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WO2025233825A1 - Enrichissement de cellules exprimant un lieur bird - Google Patents

Enrichissement de cellules exprimant un lieur bird

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Publication number
WO2025233825A1
WO2025233825A1 PCT/IB2025/054718 IB2025054718W WO2025233825A1 WO 2025233825 A1 WO2025233825 A1 WO 2025233825A1 IB 2025054718 W IB2025054718 W IB 2025054718W WO 2025233825 A1 WO2025233825 A1 WO 2025233825A1
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amino acid
seq
antigen
acid sequence
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Inventor
Michael J. Allegrezza
Jennifer Mccaffrey
Jun Chen
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Janssen Pharmaceutica NV
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Janssen Pharmaceutica NV
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Publication of WO2025233825A1 publication Critical patent/WO2025233825A1/fr
Pending legal-status Critical Current
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/17—Hinge-spacer domain
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the disclosure provided herein relates to methods of enriching cells expressing a chimeric antigen receptor (CAR) transgene comprising a BIRD linker peptide.
  • CAR chimeric antigen receptor
  • the disclosure provided herein also relates to methods of using antibodies that bind to the BIRD linker peptide.
  • iPSCs Human induced pluripotent stem cells
  • iPSC-derived cell therapies offer the potential of delivering allogeneic therapeutics that are clonally derived and well-characterized to cancer patients.
  • Allogeneic iPSC-derived cell therapy products can be genetically engineered to express a chimeric antigen receptor (CAR) to selectively target and kill cancer cells.
  • CAR chimeric antigen receptor
  • the CAR transgene can be engineered into the iPSCs using several techniques, including a CRISPR-based targeted insertion.
  • the CRISPR-edited cells are single-cell sorted, expanded, and characterized to identify clones with the desired genetic edits to be differentiated into the cell therapy product.
  • the CRISPR-edited knock-in cells can be a small percentage of the total population, so a critical challenge is the number of clones that need to be randomly screened to obtain CAR + iPSCs with appropriate genetic and phenotypic qualities.
  • One method to reduce the number of clones screened is to isolate the edited cells before single cell cloning.
  • Traditional methods for enrichment, such as antibiotic selection or fluorescent protein markers, are not viable options for iPSC-derived cell therapies because they involve the addition of selection reagents that can affect iPSC fitness and differentiation capacity and/or the expression of undesirable transgenes from a safety and regulatory perspective.
  • the method uses a monoclonal anti- BIRD linker antibody (e.g., BIRDB123) detection reagent to select for the CAR + cells.
  • the antibodies recognize the BIRD linker portion of the CAR and therefore may be used as universal detection reagents for any CAR containing that linker.
  • the CAR + cells can be enriched with a cell sorter prior to differentiation or single-cell sorting.
  • the present disclosure provides a method of enriching cells expressing a chimeric antigen receptor (CAR) transgene comprising a BIRD linker peptide, the method comprising contacting a plurality of cells with an isolated antibody or antigen-binding fragment thereof, that specifically binds to the BIRD linker peptide having the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • CAR chimeric antigen receptor
  • the enriched cells are CAR + human cells. In other embodiments, the enriched cells are CAR + non-human cells. In some embodiments, the enriched cells are CAR + iPSCs. In other embodiments, the enriched cells are CAR + primary T cells, CAR + CD34 + hematopoietic stem cells, or CAR + iPSC-derived T cells.
  • the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 90% identity thereto; and a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 90% identity thereto.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 90% identity thereto; and a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 95% identity thereto.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain (HC) comprising a heavy chain complementarity determining region 1 (HCCDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3), a light chain (LC) comprising a light chain complementarity determining region 1 (CDR1), a light chain complementarity determining region 2 (CDR2), and a light chain complementarity determining region 3 (CDR3), where
  • HC heavy chain
  • HCCDR1 heavy chain complementarity determining region 1
  • CDR2 heavy chain complementarity determining region 2
  • CDR3 heavy chain complementarity determining region 3
  • the HCDR1, HCDR2, and HCDR3 comprise amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and the LCDR1, LCDR2, and LCDR3 comprise amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively;
  • the HCDR1, HCDR2, and HCDR3 comprise amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively, and the LCDR1, LCDR2, and LCDR3 comprise amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively;
  • the HCDR1, HCDR2, and HCDR3 comprise amino acid sequences of SEQ ID NOs: 23, 24, and 25, respectively, and the LCDR1, LCDR2, and LCDR3 comprise amino acid sequences of SEQ ID NO: 26, 27, and 28, respectively;
  • the HCDR1, HCDR2, and HCDR3 comprise comprising amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively, and the LCDR1, LCDR2, and LCDR3 comprise amino acid sequences of SEQ ID NO: 32, 33, and 34, respectively; or
  • the HCDR1, HCDR2, and HCDR3 comprise amino acid sequences of SEQ ID NOs: 35, 36, and 37, respectively, and the LCDR1, LCDR2, and LCDR3 comprise amino acid sequences of SEQ ID NO: 38, 39, and 40, respectively.
  • the enriched cells in the methods disclosed herein are human stem cells. In other embodiments, the enriched cells are non-human stem cells. In some embodiments, the enriched cells are non-embryotic stem cells. In some embodiments, the enriched cells are iPSCs, primary T cells, CD34 + hematopoietic stem cells, or iPSC-derived T cells. In some embodiments, the enriched cells are human non-embryotic stem cells. In some embodiments, the enriched cells are human induced pluripotent stem cells (iPSCs). In some embodiments, the enriched cells are CAR + human cells. In other embodiments, the enriched cells are CAR + non-human cells.
  • iPSCs human induced pluripotent stem cells
  • the enriched cells are CAR + iPSCs. In other embodiments, the enriched cells are CAR + primary T cells, CAR + CD34 + hematopoietic stem cells, or CAR + iPSC-derived T cells.
  • the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by about 1% to about 99% after the enrichment. In some embodiments, the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 99% after the enrichment.
  • the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% after the enrichment.
  • the pluripotency marker is stage-specific embryonic antigen-3 (SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), SOX2, OCT3/4, or any combination thereof.
  • the non-iPSC surface marker is stage-specific embryonic antigen- 1 (SSEA-1), CD34, or both.
  • the methods disclosed herein further comprise forming a subpopulation of cells bound to the isolated antibody or antigen-binding fragment thereof.
  • the methods disclosed herein further comprise sorting the subpopulation of cells bound to the isolated antibody or antigen-binding fragment thereof, from the remainder of the cell sample, thereby generating the enriched cells.
  • the sorting is by flow cytometry.
