen receptor comprising the DLL3 binding molecule of the first aspect of the invention.
  • the invention provides an antibody conjugate comprising the DLL3 binding molecule of the first aspect of the invention or the multispecific antigen-binding construct of the second aspect of the invention conjugated to at least one therapeutic agent.
  • the present invention provides use of the DLL3 binding molecule of the first aspect of the invention, the multispecific antigen-binding construct of the second aspect of the invention, the immune effector cell of the fourth aspect of the invention, the antibody conjugate of the fifth aspect of the invention, or the pharmaceutical composition of the sixth aspect of the invention in the preparation of a medicament for treating cancer.
  • the present invention provides a polynucleotide encoding the DLL3 binding molecule of the first aspect of the invention or the multispecific antigen-binding construct of the second aspect of the invention.
  • 1A-1D show the binding activity of candidate antibodies to human DLL3 protein.
  • 2A-2D show the binding activity of candidate antibodies to cynomolgus monkey DLL3 protein.
  • Figures 4A-4D show the binding activity of candidate antibodies to CHO-K1 cells overexpressing human DLL3.
  • 10A-10B show the internalization of candidate antibodies in SHP-77 tumor cells.
  • antibody refers to an immunoglobulin or a fragment thereof that specifically binds to an antigenic epitope through at least one antigen binding site. Antibody encompasses antibody fragments.
  • the term “antibody” includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, fully humanized antibodies, heavy chain antibodies, nanobodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as but not limited to Fab fragments, Fab' fragments, F(ab') 2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies.
  • the antibodies provided herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).
  • immunoglobulin class e.g., IgG, IgM, IgD, IgE, IgA, and IgY
  • any class e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2b
  • subclass e.g., IgG2a and IgG2b.
  • the antibodies of the present invention are heavy chain antibodies, and/or are fully humanized antibodies.
  • an "antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the variable region (e.g., one or more CDRs and/or one or more antibody binding sites) of the antibody that binds to an antigen and thus retains binding specificity and at least a portion of the specific binding ability of the full-length antibody.
  • an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived.
  • Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives.
  • Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov).
  • the fragments can include multiple chains linked together, for example, by disulfide bonds and/or by peptide linkers.
  • Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids.
  • Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding regions), results in an antibody that immunospecifically binds to an antigen.
  • heavy-chain-only antibody and “heavy-chain antibody” are used interchangeably and in their broadest sense to refer to antibodies that lack conventional antibody light chains and contain only a heavy chain variable region and a heavy chain constant region that does not contain CH1 (e.g., Fc fragment).
  • Fc fragment generally refers to a crystallizable fragment of a conventional antibody or heavy chain antibody after papain digestion.
  • the Fc fragment of IgG and heavy chain antibodies can include part of the hinge region, CH2 and CH3.
  • the Fc fragment can include at least part of the hinge region (e.g., all or part of the hinge region), CH2 and CH3.
  • variable region i.e., "binding domain” allows the binding molecule to selectively recognize an epitope on an antigen and specifically bind to the epitope. That is, for example, the light chain variable region (VL) domain and the heavy chain variable region (VH) domain of the binding molecule of an antibody, or these complementary determining regions (CDR) subgroups combine to form a variable region that determines a three-dimensional antigen binding site. More specifically, the antigen binding site is determined by three CDRs on each VH and VL chain.
  • the heavy chain antibody of the present invention has an antigen binding site determined by three CDRs on the VH chain.
  • CDRs complementary determining regions
  • FR framework
  • the binding domain formed by the positioned CDRs determines a surface that is complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its complementary epitope.
  • the amino acids that make up the CDRs and framework regions, respectively, in any given heavy or light chain variable region can be identified by conventional methods (see, "Sequences of Proteins of Immunological Interest," Kabat, E.
  • the CDRs (CDRL or LCDR) of the light chain variable region may be referred to as LCDR1, LCDR2, and LCDR3, and the CDRs (CDRH or HCDR) of the heavy chain variable region may be referred to as HCDR1, HCDR2, and HCDR3.
  • the amino acid sequences of CDRs are all shown according to the Chothia definition rules (the sequences in the claims of the present invention are also shown according to the Chothia definition rules).
  • the CDR of an antibody can be defined in the art by a variety of methods, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (see, for example, Chothia, C. et al., Nature, 342, 877-883 (1989); and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)), Kabat based on antibody sequence variability (see, for example, Kabat, E.A.
  • CDR complementary determining region
  • the terms "CDR” and "complementarity determining region" of a given antibody or a region thereof (such as a variable region) should be understood to cover the complementary determining region defined by any of the above-mentioned known schemes described by the present invention.
  • the scope of protection claimed in the claims of the present invention is based on the sequences shown in the Chothia definition rules, the amino acid sequences corresponding to the definition rules of other CDRs should also fall within the scope of protection of the present invention.
  • antibodies defined by specific CDR sequences defined herein the scope of said antibodies also encompasses antibodies whose variable region sequences comprise said specific CDR sequences, but whose declared CDR boundaries differ from the specific CDR boundaries defined herein due to the application of a different scheme (e.g., a different assignment system rule or combination).
  • framework region and “framework region” are used interchangeably.
  • framework region refers to those amino acid residues in the antibody variable region excluding the CDR sequences as defined above.
  • disulfide bond includes a covalent bond formed between two sulfur atoms.
  • the amino acid cysteine contains a sulfhydryl group that can form a disulfide bond or bridge a second sulfhydryl group.
  • percent (%) sequence identity or “sequence identity” of amino acid sequences has the art-recognized definition of the percentage of identity between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a publicly known algorithm). This can be determined using methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal Omega, and FASTA software.
  • Affinity or "binding affinity” is a measure of the strength of the non-covalent binding between an antibody and an antigen. Affinity can be determined using conventional techniques known in the art, such as membrane interferometry (e.g., using the Octet Fortebio assay system), radioimmunoassay, surface plasmon resonance, enzyme-linked immunosorbent assay (ELISA), or flow cytometry (FACS).
  • membrane interferometry e.g., using the Octet Fortebio assay system
  • radioimmunoassay e.g., using the Octet Fortebio assay system
  • ELISA enzyme-linked immunosorbent assay
  • FACS flow cytometry
  • binding molecule "A” has a higher specificity for a given epitope than binding molecule "B,” or it can be said that binding molecule "A” specifically binds to epitope "C” with a higher specificity than its specificity for a related epitope "D.”
  • endocytosis refers to the process by which a substance to be internalized is enclosed by the cell membrane and then buds into a vesicle containing the internalized substance, bringing the external substance into the cell. This is one way that substances are transported across the cytoplasmic membrane.
  • antibody internalization refers to the induction of receptor-mediated endocytosis when a cell surface receptor binds to its specific antibody.
  • DLL3 antibodies can mediate the internalization of cell-surface-expressed DLL3 protein by binding to the extracellular portion of DLL3. The endocytic effect of antibodies can be measured using conventional techniques known in the art, such as labeling with internalizing antibodies.
  • polynucleotide and “nucleic acid” are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA).
  • Polynucleotide sequence “nucleic acid sequence,” and “nucleotide sequence” are used interchangeably to refer to the order of nucleotides in a polynucleotide.
  • an isolated nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule.
  • An "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized.
  • Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules that encode provided DLL3 binding molecules.
  • expression includes transcription and/or translation of a nucleotide sequence.
  • expression can involve the production of transcripts and/or polypeptides.
  • Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes virus vectors, pox virus vectors, and baculovirus vectors, etc.
  • a "host cell” is a cell that is used to receive, maintain, replicate, and amplify a vector.