  • the sorting is singlecell sorting.
  • the sorting is mechanical cartridge based sorting, microchip based sorting, or bead based sorting
  • the methods disclosed herein further comprise detecting the enriched cells bound to the isolated antibody or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment thereof comprises a detectable label.
  • the detectable label is a fluorescent tag.
  • the detecting comprises detection of the fluorescent tag.
  • the methods disclosed herein further comprise isolating the enriched cells.
  • the methods disclosed herein further comprise cloning and expanding the enriched cells.
  • the CAR binds to a cancer antigen.
  • the cancer antigen is a breast cancer antigen, a lung cancer antigen, a prostate cancer antigen, or a colorectal cancer antigen.
  • the CAR comprises a heavy chain variable region (VH) and a light chain variable region (VL) joined by a spacer region.
  • the spacer region comprises the BIRD linker peptide.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and a LCDR1, a LCDR2, and a LCDR3 comprise amino acid sequences of SEQ ID NO: 5, 6, and 7, respectively.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18 and/or a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain (HC) comprising an amino acid sequence of
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a light chain (LC) comprising an amino acid sequence of DIKMTQTPLTLSVSLGQPASFSCKSSQSLLDSDGETYLNWLFQGPGQSPKRLIYQVSK LDSRVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTFFPRTFGGGTKLEIERAD AAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQD SKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain (HC) comprising an amino acid sequence of SEQ ID NO: 14, and a light chain comprising an amino acid sequence of SEQ ID NO: 15.
  • HC heavy chain
  • SEQ ID NO: 14 amino acid sequence of SEQ ID NO: 14
  • a light chain comprising an amino acid sequence of SEQ ID NO: 15.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively, and a LCDR1, a LCDR2, and a LCDR3 comprising amino acid sequences of SEQ ID NO: 11, 12, and 13, respectively.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 20 and/or a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 21.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain (HC) comprising an amino acid sequence of
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain (HC) comprising an amino acid sequence of SEQ ID NO: 16, and a light chain comprising an amino acid sequence of SEQ ID NO: 17.
  • HC heavy chain
  • SEQ ID NO: 16 amino acid sequence of SEQ ID NO: 16
  • a light chain comprising an amino acid sequence of SEQ ID NO: 17.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises (a) a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences of SEQ ID NOs: 23, 24, and 25, respectively, and a LCDR1, a LCDR2, and a LCDR3 comprising amino acid sequences of SEQ ID NO: 26, 27, and 28, respectively; or (b) a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively, and a LCDR1, a LCDR2, and a LCDR3 comprising amino acid sequences of SEQ ID NO: 32, 33, and 34, respectively; or (c) a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences of SEQ ID NOs: 35, 36, and 37, respectively, and a LCDR1, a LCDR2, and a LCDR3 comprising amino acid sequences of SEQ ID NO: 38
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises (a) a VH comprising an amino acid sequence of SEQ ID NO: 41 and/or a VL comprising an amino acid sequence of SEQ ID NO: 42; or (b) a VH comprising an amino acid sequence of SEQ ID NO: 43 and/or a VL comprising an amino acid sequence of SEQ ID NO: 44; or (c) a VH comprising an amino acid sequence of SEQ ID NO: 45 and/or a VL comprising an amino acid sequence of SEQ ID NO: 46.
  • Fig. 1 shows bulk enrichment of CAR + iPSCs and generation of CAR + iPSC clones
  • a Flow cytometry plot of CAR expression of bulk edited population post Tyto enrichment. The flow plot is gated on live, single cells, and the CAR + cells were determined by comparing the edited sample to the unedited negative control
  • b Flow cytometry plots of CAR expression and pluripotency markers of a representative clone that was selected and single-cell sorted with BIRDB123 mAh AF488 conjugate. The flow plots are gated on live, single cells.
  • Figs. 2 shows selection of CAR + iPSCs for single-cell sorting and generation of CAR + iPSC clones
  • a Flow cytometry plot of CAR expression of bulk edited population 8 days post nucleofection. The flow plot is gated on live, single cells, and the CAR + cells were determined by comparing the edited sample to the unedited negative control
  • b Flow cytometry plots of CAR expression and pluripotency markers of a representative clone that was selected and single-cell sorted with BIRDB123 mAh AF488 conjugate. The flow plots are gated on live, single cells.
  • the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.”
  • isolated means a biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins.
  • Nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. “Isolated” nucleic acids, peptides and proteins can be part of a composition and still be isolated if such composition is not part of the native environment of the nucleic acid, peptide, or protein.
  • an “isolated” antibody or antigen-binding fragment is intended to refer to an antibody or antigen-binding fragment which is substantially free of other antibodies or antigen-binding fragments having different antigenic specificities (for instance, an isolated antibody that specifically binds to the linker peptide of SEQ ID NO: 1 is substantially free of antibodies that specifically bind antigens other than the linker peptide of SEQ ID NO: 1).
  • An isolated antibody may specifically binds to an epitope, isoform or variant of the linker peptide of SEQ ID NO: 1.
  • the term “specifically bind” or “bind specifically” or derivatives thereof when used in the context of antibodies, or antibody fragments, represents binding via domains encoded by immunoglobulin genes or fragments of immunoglobulin genes to one or more epitopes of a protein of interest, without preferentially binding other molecules in a sample containing a mixed population of molecules.
  • an antibody binds to a cognate antigen with a Kd of less than about IxlO -8 M, as measured by a surface plasmon resonance assay or a cell-binding assay.
  • Phrases such as “[antigen] -specific” antibody e.g., linkerspecific antibody
  • linkerspecific antibody are meant to convey that the recited antibody specifically binds the recited antigen.
  • antibody refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD, and IgY) including various monomeric, polymeric and chimeric forms, unless otherwise specified. Specifically encompassed by the term “antibody” are polyclonal antibodies, monoclonal antibodies (mAbs), and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies.
  • antigen-binding fragments are any proteinaceous structure that may exhibit binding affinity for a particular antigen.
  • Antigen-binding fragments include those provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. Some antigen-binding fragments are composed of portions of intact antibodies that retain antigen-binding specificity of the parent antibody molecule.
  • antigen-binding fragments may comprise at least one variable region (either a heavy chain or light chain variable region) or one or more CDRs of an antibody known to bind a particular antigen.
  • antigen-binding fragments include, without limitation diabodies and single-chain molecules as well as Fab, F(ab’)2, Fc, Fabc, and Fv molecules, single chain (Sc) antibodies (e.g., scFv), linear antibodies, single domain antibodies such as sdAb (either VE or VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains or CDRs and other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, a monovalent fragment consisting of the VL, VH, CL and CHI domains, or a monovalent antibody as described in W02007059782, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, a Fd fragment consisting essentially of the VH and CHI domains; a Fv fragment consisting essentially of the
  • antigenbinding fragments may include non-antibody proteinaceous frameworks that may successfully incorporate polypeptide segments in an orientation that confers affinity for a given antigen of interest, such as protein scaffolds.
  • Antigen-binding fragments may be recombinantly produced or produced by enzymatic or chemical cleavage of intact antibodies.
  • the phrase “an antibody or antigen-binding fragment thereof’ may be used to denote that a given antigen-binding fragment incorporates one or more amino acid segments of the antibody referred to in the phrase.
  • CDR complementarity determining region
  • LCDR1, LCDR2 and LCDR3 three make up the binding character of a light chain variable region
  • HCDR1, HCDR2 and HCDR3 three make up the binding character of a heavy chain variable region
  • CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions.
  • the exact definitional CDR boundaries and lengths are subject to different classification and numbering systems. CDRs may therefore be referred to by Kabat, Chothia, contact or any other boundary definitions.
  • CDR definitions according to these systems may therefore differ in length and boundary areas with respect to the adjacent framework region. See for example Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. NIH Publication No. 91-3242 (1991); Chothia et al., “Canonical Structures For the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196:901 (1987); and MacCallum et al., “Antibody- Antigen Interactions: Contact Analysis and Binding Site Topography,” J. Mol. Biol.
  • CDRs form a loop structure that can be classified as a canonical structure.
  • canonical structure refers to the main chain conformation that is adopted by the antigen binding (CDR) loops. From comparative structural studies, it has been found that five of the six antigen binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angles of the polypeptide backbone.
  • Correspondent loops between antibodies may, therefore, have very similar three dimensional structures, despite high amino acid sequence variability in most parts of the loops (Chothia et al., “Canonical Structures For the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196:901 (1987); Chothia et al., “Conformations of Immunoglobulin Hypervariable Regions,” I 342:877 (1989); Martin and Thornton, “Structural Families in Eoops of Homologous Proteins: Automatic Classification, Modelling and Application to Antibodies,” J. Mol. Biol. 263:800 (1996), each of which is incorporated by reference in its entirety).
  • the conformation of a particular canonical class is determined by the length of the loop and the amino acid residues residing at key positions within the loop, as well as within the conserved framework (i.e., outside of the loop). Assignment to a particular canonical class can therefore be made based on the presence of these key amino acid residues.
  • polypeptide is used interchangeably with the term “protein” and in its broadest sense refers to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics.
  • the subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and E optical isomers, amino acid analogs and peptidomimetics.
  • a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.
  • Polynucleotide synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and doublestranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.
  • the term “enriching” refers to a process whereby a cell of interest that is in a cell sample has an increased percentage in that cell cample after the enriching process as compared to before the enriching process.
  • the cell of interest is a cell that expresses a certain transgene, e.g., a chimeric antigen receptor (CAR) transgene.
  • CAR chimeric antigen receptor
  • the term “increased” refers to an increase by at least about 5% as compared to a reference level, for example an increase by at least about: 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to a reference level.
  • the percentage of the cells expressing a CAR transgene in a cell sample is increased by at least about: 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to the percentage of CAR + cells prior to the enriching process.
  • chimeric antigen receptor refers to a polypeptide comprising at least an extracellular domain that is bound by a monospecific or multispecific antibody or binds specifically to a target on a monospecific or multispecific antibody, a transmembrane domain and an intracellular T cell receptor-activating signaling domain.
  • the extracellular domain can comprise a binding domain against a linker polypeptide, a linker polypeptide alone, or a linker polypeptide fused to a recombinant polypeptide.
  • CARs redirect the specificity of immune effector cells and trigger proliferation, cytokine production, phagocytosis and/or production of molecules that can mediate cell death of the TAA-expressing cell in a major histocompatibility (MHC)- independent manner.
  • MHC major histocompatibility
  • a polypeptide linker can be used to genetically fuse different heavy chain variable (VH) and light chain variable (VL) regions to form single-chain variable fragment (scFv) molecules. These scFv molecules can subsequently be part of a bispecific antibody or a chimeric antigen receptor (CAR) on engineered immune cells and cell lines.
  • VH heavy chain variable
  • VL light chain variable
  • CAR chimeric antigen receptor
  • One example of the linker peptide is the BIRD linker peptide GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • transgene refers to a polynucleotide sequence (encoding, for example, a chimeric antigen receptor) that is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location that differs from that of the natural gene or its insertion results in a knockout).
  • the term “exogenous” is intended to mean that the referenced molecule or the referenced activity is introduced into the host cell.
  • the molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the cell.
  • the term “endogenous” refers to a referenced molecule or activity that is present in the host cell. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the cell and not exogenously.
  • the term “recombinant” polynucleotide is a polynucleotide that is not in its native state, e.g., the polynucleotide comprises a nucleotide sequence not found in nature, or the polynucleotide is in a context other than that in which it is naturally found, e.g., separated from nucleotide sequences with which it typically is in proximity in nature, or adjacent (or contiguous with) nucleotide sequences with which it typically is not in proximity.
  • the sequence at issue can be cloned into a vector, or otherwise recombined with one or more additional nucleic acid.
  • clone is a population of cells derived from a single cell or common ancestor by mitosis.
  • a “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations. In some examples provided herein, cells are transformed by transfecting the cells with DNA.
  • the term “express” refers to the biosynthesis of a gene product. These terms encompass the transcription of a gene into RNA. These terms also encompass translation of RNA into one or more polypeptides, and further encompass all naturally occurring post- transcriptional and post-translational modifications. The expression or production of an antibody or antigen-binding fragment thereof may be within the cytoplasm of the cell, or into the extracellular milieu such as the growth medium of a cell culture.
  • the term "host cell” can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line.
  • the term “host cell” refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, e.g., due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.
  • T lymphocyte and “T cell” are used interchangeably and refer to a principal type of white blood cell that completes maturation in the thymus and that has various roles in the immune system, including the identification of specific foreign antigens in the body and the activation and deactivation of other immune cells.
  • a T lymphocyte can be any T lymphocyte, such as a cultured T lymphocyte, e.g., a primary T lymphocyte, or a T lymphocyte from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T lymphocyte obtained from a mammal.
  • the T lymphocyte can be CD3+ cells.