  • a host cell can also be used to express a polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector is replicated, thereby amplifying the nucleic acid.
  • the host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells such as HEK 293 cells.
  • a "subject in need thereof” can include a subject already suffering from a disease; a subject susceptible to a disease; and a subject in need of prevention of a disease.
  • a DLL3 binding molecule can include any molecule that specifically binds to DLL3.
  • the DLL3 binding molecule is a polypeptide or protein, such as an antibody.
  • the DLL3 binding molecule is an antibody or antigen-binding fragment thereof directed against DLL3.
  • DLL3-binding molecules include synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, fully humanized antibodies, heavy chain antibodies, nanobodies, chimeric antibodies, intrabodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the foregoing.
  • multispecific antibodies e.g., bispecific antibodies
  • human antibodies e.g., non-human antibodies, humanized antibodies, fully humanized antibodies, heavy chain antibodies, nanobodies, chimeric antibodies, intrabodies, and antibody fragments, such as, but not limited to,
  • the DLL3 binding molecule is a heavy chain antibody or antigen-binding fragment thereof comprising a heavy chain variable region.
  • the DLL3 binding molecule is a heavy chain antibody. In another embodiment, the DLL3 binding molecule is a fully humanized antibody. In a preferred embodiment, the DLL3 binding molecule is a heavy chain antibody and is a fully humanized antibody.
  • the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 is substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 18 is substituted with N (SEQ ID NO: 19; GFNFSDY).
  • the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 4 is substituted. In one embodiment, the amino acid at position 4 in SEQ ID NO: 18 is substituted with L (SEQ ID NO: 20; GFTLSDY).
  • the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 4 is substituted. In one embodiment, the amino acid at position 4 in SEQ ID NO: 18 is substituted with 1 (SEQ ID NO: 21; GFTISDY).
  • the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 4 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 and the amino acid at position 4 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 18 is substituted with N, and the amino acid at position 4 in SEQ ID NO: 18 is substituted with I (SEQ ID NO: 22; GFNISDY).
  • the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 4 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 and the amino acid at position 4 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 18 is substituted with N, and the amino acid at position 4 in SEQ ID NO: 18 is substituted with L (SEQ ID NO: 23; GFNLSDY).
  • the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 5 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 and the amino acid at position 5 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 18 is substituted with N, and the amino acid at position 5 in SEQ ID NO: 18 is substituted with N (SEQ ID NO: 24; GFNFNDY).
  • the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 18 is substituted with N (SEQ ID NO: 26; GFTFSNY).
  • the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 7 is substituted. In one embodiment, the amino acid at position 7 in SEQ ID NO: 18 is substituted with H (SEQ ID NO: 27; GFTFSDH).
  • the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 18 is substituted with T (SEQ ID NO: 28; GFTFSTY).
  • the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 is substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 18 is substituted with A (SEQ ID NO: 29; GFAFSDY).
  • the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 18 is substituted with I, and the amino acid at position 6 in SEQ ID NO: 18 is substituted with N (SEQ ID NO: 30; GFIFSNY).
  • the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 18 is substituted with I, and the amino acid at position 6 in SEQ ID NO: 18 is substituted with S (SEQ ID NO: 31; GFIFSSY).
  • the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 4 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 4 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 4 in SEQ ID NO: 18 is substituted with I, and the amino acid at position 6 in SEQ ID NO: 18 is substituted with N (SEQ ID NO: 33; GFTISNY).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 2 and the amino acid at position 3 are substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO: 69 is substituted with A, and the amino acid at position 3 in SEQ ID NO: 69 is substituted with N (SEQ ID NO: 61; SANGST).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 1 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 1 and the amino acid at position 2 are substituted. In one embodiment, the amino acid at position 1 in SEQ ID NO: 69 is substituted with T, and the amino acid at position 2 in SEQ ID NO: 69 is substituted with G (SEQ ID NO: 62; TGSGST).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 5 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 2 and the amino acid at position 5 are substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO: 69 is substituted with G, and the amino acid at position 5 in SEQ ID NO: 69 is substituted with R (SEQ ID NO: 65; SGSGRT).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 2 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO: 69 is substituted with G, and the amino acid at position 6 in SEQ ID NO: 69 is substituted with I (SEQ ID NO: 68; SGSGSI).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 5 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 5 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 5 in SEQ ID NO: 69 is substituted with D, and the amino acid at position 6 in SEQ ID NO: 69 is substituted with S (SEQ ID NO: 70; SSSGDS).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 5 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 5 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 5 in SEQ ID NO: 69 is substituted with N, and the amino acid at position 6 in SEQ ID NO: 69 is substituted with S (SEQ ID NO: 73; SSSGNS).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 3 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 69 is substituted with D, and the amino acid at position 6 in SEQ ID NO: 69 is substituted with S (SEQ ID NO: 75; SSDGSS).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 2 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO: 69 is substituted with G, and the amino acid at position 6 in SEQ ID NO: 69 is substituted with D (SEQ ID NO: 76; SGSGSD).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 3 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 69 is substituted with D, and the amino acid at position 6 in SEQ ID NO: 69 is substituted with N (SEQ ID NO: 83; SSDGSN).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 2 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO: 69 is substituted with T, and the amino acid at position 6 in SEQ ID NO: 69 is substituted with P (SEQ ID NO: 89; STSGSP).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 74 (SEQ ID NO: 74; SGSDNN), wherein the amino acid at position 1 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 74 (SEQ ID NO: 74; SGSDNN), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 74 (SEQ ID NO: 74; SGSDNN), wherein the amino acid at position 1 and the amino acid at position 3 are substituted. In one embodiment, the amino acid at position 1 in SEQ ID NO: 74 is substituted with N, and the amino acid at position 3 in SEQ ID NO: 74 is substituted with N (SEQ ID NO: 72; NGNDNN).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 2 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO: 79 is substituted with G, and the amino acid at position 6 in SEQ ID NO: 79 is substituted with N (SEQ ID NO: 77; NGDGSN).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 79 is substituted with Y (SEQ ID NO: 78; NSDGSY).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 5 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 3 and the amino acid at position 5 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 79 is substituted with N, and the amino acid at position 5 in SEQ ID NO: 79 is substituted with N (SEQ ID NO: 81; NSNGNT).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 79 is substituted with D (SEQ ID NO: 84; NSDGSD).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 79 is substituted with N (SEQ ID NO: 85; NSDGSN).
  • the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 79 is substituted with S (SEQ ID NO: 88; NSDGSS).
  • the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 126 (SEQ ID NO: 126; ENGNSDY), wherein the amino acid at position 2 is substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO: 126 is substituted with S (SEQ ID NO: 131; ESGNSDY).
  • the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 127 (SEQ ID NO: 127; GLGEFSLGI), wherein the amino acid at position 9 is substituted. In one embodiment, the amino acid at position 9 in SEQ ID NO: 127 is substituted with S (SEQ ID NO: 130; GLGEFSLGS).
  • the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 127 (SEQ ID NO: 127; GLGEFSLGI), wherein the amino acid at position 5 is substituted. In one embodiment, the amino acid at position 5 in SEQ ID NO: 127 is substituted with L (SEQ ID NO: 132; GLGELSLGI).
  • the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 133 (SEQ ID NO: 133; ERGSTGGWFDP), wherein the amino acid at position 2 is substituted.
  • the amino acid at position 2 in SEQ ID NO: 133 is substituted with K (SEQ ID NO: 134; EKGSTGGWFDP).