  • the T lymphocyte can be any type of T lymphocyte and can be of any developmental stage, including but not limited to, CD4+/CD8+ double positive T cells, CD4+ helper T cells (e.g., Thl and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (ULs), memory T cells, naive T cells, regulator T cells, gamma delta T cells (y5 T cells), and the like.
  • CD4+/CD8+ double positive T cells CD4+ helper T cells (e.g., Thl and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (ULs), memory T cells, naive T cells, regulator T cells, gamma delta T cells (y5
  • a T lymphocyte can be T regulatory cell, which includes nTregs (natural Tregs), iTregs (inducible Tregs), CD8+ Treg, Tri regulatory cells, and Th3 cells. Additional types of helper T cells include cells such as Th3 (Treg), Thl 7, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (TCM cells), effector memory T cells (TEM cells and TEMRA cells).
  • T lymphocyte can also refer to a genetically engineered T lymphocyte, such as a T lymphocyte modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • the T lymphocyte can also be differentiated from a stem cell, definitive hemogenic endothelium, a CD34+ cell, an HSC (hematopoietic stem and progenitor cell), a hematopoietic multipotent progenitor cell, or a T cell progenitor cell.
  • a stem cell definitive hemogenic endothelium
  • a CD34+ cell an HSC (hematopoietic stem and progenitor cell)
  • hematopoietic multipotent progenitor cell a T cell progenitor cell.
  • iPSCs induced pluripotent stem cells
  • reprogramming refers to a process that alters or reverses the differentiation state of a somatic cell.
  • the cell can be either partially or terminally differentiated prior to reprogramming.
  • Reprogramming encompasses complete reversion of the differentiation state of a somatic cell (e.g., a T cell) to a pluripotent state.
  • Reprogramming also encompasses partial reversion of the differentiation state of a somatic cell to a state that renders the cell more susceptible to complete reprogramming to a pluripotent state when subjected to additional manipulations such as those described herein.
  • Such contacting may result in expression of particular genes by the cells, which expression contributes to reprogramming.
  • reprogramming of a somatic cell causes the somatic cell to be a pluripotent and ES-like state.
  • the resulting cells are referred to herein as reprogrammed pluripotent somatic cells or induced pluripotent stem cells (iPSCs).
  • reprogramming also encompasses partial reversion of the differentiation state of a somatic cell to a multipotent state.
  • Reprogramming is distinct from simply maintaining the existing undifferentiated state of a cell that is already pluripotent or maintaining the existing less than fully differentiated state of a cell that is already a multipotent cell (e.g., a hematopoietic stem cell). Reprogramming is also distinct from promoting the self-renewal or proliferation of cells that are already pluripotent or multipotent.
  • the methods described herein contribute to establishing the pluripotent state by reprogramming.
  • the methods described herein may be practiced on cells that fully differentiated and/or particular types of cells (e.g., y5 T cells), rather than on cells that are already multipotent or pluripotent.
  • pluripotent refers to the ability of a cell to form all lineages of the body or soma (i.e., the embryo proper).
  • embryonic stem cells and induced pluripotent stem cells are pluripotent stem cells that are able to form cells from each of the three germs layers, the ectoderm, the mesoderm, and the endoderm.
  • Pluripotency is a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell or EpiSC), which is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e.g., an embryonic stem cell).
  • the incompletely or partially pluripotent cell e.g., an epiblast stem cell or EpiSC
  • EpiSC epiblast stem cell
  • a complete organism e.g., an embryonic stem cell
  • pluripotency marker refers to a gene or protein whose expression under normal conditions (e.g., in the absence of genetic engineering or other manipulation designed to alter gene expression) occurs in and is typically restricted to pluripotent stem cells, and is crucial for their functional identity as such.
  • pluripotency markers used for identifying induced pluripotent stem cells include stage-specific embryonic antigens-3 and -4 (SSEA-3 and SSEA-4), SOX2, and OCT3/4.
  • non-iPSC surface marker refers to a surface moledule that is used to identify non-iPSC cells.
  • Non-limiting examples of non-iPSC surface marker include SSEA-1 and CD34.
  • cell culture medium (also referred to herein as a “culture medium” or “culture” or “medium”) is a medium for culturing cells containing nutrients that maintain cell viability and support proliferation.
  • the cell culture medium may contain any of the following in any appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc.
  • Cell culture media ordinarily used for particular cell types are known to those skilled in the art. Some non-limiting examples are provided herein.
  • cell line refers to a population of largely or substantially identical cells that has typically been derived from a single ancestor cell or from a defined and/or substantially identical population of ancestor cells.
  • the cell line may have been or may be capable of being maintained in culture for an extended period (e.g., months, years, for an unlimited period of time). It may have undergone a spontaneous or induced process of transformation conferring an unlimited culture lifespan on the cells.
  • Cell lines include all those cell lines recognized in the art as such. It will be appreciated that cells acquire mutations and possibly epigenetic changes over time such that at least some properties of individual cells of a cell line may differ with respect to each other.
  • differentiate refers to the process by which an unspecialized (or uncommitted) or less specialized cell acquires the features of a specialized cell such as, for example, a blood cell or a muscle cell.
  • a differentiated or differentiation-induced cell is one that has taken on a more specialized (or committed) position within the lineage of a cell.
  • a cell is committed when it has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type.
  • the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or a mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the noncoding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • the term “genetic modification” generally refers to a site of genomic DNA that has been genetically edited or manipulated using any molecular biological method, e.g., methods described herein or known in the art, e.g., by delivering to a site of genomic DNA an endonuclease and at least one gRNA.
  • Example of genetic modifications include insertions, deletions, duplications, inversions, and translocations, and combinations thereof.
  • a genetic modification is a deletion.
  • a genetic modification is an insertion.
  • a genetic modification is an insertiondeletion mutation (or indel), such that the reading frame of the target gene is shifted leading to an altered gene product or no gene product.
  • knock-in refers to a genetic modification that replaces the genetic information encoded at a chromosomal locus with a different DNA sequence.
  • an endonuclease generally refers to an enzyme that cleaves phosphodiester bonds within a polynucleotide.
  • an endonuclease specifically cleaves phosphodiester bonds within a DNA polynucleotide.
  • an endonuclease is a zinc finger nuclease (ZFN), transcription activator like effector nuclease (TALEN), homing endonuclease (HE), meganuclease, MegaTAL, or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated endonuclease.
  • ZFN zinc finger nuclease
  • TALEN transcription activator like effector nuclease
  • HE homing endonuclease
  • meganuclease MegaTAL
  • CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids.
  • CRISPR clusters are tran
  • vector refers to a replicon, such as plasmid, phage, cosmid, or virus in which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.
  • sample refers to any composition or mixture that can contain an analyte of interest.