  • the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 139 (SEQ ID NO: 139; GGDTVVVPEFDY), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 139 (SEQ ID NO: 139; GGDTVVVPEFDY), wherein the amino acid at position 7 is substituted. In some embodiments, the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 139 (SEQ ID NO: 139; GGDTVVVPEFDY), wherein the amino acid at position 2 and the amino acid at position 7 are substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO: 139 is substituted with D, and the amino acid at position 7 in SEQ ID NO: 139 is substituted with L (SEQ ID NO: 145; GDDTVVLPEFDY).
  • HCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34.
  • HCDR2 comprises an amino acid sequence as shown in SEQ ID NO: 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90.
  • HCDR3 comprises an amino acid sequence as shown in SEQ ID NO:126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 or 152.
  • HCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34
  • HCDR2 comprises an amino acid sequence as shown in SEQ ID NO: 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 2, 83, 84, 85, 86, 87, 88, 89 or 90
  • HCDR3 comprises the amino acid sequence shown in SEQ ID NO:126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 or 152.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO:18, 60, 126, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO:19, 60, 126, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO:18, 61, 126, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 20, 62, 127, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO:18, 63, 128, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 22, 65, and 126, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 23, 65, and 126, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO:18, 66, 126, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO:18, 67, 130, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO:18, 64, 131, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO:19, 68, and 126, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO:18, 68, 126, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 20, 69, 132, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 24, 66, 126, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO:18, 71, 134, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 25, 74, 135, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 26, 75, 136, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 25, 77, 138, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 25, 78, 139, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 25, 82, 143, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 29, 84, 143, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO:32, 86, and 147, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO:18, 85, 148, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 33, 87, 149, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 26, 88, and 150, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO:18, 89, 151, respectively.
  • the heavy chain variable region comprises 1) SEQ ID NO: 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210 , 211, 212, 213, 214, 215, 216 or 217 having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%
  • the heavy chain variable region is fused to another molecule.
  • the other molecule is the Fc domain of an immunoglobulin.
  • the other molecule is the Fc domain of human immunoglobulin G1 (IgG1).
  • the DLL3 binding molecule comprises 1) SEQ ID NO: 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260 , 261, 262, 263, 264 or 265; 2) an amino acid sequence having the same amino acid sequence as SEQ ID NO: 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258 8) an amino acid sequence having at least 80%, at least 85%,
  • the DLL3 binding molecule has at least one of the following characteristics:
  • the DLL3 binding molecules include antibodies to DLL3, such as PR013234, PR013235, PR013236, PR013239, PR013240, PR013241, PR013242, PR013244, PR013246, PR013249, PR013250, PR013252, PR013253, PR013254, PR013256, PR013257, PR013258, PR013259, PR013260, PR013261, PR013262, PR013263, PR013264, PR013363, PR013364, 13240, PR013254, PR013371, PR013377, PR013393, and PR013397.
  • the DLL3 antibody PR013241 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 179 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 179.
  • the DLL3 antibody PR013241 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013241 comprises the amino acid sequence shown in SEQ ID NO:227 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:227.
  • the antibody comprises a heavy chain variable region, wherein
  • the heavy chain variable region comprises:
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:64, and
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:129.
  • the DLL3 antibody PR013242 comprises a heavy chain variable region (VH),
  • the DLL3 antibody PR013242 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013242 comprises the amino acid sequence shown in SEQ ID NO:228 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:228.
  • the present invention provides an antibody PR013244 or an antigen-binding fragment thereof against DLL3,
  • the antibody comprises a heavy chain variable region, wherein
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 181 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 181.
  • the DLL3 antibody PR013244 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013244 comprises the amino acid sequence shown in SEQ ID NO:229 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:229.
  • the present invention provides an antibody PR013246 or an antigen-binding fragment thereof against DLL3,
  • the DLL3 antibody PR013246 comprises a heavy chain variable region (VH),
  • the DLL3 antibody PR013246 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013246 comprises the amino acid sequence shown in SEQ ID NO:230 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:230.
  • the present invention provides an antibody PR013249 or an antigen-binding fragment thereof against DLL3,
  • the antibody comprises a heavy chain variable region, wherein
  • the heavy chain variable region comprises:
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:66, and
  • the DLL3 antibody PR013249 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 183 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 183.
  • the DLL3 antibody PR013249 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013249 comprises the amino acid sequence shown in SEQ ID NO:231 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:231.
  • the antibody comprises a heavy chain variable region, wherein
  • the heavy chain variable region comprises:
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:67, and
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 184 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 184.
  • the DLL3 antibody PR013250 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013250 comprises the amino acid sequence shown in SEQ ID NO:232 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:232.
  • the antibody comprises a heavy chain variable region, wherein
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:18,
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:131.
  • the DLL3 antibody PR013252 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 185 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 185.
  • the DLL3 antibody PR013252 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013252 comprises the amino acid sequence shown in SEQ ID NO:233 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:233.
  • the present invention provides an antibody PR013253 or an antigen-binding fragment thereof against DLL3,
  • the heavy chain variable region comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:19,
  • the DLL3 antibody PR013253 comprises a heavy chain variable region (VH),
  • the DLL3 antibody PR013253 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013253 comprises the amino acid sequence shown in SEQ ID NO:234 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:234.
  • the present invention provides an antibody PR013254 or an antigen-binding fragment thereof against DLL3,
  • the antibody comprises a heavy chain variable region, wherein
  • the DLL3 antibody PR013254 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 187 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 187.
  • the DLL3 antibody PR013254 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013254 comprises the amino acid sequence shown in SEQ ID NO:235 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:235.
  • the present invention provides an antibody PR013256 or an antigen-binding fragment thereof against DLL3,
  • the heavy chain variable region comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:18,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:68, and
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:126.
  • the DLL3 antibody PR013256 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 188 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 188.
  • the DLL3 antibody PR013256 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013256 comprises the amino acid sequence shown in SEQ ID NO:236 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:236.
  • the present invention provides an antibody PR013257 or an antigen-binding fragment thereof against DLL3,
  • the antibody comprises a heavy chain variable region, wherein
  • the heavy chain variable region comprises:
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 189 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 189.
  • the present invention provides an antibody PR013258 or an antigen-binding fragment thereof against DLL3,
  • the antibody comprises a heavy chain variable region, wherein
  • the heavy chain variable region comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:24,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:66, and
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:126.
  • the DLL3 antibody PR013258 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 190 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 190.
  • the DLL3 antibody PR013258 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013258 comprises the amino acid sequence shown in SEQ ID NO:238 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:238.
  • the present invention provides an antibody PR013259 or an antigen-binding fragment thereof against DLL3,
  • the heavy chain variable region comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:18,
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:133.
  • the DLL3 antibody PR013259 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 191 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 191.
  • the present invention provides an antibody PR013260 or an antigen-binding fragment thereof against DLL3,
  • the antibody comprises a heavy chain variable region, wherein
  • the heavy chain variable region comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:18,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:70, and
  • the DLL3 antibody PR013260 comprises a heavy chain variable region (VH),
  • the DLL3 antibody PR013260 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013260 comprises the amino acid sequence shown in SEQ ID NO: 240 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 240.
  • the present invention provides an antibody PR013261 or an antigen-binding fragment thereof against DLL3,
  • the antibody comprises a heavy chain variable region, wherein
  • the heavy chain variable region comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:18,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:71, and
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:134.
  • the DLL3 antibody PR013261 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 193 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 193.
  • the DLL3 antibody PR013261 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013261 comprises the amino acid sequence shown in SEQ ID NO:241 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:241.
  • the antibody comprises a heavy chain variable region, wherein
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:25,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:72, and
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 194 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 194.