  • the sample may be a collection of similar fluids, cells, or tissues (e.g., surgically resected tumor tissue, biopsies, including fine needle aspiration), isolated from a subject, as well as fluids, cells, or tissues present within a subject.
  • subject refers to human and non-human animals, including all vertebrates, e.g., mammals and nonmammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In many embodiments of the described methods, the subject is a human.
  • the present disclosure provides methods using isolated antibodies or antigen-binding fragments that specifically bind to the BIRD linker peptide comprising the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • the described isolated antibodies or antigen-binding fragments thereof specifically bind to a linker peptide consisting of the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • the general structure of an antibody molecule comprises an antigen binding domain, which includes heavy and light chains, and the Fc domain, which serves a variety of functions, including complement fixation and binding antibody receptors.
  • the isolated antibodies or antigen-binding fragments antibody may specifically binds to a fragment or a variant of the linker peptide of SEQ ID NO: 1.
  • the described linker-specific antibodies or antigen-binding fragments include all isotypes, IgA, IgD, IgE, IgG and IgM, and synthetic multimers of the four-chain immunoglobulin structure.
  • the described antibodies or antigen-binding fragments also include the IgY isotype generally found in hen or turkey serum and hen or turkey egg yolk.
  • the linker-specific antibodies and antigen-binding fragments may be derived from any species by recombinant means.
  • the antibodies or antigen-binding fragments may be mouse, rat, goat, horse, swine, bovine, chicken, rabbit, camelid, donkey, human, or chimeric versions thereof.
  • non-human derived antibodies or antigen-binding fragments may be genetically or structurally altered to be less antigenic upon administration to a human patient.
  • the antibodies or antigen-binding fragments are chimeric.
  • the term “chimeric” refers to an antibody, or antigen-binding fragment thereof, having at least some portion of at least one variable domain derived from the antibody amino acid sequence of a non-human mammal, a rodent, or a reptile, while the remaining portions of the antibody, or antigen-binding fragment thereof, are derived from a human.
  • the antibodies may include monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, polyclonal, antigen-binding fragments, bispecific or multispecific antibodies, monomeric, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity.
  • the antibody can be a naturally-occurring antibody, e.g., an antibody isolated and/or purified from a mammal, e.g., a murine, primate, mouse, rabbit, goat, horse, chicken, hamster, human, etc.
  • the antibody can be an engineered (e.g., genetically-engineered) antibody.
  • the antibodies are humanized antibodies.
  • Humanized antibodies may be chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementary-determining region
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence.
  • the humanized antibody may include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Humanized antibodies have antigen binding sites derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Human antibodies have heavy and light chain variable regions in which both the framework and the antigen binding site are derived from sequences of human origin.
  • the antibodies or antigen-binding fragments that can be used in the methods disclosed herein can occur in a variety of forms.
  • Non-limiting examples of the anti-BIRD linker antibodies and the antigen-binding fragments thereof include one or more of the antibody CDRs disclosed in Table 1 of WO 2022/177902, which is incorporated by reference herein in its entirety.
  • a linker-specific antibody, or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 1 of WO 2022/177902.
  • a linker-specific antibody, or an antigen-binding fragment thereof comprising a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 1 of WO 2022/177902 and a light chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 1 of WO 2022/177902.
  • a linker-specific antibody, or an antigen-binding fragment thereof comprising a heavy chain comprising a heavy chain variable domain of any one of the antibodies described in Table 2 of WO 2022/177902.
  • a linker-specific antibody, or an antigen-binding fragment thereof comprising a light chain comprising a light chain variable domain of any one of the antibodies described in Table 2 of WO 2022/177902.
  • a linker-specific antibody, or an antigen-binding fragment thereof comprising a heavy chain comprising a heavy chain variable domain of any one of the antibodies described in Table 2 of WO 2022/177902 and a light chain comprising a light chain variable domain of any one of the antibodies described in Table 2 of WO 2022/177902.
  • the heavy chain variable domain of a linker-specific antibody described herein, or an antigen-binding fragment thereof is encoded by a VH-encoding nucleotide sequence described in Table 2 of WO 2022/177902.
  • the light chain variable domain of a linker-specific antibody described herein, or an antigen-binding fragment thereof is encoded by a VL-encoding nucleotide sequence described in Table 2 of WO 2022/177902.
  • the isolated antibody, or antigen-binding fragment thereof comprises a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18,20, 41, 43, or 45, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 90% identity thereto.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 95% identity thereto. In some embodiments, the isolated antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45. In some embodiments, the isolated antibody, or antigenbinding fragment thereof, comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45. In some embodiments, the isolated antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody, or antigen-binding fragment thereof comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • VL light chain variable region
  • the isolated antibody, or antigenbinding fragment thereof comprises an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 90% identity thereto.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 95% identity thereto. In some embodiments, the isolated antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46. In some embodiments, the isolated antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46. In some embodiments, the isolated antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody, or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto; and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • VH heavy chain variable region
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 90% identity thereto.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 95% identity thereto.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45, and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45, and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46.
  • the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof.
  • the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody, or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45; and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or
  • the isolated antibody, or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto; and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody, or antigen- binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 18, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 19, or a sequence having at least 90% identity thereto.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 18, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 19, or a sequence having at least 95% identity thereto.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 18 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 19.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 18 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 19. In some embodiments, the isolated antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 18 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 19. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody, or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 20, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto; and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 21, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody, or antigenbinding fragment thereof comprises an amino acid sequence of SEQ ID NO: 20, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 21, or a sequence having at least 90% identity thereto.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 20, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 21, or a sequence having at least 95% identity thereto.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 20 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 21.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 20 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 21. In some embodiments, the isolated antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 20 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 21. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody, or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 41, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto; and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 42, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody, or antigenbinding fragment thereof comprises an amino acid sequence of SEQ ID NO: 41, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 42, or a sequence having at least 90% identity thereto.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 41, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 42, or a sequence having at least 95% identity thereto.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 41 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 42. In some embodiments, the isolated antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 41 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 42. In some embodiments, the isolated antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 41 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 42.
  • the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody, or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 43, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto; and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 44, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 43, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 44, or a sequence having at least 90% identity thereto.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 43, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 44, or a sequence having at least 95% identity thereto.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 43 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 44. In some embodiments, the isolated antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 43 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 44. In some embodiments, the isolated antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 43 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 44. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody, or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 45, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto; and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 46, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody, or antigenbinding fragment thereof comprises an amino acid sequence of SEQ ID NO: 45, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 46, or a sequence having at least 90% identity thereto.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 45, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 46, or a sequence having at least 95% identity thereto.
  • the isolated antibody, or antigen-binding fragment thereof comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 45 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 46. In some embodiments, the isolated antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 45 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 46. In some embodiments, the isolated antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 45 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 46. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody, or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18 and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody, or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 20 and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 21.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody, or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 41 and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 42.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody, or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 43 and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 44.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody, or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 45 and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 46.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 4; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10; or.
  • a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 31; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 37.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a heavy chain CDR1 of SEQ ID NO: 2, 8, 23, 29, or 35, and conservative modifications thereof, wherein the anti-BIRD linker antibody or antigen-binding fragment binds the linker peptide comprising the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a heavy chain CDR2 of SEQ ID NO: 3, 9, 24, 30, or 36, and conservative modifications thereof, wherein the anti-BIRD linker antibody or antigen-binding fragment binds the linker peptide comprising the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a heavy chain CDR3 of SEQ ID NO: 4, 10, 25, 31, or 37, and conservative modifications thereof, wherein the anti-BIRD linker antibody or antigen-binding fragment binds the linker peptide comprising the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7; or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11 , a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13; or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 28; or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a light chain CDR1 of SEQ ID NO: 5, 11, 26, 32, or 38, and conservative modifications thereof, wherein the anti-BIRD linker antibody or antigen-binding fragment binds the linker peptide comprising the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a light chain CDR2 of SEQ ID NO: 6, 12, 27, 33, or 39, and conservative modifications thereof, wherein the anti-BIRD linker antibody or antigen-binding fragment binds the linker peptide comprising the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a light chain CDR3 of SEQ ID NO: 7, 13, 28, 34, or 40, and conservative modifications thereof, wherein the anti-BIRD linker antibody or antigen-binding fragment binds the linker peptide comprising the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • the anti-BIRD linker antibody or antigen-binding fragment comprises the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2 and the light chain CDR3 comprising the amino acid sequence of a) SEQ ID NOs: 2, 3, 4, 8, 9, and 10, respectively; b) SEQ ID NOs: 5, 6, 7, 11, 12, and 13, respectively; c) SEQ ID NOs: 23-28, respectively; d) SEQ ID NOs: 29-34, respectively; or e) SEQ ID NOs: 35-40, respectively.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto; and/or a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-BIRD linker antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 90% identity thereto. In some embodiments, the anti-BIRD linker antibody or antigenbinding fragment comprises an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 95% identity thereto. In some embodiments, the anti-BIRD linker antibody or antigen-binding fragment comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45. In some embodiments, the anti-BIRD linker antibody or antigen-binding fragment comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 90% identity thereto. In some embodiments, the anti-BIRD linker antibody or antigenbinding fragment comprises an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 95% identity thereto. In some embodiments, the anti-BIRD linker antibody or antigen-binding fragment comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46. In some embodiments, the anti-BIRD linker antibody or antigen-binding fragment comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO:
  • amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 90% identity thereto. In some embodiments, the anti-BIRD linker antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 18,
  • the anti-BIRD linker antibody or antigen-binding fragment comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45, and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45, and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45, and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46.
  • the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof.
  • the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45,; and/or a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45,; and/or a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45,; and/or a light chain variable region (VL) comprising an amino acid sequence of SEQ ID
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 18, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 19, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the anti-BIRD linker antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 18, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 19, or a sequence having at least 90% identity thereto.
  • the anti-BIRD linker antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 18, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 19, or a sequence having at least 95% identity thereto.
  • the anti-BIRD linker antibody, or antigenbinding fragment thereof comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 18 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 19. In some embodiments, the anti-BIRD linker antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 18 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 19. In some embodiments, the anti-BIRD linker antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 18 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 19. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 20, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 21, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the anti-BIRD linker antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 20, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 21, or a sequence having at least 90% identity thereto.
  • the anti-BIRD linker antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 20, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 21, or a sequence having at least 95% identity thereto.
  • the anti-BIRD linker antibody, or antigenbinding fragment thereof comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 20 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 21. In some embodiments, the anti-BIRD linker antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 20 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 21. In some embodiments, the anti-BIRD linker antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 20 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 21. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 41, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 42, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the anti-BIRD linker antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 41, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 42, or a sequence having at least 90% identity thereto.
  • the anti-BIRD linker antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 41, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 42, or a sequence having at least 95% identity thereto.
  • the anti-BIRD linker antibody, or antigenbinding fragment thereof comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 41 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 42.
  • the anti-BIRD linker antibody, or antigen-binding fragment thereof comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 41 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 42.
  • the anti-BIRD linker antibody, or antigen-binding fragment thereof comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 41 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 42.
  • the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof.
  • the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 43, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 44, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the anti-BIRD linker antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 43, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 44, or a sequence having at least 90% identity thereto.
  • the anti-BIRD linker antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 43, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 44, or a sequence having at least 95% identity thereto.
  • the anti-BIRD linker antibody, or antigenbinding fragment thereof comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 43 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 44. In some embodiments, the anti-BIRD linker antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 43 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 44. In some embodiments, the anti-BIRD linker antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 43 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 44. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 45, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 46, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the anti-BIRD linker antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 45, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 46, or a sequence having at least 90% identity thereto.
  • the anti-BIRD linker antibody, or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 45, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 46, or a sequence having at least 95% identity thereto.
  • the anti-BIRD linker antibody, or antigenbinding fragment thereof comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 45 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 46.
  • the anti-BIRD linker antibody, or antigen-binding fragment thereof comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 45 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 46.
  • the anti-BIRD linker antibody, or antigen-binding fragment thereof comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 45 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 46.
  • the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof.
  • the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-BIRD linker antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 21.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 41 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 42.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 43 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 44.
  • the anti-BIRD linker antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 45 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 46.
  • the anti-BIRD linker antibody or antigen-binding fragment does not bind to the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22).
  • the anti-BIRD linker antibody or antigen-binding fragment does not have non-specific binding to Immunoglobulin G (IgG). In some embodiments, the anti-BIRD linker antibody or antigen-binding fragment does not bind to the constant region of Immunoglobulin G (IgG).
  • the BIRD linker-specific antibodies or antigen-binding fragments used in the methods described herein include variants having single or multiple amino acid substitutions, deletions, or additions that retain the biological properties (e.g., binding affinity or immune effector activity) of the described linker -specific antibodies or antigen-binding fragments.
  • the skilled person may produce variants having single or multiple amino acid substitutions, deletions, or additions.