  • the present invention provides an antibody PR013263 or an antigen-binding fragment thereof against DLL3,
  • the antibody comprises a heavy chain variable region, wherein
  • the heavy chain variable region comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:18,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:73, and
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:134.
  • the DLL3 antibody PR013263 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 195 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 195.
  • the DLL3 antibody PR013263 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013263 comprises the amino acid sequence shown in SEQ ID NO:243 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:243.
  • the present invention provides an antibody PR013264 or an antigen-binding fragment thereof against DLL3,
  • the antibody comprises a heavy chain variable region, wherein
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:74, and
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 196 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 196.
  • the DLL3 antibody PR013264 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013264 comprises the amino acid sequence shown in SEQ ID NO:244 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:244.
  • the antibody comprises a heavy chain variable region, wherein
  • the heavy chain variable region comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:26,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:75, and
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:136.
  • the DLL3 antibody PR013363 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 197 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 197.
  • the DLL3 antibody PR013363 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013363 comprises the amino acid sequence shown in SEQ ID NO:245 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:245.
  • the present invention provides an antibody PR013364 or an antigen-binding fragment thereof against DLL3,
  • the heavy chain variable region comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:27,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:76, and
  • the DLL3 antibody PR013364 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 198 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 198.
  • the antibody comprises a heavy chain variable region, wherein
  • the heavy chain variable region comprises:
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:77, and
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:138.
  • the DLL3 antibody PR013367 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 199 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 199.
  • the DLL3 antibody PR013367 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013367 comprises the amino acid sequence shown in SEQ ID NO:247 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:247.
  • the present invention provides an antibody PR013368 or an antigen-binding fragment thereof against DLL3,
  • the heavy chain variable region comprises:
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:139.
  • the present invention provides an antibody PR013370 or an antigen-binding fragment thereof against DLL3,
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:18,
  • the DLL3 antibody PR013370 comprises a heavy chain variable region (VH),
  • the DLL3 antibody PR013370 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013370 comprises the amino acid sequence shown in SEQ ID NO:249 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:249.
  • the present invention provides an antibody PR013371 or an antigen-binding fragment thereof against DLL3.
  • the antibody comprises a heavy chain variable region, wherein
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:26,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:80, and
  • the DLL3 antibody PR013371 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 202 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 202.
  • the DLL3 antibody PR013371 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013371 comprises the amino acid sequence shown in SEQ ID NO: 250 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 250.
  • the antibody comprises a heavy chain variable region, wherein
  • the heavy chain variable region comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:26,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:81, and
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:142.
  • the DLL3 antibody PR013372 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 203 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 203.
  • the DLL3 antibody PR013372 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013372 comprises the amino acid sequence shown in SEQ ID NO:251 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:251.
  • the present invention provides an antibody PR013376 or an antigen-binding fragment thereof against DLL3,
  • the antibody comprises a heavy chain variable region, wherein
  • the heavy chain variable region comprises:
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:82, and
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:143.
  • the DLL3 antibody PR013376 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 204 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 204.
  • the DLL3 antibody PR013376 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013376 comprises the amino acid sequence shown in SEQ ID NO:252 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:252.
  • the present invention provides an antibody PR013377 or an antigen-binding fragment thereof against DLL3,
  • the antibody comprises a heavy chain variable region, wherein
  • the heavy chain variable region comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:18,
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:126.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 205 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 205.
  • the DLL3 antibody PR013377 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013377 comprises the amino acid sequence shown in SEQ ID NO:253 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:253.
  • the present invention provides an antibody PR013378 or an antigen-binding fragment thereof against DLL3,
  • the antibody comprises a heavy chain variable region, wherein
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:28,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:83, and
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 206 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 206.
  • the DLL3 antibody PR013378 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013378 comprises the amino acid sequence shown in SEQ ID NO:254 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:254.
  • the present invention provides an antibody PR013381 or an antigen-binding fragment thereof against DLL3,
  • the antibody comprises a heavy chain variable region, wherein
  • the heavy chain variable region comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:29,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:84, and
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:143.
  • the DLL3 antibody PR013381 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 207 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 207.
  • the DLL3 antibody PR013381 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013381 comprises the amino acid sequence shown in SEQ ID NO:255 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:255.
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:26,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:85, and
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 208 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 208.
  • the present invention provides an antibody PR013384 or an antigen-binding fragment thereof against DLL3,
  • the antibody comprises a heavy chain variable region, wherein
  • the heavy chain variable region comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:30,
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:136.
  • the DLL3 antibody PR013384 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 209 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 209.
  • the DLL3 antibody PR013384 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013384 comprises the amino acid sequence shown in SEQ ID NO:257 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:257.
  • the present invention provides an antibody PR013387 or an antigen-binding fragment thereof against DLL3,
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:146.
  • the DLL3 antibody PR013387 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 210 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 210.
  • the present invention provides an antibody PR013392 or an antigen-binding fragment thereof against DLL3,
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:18,
  • the DLL3 antibody PR013392 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013392 comprises the amino acid sequence shown in SEQ ID NO:259 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:259.
  • the heavy chain variable region comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:32,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:86, and
  • the DLL3 antibody PR013393 comprises a heavy chain variable region (VH),
  • the DLL3 antibody PR013393 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013393 comprises the amino acid sequence shown in SEQ ID NO:260 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:260.
  • the heavy chain variable region comprises:
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 214 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 214.
  • the DLL3 antibody PR013397 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013397 comprises the amino acid sequence shown in SEQ ID NO:262 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:262.
  • the present invention provides an antibody PR013399 or an antigen-binding fragment thereof against DLL3.
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO:150.
  • the DLL3 antibody PR013399 comprises a heavy chain variable region (VH),
  • the DLL3 antibody PR013399 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013399 comprises the amino acid sequence shown in SEQ ID NO:263 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:263.
  • the antibody comprises a heavy chain variable region, wherein
  • the DLL3 antibody PR013402 comprises a heavy chain variable region (VH),
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 216 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 216.
  • the DLL3 antibody PR013402 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013402 comprises the amino acid sequence shown in SEQ ID NO:264 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:264.
  • the antibody comprises a heavy chain variable region, wherein
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:34,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO:90, and
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 217 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 217.
  • modification refers to the presence of changes to a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. Such changes or modifications can be obtained by post-synthetic modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide; or by co-translational or post-translational modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide.
  • the invention provides a multispecific antigen-binding construct comprising a first antigen-binding moiety that binds DLL3, wherein the first antigen-binding moiety comprises the DLL3-binding molecule of the first aspect of the invention.
  • the multispecific antigen-binding construct comprises a first antigen-binding moiety that binds DLL3 and a second antigen-binding moiety that binds a second antigen, wherein the first antigen-binding moiety comprises the DLL3-binding molecule of the first aspect of the invention.
  • first antigen-binding portion and “second antigen-binding portion” refer to amino acid sequences that contain an antigen-binding site and are capable of binding to an antigen epitope, and are defined within the meaning of an antibody or an antigen-binding fragment.
  • the second antigen binding portion can be an antibody or antigen binding fragment that binds to any antigenic epitope of interest.
  • the second antigen is an antigen other than DLL3.
  • Antigens that the second antigen binding portion can specifically bind to may include: tumor antigens (e.g., tumor-associated antigens and tumor-specific antigens), immunomodulatory receptors, and immune checkpoint molecules.
  • tumor antigens e.g., tumor-associated antigens and tumor-specific antigens
  • immunomodulatory receptors e.g., immunomodulatory receptors, and immune checkpoint molecules.
  • tumor antigens e.g., tumor-associated antigens and tumor-specific antigens
  • immunomodulatory receptors e.g., antigen-associated antigens and tumor-specific antigens
  • immune checkpoint molecules e.g., tumor-associated antigens and tumor-specific antigens
  • tumor-associated antigen refers to an antigen that is highly expressed in tumor cells and also present in healthy cells but
  • the second antigen binding portion specifically binds SIRP ⁇ , PD-1, PD-L1, LAG3, TIM-3, CTLA-4, VISTA, GPC3, EGFR, HER-2, CD19, CD20, CD33, CD40, CD73, OX40, CD3, TIP-1, folate receptor alpha (FOLR1) and/or other antigens.
  • the first antigen binding portion and the second antigen binding portion can optionally be connected by a linker. In some embodiments, the first antigen binding portion and the second antigen binding portion are not connected by a linker. In other embodiments, the first antigen binding portion and the second antigen binding portion are connected by a linker, such as a peptide linker or a chemical bond. Preferably, the first antigen binding portion and the second antigen binding portion are connected by a peptide linker.
  • Exemplary peptide linkers can include but are not limited to polyglycine (G), polyalanine (A), polyserine (S) or a combination thereof, such as GGAS, GGGS, GGGSG or (G 4 S) n , wherein n is an integer of 1-20.
  • the present invention also provides a chimeric antigen receptor (CAR) comprising a DLL3 binding molecule of the present invention (e.g., an anti-DLL3 antibody of the present invention or an antigen-binding fragment thereof).
  • CAR chimeric antigen receptor
  • the term "chimeric antigen receptor (CAR)" refers to a polypeptide comprising at least an extracellular domain, a transmembrane domain, and an intracellular T cell receptor activation signaling domain, wherein the extracellular domain binds to or specifically binds to a target on a monospecific or multispecific antibody through a monospecific or multispecific antibody.
  • CAR is a recombinant receptor that simultaneously provides antigen binding and activates T cell function.
  • CAR structure and engineering are described in, for example, Dotti G. et al., (2014) Immunol Rev. 257(1):107-126, which is incorporated herein by reference.
  • the extracellular region comprises a DLL3 binding molecule of the invention (e.g., an anti-DLL3 antibody of the invention or an antigen-binding fragment thereof), such that the CAR of the invention binds to cells expressing DLL3 (e.g., cancer cells).
  • a DLL3 binding molecule of the invention e.g., an anti-DLL3 antibody of the invention or an antigen-binding fragment thereof
  • the CAR of the invention binds to cells expressing DLL3 (e.g., cancer cells).
  • the intracellular signaling region further comprises at least one co-stimulatory domain.
  • the co-stimulatory domain can promote the activation of CAR-expressing immune effector cells after binding to the antigen targeted by the extracellular region.
  • the present invention also relates to a polynucleotide encoding the CAR of the present invention and an expression vector comprising the polynucleotide.
  • the present invention also provides an immune effector cell that expresses the CAR of the present invention on the cell surface.
  • the immune effector cell is selected from T lymphocytes (e.g., cytotoxic T cells (CTL)), macrophages, natural killer cells (NK) and natural killer T cells (NKT).
  • TTL cytotoxic T cells
  • NK natural killer cells
  • NKT natural killer T cells
  • Immune effector cells can target DLL3-positive diseased cells (e.g., DLL3-positive cancer cells) and be activated to start effector functions, for example, causing the death of DLL3-positive cancer cells.
  • the present invention further provides an antibody conjugate comprising a DLL3-binding molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof) or a multispecific antigen-binding construct of the invention conjugated to at least one therapeutic agent.
  • An antibody-drug conjugate is a typical antibody conjugate, wherein the therapeutic agent can be, for example, a cytotoxic agent.
  • the therapeutic agent can be selected from cytotoxic agents, therapeutic antibodies (e.g., antibodies or antigen-binding fragments thereof that specifically bind to another antigen), radioactive isotopes, oligonucleotides and their analogs (e.g., interfering RNA), biologically active peptides, protein toxins (e.g., diphtheria toxin, ricin toxin), and enzymes (e.g., urease).
  • therapeutic antibodies e.g., antibodies or antigen-binding fragments thereof that specifically bind to another antigen
  • radioactive isotopes e.g., oligonucleotides and their analogs (e.g., interfering RNA), biologically active peptides, protein toxins (e.g., diphtheria toxin, ricin toxin), and enzymes (e.g., urease).
  • therapeutic antibodies e.g., antibodies or antigen-binding fragments thereof that specifically bind to another
  • Cytotoxic agents refer to substances that inhibit or reduce the activity, function and/or kill cells.
  • examples of cytotoxic agents may include, but are not limited to, maytansinoids (e.g., maytansine), auristatins (e.g., MMAF, MMAE, MMAD), duostatins, cryptophycins, vinca alkaloids (e.g., vinblastine, vincristine), colchicines, dolastatins, taxanes, paclitaxel, docetaxel, cabazitaxel, enediyne antibiotics, cytochalasins, camptothecins, anthracyclines (e.g., daunorubicin, dihydroxyanthracindione, doxorubicin), cytotoxic antibiotics (e.g., mitomycin, actinomycin), dactinomycin, dactin, dactycin, dactin ... [0013] Examples of the
  • the therapeutic agent is selected from cytotoxic agents, chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, antibodies targeting tumor-specific antigens, and other anti-tumor drugs.
  • the therapeutic agent is a cytotoxic agent.
  • the therapeutic agent is a radioisotope.
  • the therapeutic agent is an immune checkpoint inhibitor.
  • the therapeutic agent can be conjugated to the DLL3-binding molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof) or the multispecific antigen-binding construct of the invention via a linker using any technique known in the art.
  • the linker can comprise a reactive group for covalent conjugation, such as an amine, hydroxylamine, maleimido, carboxyl, phenyl, thiol, sulfhydryl, or hydroxyl group.
  • the present invention also provides a pharmaceutical composition
  • a DLL3 binding molecule of the present invention e.g., an anti-DLL3 antibody or antigen-binding fragment thereof of the present invention
  • a multispecific antigen-binding construct e.g., an immune effector cell or an antibody conjugate, and a pharmaceutically acceptable carrier.
  • suitable carriers can be selected from a buffer (e.g., citrate buffer, acetate buffer, phosphate buffer, histidine buffer, histidine salt buffer), an isotonic agent (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), a non-ionic surfactant (e.g., polysorbate 80, polysorbate 20, poloxamer), or a combination thereof.
  • a buffer e.g., citrate buffer, acetate buffer, phosphate buffer, histidine buffer, histidine salt buffer
  • an isotonic agent e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose
  • a non-ionic surfactant e.g., polysorbate 80, polysorbate 20, poloxamer
  • compositions provided herein may be in a variety of dosage forms, including but not limited to solid, semisolid, liquid, powder, or lyophilized forms.
  • preferred dosage forms include, for example, injection solutions and lyophilized powders.
  • compositions provided herein can be administered to a subject by any method known in the art, for example, by systemic or topical administration.
  • Routes of administration include, but are not limited to, parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous, or intracavitary), topical (e.g., intratumoral), epidural, or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual, or topical).
  • parenteral e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous, or intracavitary
  • topical e.g., intratumoral
  • epidural e.g., intranasal, oral, vaginal, rectal, sublingual, or topical
  • the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion).
  • the exact dosage administered will depend on various factors, such as the metabolic kinetic properties of the pharmaceutical composition, the duration of treatment, the excretion rate of the specific compound, the purpose of the treatment, the route of administration, and the condition of the subject, such as the patient's age, health status, weight, sex, diet, medical history, and other factors well known in the medical field.