  • variants may include: (a) variants in which one or more amino acid residues are substituted with conservative or nonconservative amino acids, (b) variants in which one or more amino acids are added to or deleted from the polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the polypeptide, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like.
  • Antibodies or antigen-binding fragments described herein may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or nonconserved positions. In other embodiments, amino acid residues at nonconserved positions are substituted with conservative or nonconservative residues.
  • the techniques for obtaining these variants, including genetic (deletions, mutations, etc.), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art.
  • Amino acid substitutions may be conservative, by which it is meant the substituted amino acid has similar chemical properties to the original amino acid.
  • a skilled person would understand which amino acids share similar chemical properties.
  • the following groups of amino acids share similar chemical properties such as size, charge and polarity: Group 1 - Ala, Ser, Thr, Pro, Gly; Group 2 - Asp, Asn, Glu, Gin; Group 3 - His, Arg, Lys; Group 4 - Met, Leu, He, Vai, Cys; Group 5 - Phe, Thy, Trp.
  • the BIRD linker-specific antibodies or antigen-binding fragments used in the methods described herein may embody several antibody isotypes, such as IgM, IgD, IgG, IgA and IgE.
  • the antibody isotype is IgGl, IgG2, IgG3, or IgG4 isotype, preferably IgGl or IgG4 isotype.
  • the antibody isotype is IgAl or IgA2.
  • Antibody or antigen-binding fragment thereof specificity is largely determined by the amino acid sequence, and arrangement, of the CDRs. Therefore, the CDRs of one isotype may be transferred to another isotype without altering antigen specificity.
  • antibody isotypes are within the scope of the described antibodies or antigen-binding fragments.
  • the antibody or antigen-binding fragment can have any level of affinity or avidity for the linker peptide comprising the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • the antibody or antigen-binding fragment may bind the linker peptide of SEQ ID NO: 1 with a range of affinities (KD).
  • the antibody or antigen-binding fragment binds to the linker peptide of SEQ ID NO: 1 with high affinity, for example, with a KD equal to or less than about 10 -7 M, such as but not limited to, 1-9.9 (or any range or value therein, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9)xlO -8 M, 10 -9 M, IO -10 M, IO -11 M, 10“ 12 M, 10“ 13 M, 10“ 14 M, 10“ 15 M or any range or value therein, as determined by surface plasmon resonance or the Kinexa method, as practiced by those of skill in the art.
  • a KD equal to or less than about 10 -7 M, such as but not limited to, 1-9.9 (or any range or value therein, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9)xlO -8 M, 10 -9 M, IO -10 M, IO -11 M, 10“ 12 M, 10“ 13 M, 10“ 14 M, 10“ 15 M or any
  • Methods of testing antibodies for the ability to bind to the target peptide or any portion thereof include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and competitive inhibition assays.
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • competitive inhibition assays such as, for example, radioimmunoassay (RIA), Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and competitive inhibition assays.
  • Suitable methods of making antibodies are known in the art. For instance, standard hybridoma methods are described in, e.g., Kohler and Milstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)). Alternatively, other methods, such as EBV-hybridoma methods (Haskard and Archer, J. Immunol.
  • Antibodies can be produced by transgenic mice that are transgenic for specific heavy and light chain immunoglobulin genes. Such methods are known in the art and described in, for example U.S. Patent Nos. 5,545,806 and 5,569,825, and Janeway et al., supra.
  • Humanized antibodies can also be generated using the antibody resurfacing technology described in U.S. Patent No. 5,639,641 and Pedersen et al., J. Mol. Biol., 235, 959-973 (1994).
  • Antibodies can be multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.
  • the antibody is a bispecific antibody.
  • the VL and/or the VH regions of existing antibodies or the VL and VH regions identified as described herein may be engineered into bispecific full-length antibodies.
  • Such bispecific antibodies may be made by modulating the CH3 interactions in antibody Fc to form bispecific antibodies using technologies such as those described in U.S. Pat. No. 7,695,936; Int. Pat. Publ. No.
  • bispecific antibodies of the present disclosure may be generated in vitro in a cell-free environment by introducing asymmetrical mutations in the CH3 regions of two monospecific homodimeric antibodies and forming the bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies in reducing conditions to allow disulfide bond isomerization according to methods described in Inti. Pat. Publ. No. WO2011/131746.
  • the first monospecific bivalent antibody and the second monospecific bivalent antibody are engineered to have certain substitutions at the CH3 domain that promote heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; thereby generating the bispecific antibody by Fab arm exchange.
  • the incubation conditions may optimally be restored to non-reducing.
  • Example reducing agents that may be used are 2- mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol, preferably a reducing agent selected from the group consisting of: 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine.
  • incubation for at least 90 min at a temperature of at least 20 °C in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH of from 5-8, for example at pH of 7.0 or at pH of 7.4 may be used.
  • the antigen-binding regions of the bispecific antibody can take any form that allows specific recognition of the target, for example the binding region may be or may include a heavy chain variable domain, an Fv (combination of a heavy chain variable domain and a light chain variable domain), a binding domain based on a fibronectin type III domain (such as from fibronectin, or based on a consensus of the type III domains from fibronectin, or from tenascin or based on a consensus of the type III domains from tenascin, such as the Centyrin molecules from Janssen Biotech, Inc., see e.g. W02010/051274 and W02010/093627). Accordingly, bispecific molecules comprising two different antigen-binding regions which bind the linker peptide and another antigen, are provided.
  • a fibronectin type III domain such as from fibronectin, or based on a consensus of the type III domains from fibronectin, or from tenascin
  • the bispecific antibody of the present disclosure is a diabody, a crossbody, or a bispecific antibody obtained via a controlled Fab arm exchange as those described in the present disclosure.
  • the bispecific antibodies include IgG-like molecules with complementary CH3 domains to force heterodimerization; recombinant IgG-like dual targeting molecules, wherein the two sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different antibodies; IgG fusion molecules, wherein full length IgG antibodies are fused to an extra Fab fragment or parts of Fab fragment; Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to heavy-chain constant-domains, Fc-regions or parts thereof; Fab fusion molecules, wherein different Fab-fragments are fused together; ScFv- and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) wherein different single chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g. domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.
  • IgG fusion molecules wherein full length IgG antibodies are fused to an extra
  • IgG-like molecules with complementary CH3 domains molecules include the Triomab/Quadroma (Trion Pharma/Fresenius Biotech), the Knobs-into-Holes (Genentech), CrossMAbs (Roche) and the electrostatically-matched (Amgen), the LUZ-Y (Genentech), the Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), the Biclonic (Merus) and the DuoBody (Genmab A/S).