  • the DLL3-binding molecules of the invention e.g., anti-DLL3 antibodies or antigen-binding fragments thereof), multispecific antigen-binding constructs, or antibody conjugates can be administered at a dosage ranging from about 0.0001 to 100 mg/kg, more typically 0.01 to 20 mg/kg of the subject's body weight.
  • the dosage can be 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, or 20 mg/kg, or within the range of 1-20 mg/kg.
  • Exemplary treatment regimens entail dosing once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, once every three to six months, or initially with a shorter dosing interval followed by a longer dosing interval.
  • the present invention relates to use of a DLL3 binding molecule (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof), a multispecific antigen-binding construct, an immune effector cell, an antibody conjugate, or a pharmaceutical composition of the invention in the preparation of a medicament for treating a disease in a subject.
  • a DLL3 binding molecule e.g., an anti-DLL3 antibody or antigen-binding fragment thereof
  • a multispecific antigen-binding construct e.g., an immune effector cell, an antibody conjugate, or a pharmaceutical composition of the invention in the preparation of a medicament for treating a disease in a subject.
  • the present invention also relates to the DLL3 binding molecules of the present invention (eg, the anti-DLL3 antibodies or antigen-binding fragments thereof), multispecific antigen-binding constructs, immune effector cells, antibody conjugates or pharmaceutical compositions of the present invention for use in treating diseases.
  • DLL3 binding molecules of the present invention eg, the anti-DLL3 antibodies or antigen-binding fragments thereof
  • multispecific antigen-binding constructs eg. the antigen-binding constructs, immune effector cells, antibody conjugates or pharmaceutical compositions of the present invention for use in treating diseases.
  • the present invention also provides a method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of a DLL3-binding molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof), a multispecific antigen-binding construct, an immune effector cell, an antibody conjugate, or a pharmaceutical composition.
  • a DLL3-binding molecule of the invention e.g., an anti-DLL3 antibody or antigen-binding fragment thereof
  • a multispecific antigen-binding construct e.g., an immune effector cell, an antibody conjugate, or a pharmaceutical composition.
  • a "therapeutically effective dose” refers to a dose that results in a decrease in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevents damage or disability caused by the suffering of the disease.
  • a therapeutically effective dose is used for anti-proliferative effects, preventing further tumor development, reducing tumor size, reducing tumor vascularity, reducing the number of cancer cells, inhibiting, delaying or reducing tumor and/or malignant cell growth and/or metastasis in cancer patients, and/or reducing one or more symptoms associated with the disease that can be observed.
  • the therapeutically effective dose can vary depending on many different factors, including the mode of administration, the target site, the patient's physiological state, whether the patient is human or other animal, other drugs administered, and whether the treatment is prophylactic or therapeutic.
  • the patient is human, but non-human mammals, including transgenic animals, can also be treated.
  • the therapeutic dose can be titrated using conventional methods known to those skilled in the art to optimize safety and efficacy.
  • a "therapeutically effective dose" of a DLL3 binding molecule e.g., an anti-DLL3 antibody or antigen-binding fragment thereof), multispecific antigen-binding construct, immune effector cell, antibody conjugate, or pharmaceutical composition of the invention preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%.
  • the ability to inhibit tumor growth can be evaluated in animal model systems predictive of efficacy against human tumors.
  • an effective amount of a DLL3 binding molecule e.g., an anti-DLL3 antibody or antigen-binding fragment thereof
  • multispecific antigen-binding construct e.g., immune effector cell, antibody conjugate, or pharmaceutical composition of the invention is capable of reducing tumor size or otherwise alleviating symptoms in a subject, such as preventing and/or treating metastasis or recurrence.
  • Such an amount can be determined by one skilled in the art based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.
  • the disease is associated with abnormal DLL3 expression.
  • abnormal expression refers to excessively high or low protein expression levels in a sample compared to a normal sample (or a standard sample, such as a sample from a subject not suffering from a disease associated with abnormal DLL3 expression).
  • the disease is characterized by high DLL3 expression.
  • DLL3 is highly expressed in tissues (e.g., tumor tissue and adjacent tissues) of subjects suffering from or suspected of suffering from a disease (e.g., small cell lung cancer), while DLL3 is low in the corresponding tissues of subjects not suffering from the disease.
  • the disease as described above is cancer.
  • the cancer is a DLL3-positive tumor.
  • the cancer is selected from small cell lung cancer (SCLC); large cell neuroendocrine carcinoma (LCNEC); neuroendocrine tumors of various tissues including the kidney, genitourinary tract (e.g., bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (e.g., stomach, colon), thyroid (e.g., medullary thyroid carcinoma), pancreas, and lung; glioma or pseudoneuroendocrine tumor (pNET).
  • SCLC small cell lung cancer
  • LNEC large cell neuroendocrine carcinoma
  • pNET pseudoneuroendocrine tumor
  • the DLL3-binding molecules of the invention e.g., anti-DLL3 antibodies or antigen-binding fragments thereof
  • multispecific antigen-binding constructs e.g., immune effector cells, antibody conjugates, or pharmaceutical compositions of the invention
  • other treatment methods including but not limited to surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, hormone therapy, angiogenesis inhibition, and palliative care.
  • the DLL3 binding molecules of the present invention are further used in combination with one or more therapeutic agents selected from the group consisting of chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, and tumor antigen-targeted drugs.
  • chemotherapeutic agents may include, for example, antimetabolites, alkylating agents, cytotoxic agents, topoisomerase inhibitors, and microtubule inhibitors.
  • Tumor antigen-targeted drugs include, but are not limited to, drugs that target tumor-associated antigens and tumor-specific antigens.
  • therapeutic agents may include, for example, angiogenesis inhibitors, heparin deacetylase (HDAC) inhibitors, hedgehog signaling pathway blockers, mTOR inhibitors, p53/mdm2 inhibitors, PARP inhibitors, proteasome inhibitors (e.g., bortezomib, carfilzomib, ixazomib, marizomib, oprozomib), and tyrosine kinase inhibitors (e.g., BTK inhibitors).
  • HDAC heparin deacetylase
  • the DLL3 binding molecules e.g., anti-DLL3 antibodies or antigen-binding fragments thereof), multispecific antigen-binding constructs, immune effector cells, antibody conjugates, or pharmaceutical compositions of the present invention are used in combination with a radioisotope.
  • the DLL3 binding molecules e.g., anti-DLL3 antibodies or antigen-binding fragments thereof
  • multispecific antigen-binding constructs, immune effector cells, antibody conjugates, or pharmaceutical compositions of the present invention are used in combination with a tumor antigen-targeted drug.
  • the present invention provides a polynucleotide encoding a DLL3 binding molecule of the present invention (eg, an anti-DLL3 antibody or antigen-binding fragment thereof of the present invention) or a multispecific antigen-binding construct.
  • the present invention also provides an expression vector comprising a polynucleotide of the present invention.
  • the expression vector may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes.
  • the polynucleotide of the present invention may be present in one or more expression vectors.
  • the polynucleotide of the present invention is prepared as a recombinant nucleic acid. Recombinant nucleic acids can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination techniques (e.g., polymerase chain reaction (PCR) technology), etc.
  • PCR polymerase chain reaction
  • the present invention further provides a host cell comprising a polynucleotide or expression vector of the present invention.
  • the polynucleotide or expression vector of the present invention can be introduced into a suitable host cell using various methods known in the art. Such methods include, but are not limited to, liposome transfection, electroporation, viral transduction, and calcium phosphate transfection.
  • host cells are used to express the DLL3-binding molecules of the invention (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof) or multispecific antigen-binding constructs of the invention.
  • host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, such as E. coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells).
  • prokaryotic cells e.g., bacteria, such as E. coli
  • eukaryotic cells e.g., yeast, insect cells, mammalian cells.
  • Mammalian host cells suitable for antibody expression include, but are not limited to, human cervical carcinoma cells (HeLa cells), human embryonic kidney cells (HEK cells, such as HEK 293 cells), Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for antibody expression.
  • HeLa cells human cervical carcinoma cells
  • HEK cells human embryonic kidney cells
  • CHO Chinese ham
  • the present invention further provides a method for producing a DLL3-binding molecule of the present invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof of the present invention) or a multispecific antigen-binding construct of the present invention, the method comprising:
  • DLL3-binding molecules of the invention e.g., anti-DLL3 antibodies or antigen-binding fragments thereof
  • multispecific antigen-binding constructs of the invention e.g., multispecific antigen-binding constructs of the invention
  • kits comprising a DLL3-binding molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof), a multispecific antigen-binding construct, an immune effector cell, an antibody conjugate, or a pharmaceutical composition, and instructions for use.
  • the kit may also comprise a suitable container.
  • the kit further comprises a device for administration.
  • Kits generally include a label indicating the intended use and/or instructions for use of the kit contents.
  • label includes any written or recorded material provided on or with the kit, or otherwise accompanying the kit.
  • Example 1 Obtaining a fully human anti-DLL3 HCAb antibody
  • Antibodies specifically binding to DLL3 can be obtained by immunizing experimental animals (e.g., mice, rats, rabbits, sheep, camels, etc.) with the DLL3 antigen.
  • the resulting antibodies are non-human.
  • these molecules need to be humanized using antibody engineering techniques to reduce immunogenicity and improve drugability.
  • the humanization process is technically complex, and humanized molecules often have reduced affinity for the antigen.
  • advances in transgenic technology have enabled the creation of genetically engineered mice that carry a human immunoglobulin repertoire and lack the endogenous murine immune repertoire.
  • the antibodies produced by these transgenic mice possess fully human sequences, eliminating the need for further humanization and significantly improving the efficiency of therapeutic antibody development.
  • HCAb mice (Harbour Antibodies BV, WO2002/085945A3) are transgenic mice carrying a human immunoglobulin repertoire, capable of producing novel heavy-chain-only antibodies that are half the size of traditional IgG antibodies.
  • the antibodies it produces only have human antibody "heavy chain” variable domains and mouse Fc constant domains. Due to the absence of light chains, the antibodies almost solve the problems of light chain mispairing and heterodimerization, making this technology platform capable of developing products that are difficult to achieve with traditional antibody platforms.
  • mice transgenic mice expressing human DLL3-his tags, using recombinant human DLL3-his tagged protein (Acro, #DL3-H52H4).
  • each mouse was injected with 50 ⁇ g of immunogen protein and 0.2 mL of complete Freund's adjuvant (CFA, Sigma, #F5881) into the abdominal cavity, axillary lymph nodes, and inguinal lymph nodes, respectively.
  • CFA complete Freund's adjuvant
  • mouse serum was collected and diluted 10-fold to obtain six concentrations (1:100, 1:1000, 1:10,000, 1:100,000, and 1:1,000,000).
  • ELISA titers were determined using ELISA plates coated with huDLL3-his protein (Acro, #DL3-H52H4), cynoDLL3-his protein (Acro, #DL3-C52H9), or proteins unrelated to DLL3.
  • Flow cytometry was used to analyze the specific reactivity of two concentrations of mouse serum (1:100 and 1:1000) against CHO-K1/humanDLL3 cells, CHO-K1/cynoDLL3 cells, and CHO-K1 blast cells expressing high DLL3 expression.
  • a blank control (PB) consisted of serum from pre-immunized mice.
  • CHO-K1/human DLL3, CHO-K1/cyno DLL3, or CHO-K1 cells were incubated with serially diluted mouse serum for 1 hour at 4°C. After washing twice, the cells were incubated with a secondary antibody, Alexa FluorTM 488 goat anti-mouse IgG Fc ⁇ specific (Jackson, #115-545-071), for 1 hour at 4°C. After washing twice, the cells were resuspended and analyzed by flow cytometry (BD Canto II). CHO-K1 cells served as a background control.
  • the plasmid encoding the HCAb antibody is transfected into mammalian host cells (such as human embryonic kidney cells HEK293), and the purified anti-DLL3 recombinant heavy chain antibody can be obtained using conventional recombinant protein expression and purification techniques.
  • the plasmid was purified using a large extraction kit (Macherey-Nagel, Plasmids were extracted using Xtra Midi (Extra Midi) to improve their purity and quality, and then filtered through a 0.22 ⁇ m filter (millpore). The purified plasmids were used for transfection.
  • HEK293 cells were cultured in FreeStyle TM F17 Expression Medium (Thermo, A1383504).
  • the target protein is then eluted with 0.1 M glycine (pH 3.5), adjusted to neutrality with Tris-HCl (pH 8.0), and concentrated using an ultrafiltration tube (Millipore, UFC901024) to PBS buffer to obtain a purified anti-DLL3 heavy chain antibody solution.
  • the antibody concentration was determined by NanoDrop assay at 280 nm absorbance, and the antibody purity was determined by SEC-HPLC and SDS-PAGE.
  • the plate was washed three times with PBST buffer (pH 7.4, containing 0.05% tween-20). 250 mL of 2% BSA blocking buffer was added, and the plate was incubated at 37°C for 1 hour. The blocking buffer was discarded and the plate was washed three times with PBST buffer (pH 7.4, containing 0.05% Tween-20). Then, 100 mL of the antibody to be tested was added to each well in a 5-fold serial dilution. The plate was incubated at 37°C for 1 hour. An isotype antibody was used as a control.
  • 18 antibodies such as PR013234, PR013235, PR013236, PR013239, PR013240, PR013241, PR013244, PR013246, PR013254, PR013256, PR013257, PR013258, PR013259, PR013261, PR013364, PR013377, PR013392, and PR013402 have excellent binding activity to human DLL3, monkey DLL3, and mouse DLL3, especially PR013364, PR013377, and PR013402, with maximum values and EC50 values comparable to those of the reference antibody PR012621.
  • 11 antibodies have binding activity to human DLL3 and monkey DLL3, but do not bind to mouse DLL3, such as PR013363, PR013367, PR013368, PR013370, PR013371, PR013376, PR013378, PR013382, PR013384, PR013387, and PR013399.
  • PR013372 and PR013394 only bind to human DLL3.
  • Example 3 FACS detection of the binding ability of anti-DLL3 HCAb monoclonal antibody at the cellular level
  • CHO-K1 cell line hamster ovary cell subline
  • human DLL3 CHO-K1/humanDLL3, also referred to as CHO-K1-humanDLL3, Nona Bio
  • CHO-K1 cell line overexpressing cynomolgus monkey DLL3 CHO-K1/cynoDLL3, also referred to as CHO-K1-cynoDLL3, Nona Bio
  • CHO-K1 mother cell line CCL-61) or the cell line SHP-77 (human small cell lung cancer cells) that highly expresses human DLL3 ( CRL-2195), DMS79 (human small cell lung cancer cells) CRL-2049) was used for antibody binding experiments at the cellular level.
  • CHO-K1/humanDLL3 cells, CHO-K1/cynoDLL3 cells, CHO-K1 cells, SHP-77 cells or DMS79 cells were digested and collected, and resuspended in PBS containing 2% FBS. The cell density was adjusted to 1 ⁇ 10 6 cells/mL. 100 ⁇ L cells/well were seeded in a 96-well V-bottom plate (Corning, #3894), followed by the addition of 100 ⁇ L/well of a 5-fold concentration gradient dilution of the antibody to be tested at 2 times the final concentration. The cells were placed at 4°C and incubated in the dark for 2 hours.
  • the PCR plate was sealed and placed in a real-time fluorescence quantitative PCR instrument (Bio-Rad CFX96 PCR System), first incubated at 25°C for 5 minutes, then gradually heated from 25°C to 95°C with a gradient of 0.2°C/0.2 minutes, and the temperature was lowered to 25°C at the end of the test.
  • the FRET scanning mode was used and Bio-Rad CFX Maestro software was used for data analysis and calculation of the sample Tm.
  • the extracellular domain of DLL3 consists of several distinct subdomains:
  • Human DLL3EGF2 aa 274-310
  • Detailed information about the recombinant plasmids overexpressing the DLL3 subdomain is shown in Table 14 and Figure 11A. These plasmids were transiently transfected into HEK239T cells. Cells were harvested 48 hours later, and the transfection efficiency of the recombinant cell lines was determined using a FITC-labeled anti-FLAG antibody. Untransfected HEK93T cells served as a negative control for transfection efficiency. Primary antibody staining was performed using 1 mg/mL DLL3 antibody. hIgG1 was used as an isotype negative reference antibody.
  • the binding of the anti-DLL3 antibody to the transiently transfected cells containing the subdomain was measured by flow cytometry using a fluorescent secondary antibody (Alexa Fluor 647-labeled affinity-purified goat anti-human IgG, specifically reactive with Fc ⁇ fragments, Jackson, #109-605-098, 1:1000 dilution) as a secondary antibody.
  • a fluorescent secondary antibody Alexa Fluor 647-labeled affinity-purified goat anti-human IgG, specifically reactive with Fc ⁇ fragments, Jackson, #109-605-098, 1:1000 dilution
  • FIG 11B shows that recombinant plasmids overexpressing DLL3 subdomains were transiently transfected into HEK293T cells.
  • Figures 12A-12J show that DLL3 antibodies bind to different regions of the extracellular end of human DLL3, and the results are shown in Table 15.
  • PR013234 anti-DLL3 N1003P003A12 HCAb hIgG1(C220S)
  • PR013235 anti-DLL3 N1003P003B12 HCAb hIgG1(C220S)
  • PR013236 anti-DLL3 N1003P003D01 HCAb hIgG1(C220S)
  • PR013240 anti-DLL3 N1003P005G03 HCAb hIgG1(C220S)
  • PR013242 anti-DLL3 N1003P008A11 HCAb hIgG1(C220S)
  • PR013249 anti-DLL3 N1003P013C05 HCAb hIgG1(C220S)
  • PR013253 anti-DLL3 N1003P018C04 HCAb hIgG1(C220S)
  • PR013260 anti-DLL3 N1003P024E01 HCAb hIgG1(C220S)
  • PR013261 anti-DLL3 N1003P024G01 HCAb hIgG1(C220S)
  • PR013263 anti-DLL3 N1003P028F03 HCAb hIgG1(C220S)
  • PR013363 anti-DLL3 N1003P041D05 HCAb hIgG1(C220S)
  • PR013364 anti-DLL3 N1003P041G02 HCAb hIgG1(C220S)
  • PR013368 anti-DLL3 N1003P044G07 HCAb hIgG1(C220S)
  • PR013370 anti-DLL3 N1003P046B02 HCAb hIgG1(C220S)
  • PR013372 anti-DLL3 N1003P046G02 HCAb hIgG1(C220S)
  • PR013376 anti-DLL3 N1003P047C08 HCAb hIgG1(C220S)
  • PR013377 anti-DLL3 N1003P047D04 HCAb hIgG1(C220S)
  • PR013378 anti-DLL3 N1003P047D06 HCAb hIgG1(C220S)
  • PR013381 anti-DLL3 N1003P048B11 HCAb hIgG1(C220S)
  • PR013382 anti-DLL3 N1003P049A11 HCAb hIgG1(C220S)
  • PR013384 anti-DLL3 N1003P051B09 HCAb hIgG1(C220S)
  • PR013387 anti-DLL3 N1003P052G11 HCAb hIgG1(C220S)
  • PR013393 anti-DLL3 N1003P054C07 HCAb hIgG1(C220S)
  • PR013397 anti-DLL3 N1003P054E04 HCAb hIgG1(C220S)
  • PR013406 anti-DLL3 N1003P058H08 HCAb hIgG1(C220S)

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Abstract

L'invention concerne un anticorps anti-DLL3 ou un fragment de liaison à l'antigène, une construction de liaison à l'antigène multispécifique, une cellule effectrice immunitaire, un conjugué d'anticorps ou une composition pharmaceutique de celui-ci, et une utilisation associée. L'invention concerne également un polynucléotide, un vecteur d'expression et un procédé de production de l'anticorps anti-DLL3 ou du fragment de liaison à l'antigène de celui-ci.
PCT/CN2025/085579 2024-03-29 2025-03-28 Molécule de liaison à dll3 et son utilisation Pending WO2025201493A1 (fr)

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Citations (7)

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Publication number Priority date Publication date Assignee Title
CN113166263A (zh) * 2018-10-11 2021-07-23 印希比股份有限公司 Dll3单域抗体及其治疗性组合物
CN113286817A (zh) * 2018-09-25 2021-08-20 哈普恩治疗公司 Dll3结合蛋白及使用方法
WO2021226204A2 (fr) * 2020-05-05 2021-11-11 Oncorus, Inc. Anticorps anti-dll3 et méthodes d'utilisation
CN114072427A (zh) * 2019-07-17 2022-02-18 南京传奇生物科技有限公司 抗dll3嵌合抗原受体及其用途
WO2022256500A2 (fr) * 2021-06-03 2022-12-08 Harpoon Therapeutics, Inc. Protéines trispécifiques ciblant dll3 et méthodes d'utilisation
WO2023246885A1 (fr) * 2022-06-23 2023-12-28 江苏恒瑞医药股份有限公司 Molécule de liaison à l'antigène se liant spécifiquement à dll3 et cd3, et son utilisation pharmaceutique
WO2024044550A1 (fr) * 2022-08-22 2024-02-29 Abdera Therapeutics Inc. Molécules de liaison à dll3 et leurs utilisations

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113286817A (zh) * 2018-09-25 2021-08-20 哈普恩治疗公司 Dll3结合蛋白及使用方法
CN113166263A (zh) * 2018-10-11 2021-07-23 印希比股份有限公司 Dll3单域抗体及其治疗性组合物
CN114072427A (zh) * 2019-07-17 2022-02-18 南京传奇生物科技有限公司 抗dll3嵌合抗原受体及其用途
WO2021226204A2 (fr) * 2020-05-05 2021-11-11 Oncorus, Inc. Anticorps anti-dll3 et méthodes d'utilisation
WO2022256500A2 (fr) * 2021-06-03 2022-12-08 Harpoon Therapeutics, Inc. Protéines trispécifiques ciblant dll3 et méthodes d'utilisation
WO2023246885A1 (fr) * 2022-06-23 2023-12-28 江苏恒瑞医药股份有限公司 Molécule de liaison à l'antigène se liant spécifiquement à dll3 et cd3, et son utilisation pharmaceutique
WO2024044550A1 (fr) * 2022-08-22 2024-02-29 Abdera Therapeutics Inc. Molécules de liaison à dll3 et leurs utilisations

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