  • recombinant IgG-like dual targeting molecules include Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-one Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star) and CovX-body (CovX/Pfizer).
  • DT Dual Targeting
  • Genentech Two-in-one Antibody
  • Cross-linked Mabs Karmanos Cancer Center
  • mAb2 F-Star
  • CovX-body CovX/Pfizer
  • IgG fusion molecules include Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecific (InnClone/Eli Lilly), Ts2Ab (Medlmmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen pout) and Tv Ab (Roche).
  • DVD Dual Variable Domain
  • IgG-like Bispecific InnClone/Eli Lilly
  • Ts2Ab Medlmmune/AZ
  • BsAb Zymogenetics
  • HERCULES Biogen personal
  • Tv Ab Roche
  • Fc fusion molecules include to ScFv/Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics) and Dual(ScFv).sub.2-Fab (National Research Center for Antibody Medicine-China).
  • Fab fusion bispecific antibodies include F(ab)2 (Medarex/ AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).
  • ScFv-, diabody-based and domain antibodies include but are not limited to Bispecific T Cell Engager (BiTE) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) and COMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), dual targeting heavy chain only domain antibodies.
  • BiTE Bispecific T Cell Engager
  • Tiandab Tandem Diabody
  • DART Dual Affinity Retargeting Technology
  • AIT TCR-like Antibodies
  • AIT ReceptorLogics
  • Human Serum Albumin ScFv Fusion Merrimack
  • COMBODY Epigen Biotech
  • dual targeting nanobodies Ablynx
  • dual targeting heavy chain only domain antibodies dual targeting heavy chain only domain antibodies.
  • Full length bispecific antibodies of the present disclosure may be generated for example using Fab arm exchange (or half molecule exchange) between two mono specific bivalent antibodies by introducing substitutions at the heavy chain CH3 interface in each half molecule to favor heterodimer formation of two antibody half molecules having distinct specificity either in vitro in cell-free environment or using co-expression.
  • the Fab arm exchange reaction is the result of a disulfide-bond isomerization reaction and dissociationassociation of CH3 domains. The heavy-chain disulfide bonds in the hinge regions of the parent mono specific antibodies are reduced.
  • the resulting free cysteines of one of the parent monospecific antibodies form an inter heavy-chain disulfide bond with cysteine residues of a second parent mono specific antibody molecule and simultaneously CH3 domains of the parent antibodies release and reform by dissociation-association.
  • the CH3 domains of the Fab arms may be engineered to favor heterodimerization over homodimerization.
  • the resulting product is a bispecific antibody having two Fab arms or half molecules which each bind a distinct epitope.
  • Homodimerization refers to an interaction of two heavy chains having identical CH3 amino acid sequences.
  • Homodimer refers to an antibody having two heavy chains with identical CH3 amino acid sequences.
  • Heterodimerization refers to an interaction of two heavy chains having nonidentical CH3 amino acid sequences.
  • Heterodimer refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.
  • the "knob-in-hole” strategy may be used to generate full length bispecific antibodies. Briefly, selected amino acids forming the interface of the CH3 domains in human IgG can be mutated at positions affecting CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into a heavy chain of an antibody specifically binding a first antigen and an amino acid with a large side chain (knob) is introduced into a heavy chain of an antibody specifically binding a second antigen.
  • a heterodimer is formed as a result of the preferential interaction of the heavy chain with a "hole” with the heavy chain with a "knob".
  • Exemplary CH3 substitution pairs forming a knob and a hole are (expressed as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): T366Y/F405A, T366W/ F405W, F405W/Y407A, T394W/Y407T, T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S_L368A_Y407V.
  • heterodimerization may be promoted by the following substitutions (expressed as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): L351Y_F405AY407V/T394W, T366I_K392M_T394W/F405A_Y407V, T366L_K392M_T394W/F405A_Y407V, L351 Y_Y407A/T366A_K409F, L351Y_Y407A/T366V K409F Y407A/T366A_K409F, or T350V_L351Y_F405A Y407V/T350V_T366L_K392L_T394W as described in U.S. Pat. Publ. No. US2012/0149876 or U.S. Pat. Publ. No. US2013/0195849.
  • the anti-BIRD linker antibody, or antigen binding fragments thereof can be modified to comprise a detectable label, such as, for instance, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and element particles (e.g., gold particles).
  • a detectable label such as, for instance, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and element particles (e.g., gold particles).
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • an enzyme e.g., alkaline phosphat
  • Exemplary detectable labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorochromes, fluorophores, fluorescent quenching agents, colored molecules, radioactive isotopes, scintillates, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni 2+ , Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donors/acceptors, acridinium esters, and colorimetric substrates.
  • enzymes for example, as commonly used in an ELISA
  • biotin digoxigenin
  • haptens luminescent molecules
  • chemiluminescent molecules chemiluminescent molecules
  • a detectable label may emit a signal spontaneously, such as when the detectable label is a radioactive isotope. In other cases, the detectable label emits a signal as a result of being stimulated by an external field.
  • Suitable dyes include any commercially available dyes such as, for example, 5(6)-carboxyfluorescein, IRDye 680RD maleimide or IRDye 800CW, ruthenium polypyridyl dyes, and the like.
  • Suitable fluorophores are fluorescein isothiocyanate (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDyes (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594; Alexa647), near infrared (NIR) (700-900 nm) fluorescent dyes, and carbocyanine and aminostyryl dyes.
  • FITC fluorescein isothiocyanate
  • fluorescein thiosemicarbazide e.g., Texas Red
  • CyDyes e.g., Cy3, Cy5, Cy5.5
  • Alexa Fluors e.g., Alexa488, Alexa555, Alexa594; Alexa647
  • NIR near infrared
  • linker-specific antibodies or antigen-binding fragments thereof for use in biological detection and measurement.
  • these antibodies or antigen-binding fragments may be useful in detecting a polypeptide comprising a linker peptide comprising the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1) in a sample.
  • the present disclosure provides a method of detecting a polypeptide comprising a linker peptide comprising the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1) in a sample, which may include (i) contacting the sample with an isolated antibody or antigen-binding fragment described herein, and (ii) detecting the binding of the polypeptide to said isolated antibody or antigen-binding fragment.
  • the antibodies for use in these methods include those described herein above, for example a linker-specific antibody or antigen-binding fragment with the features set out in Tables 1 or 2, for example the CDRs or variable domain sequences, and in the further discussion of these antibodies.
  • the sample may be a cell sample comprising cells expressing a polypeptide comprising the BIRD linker peptide GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • the polypeptide is an antibody, antigen-binding fragment, or a chimeric antigen receptor (CAR).
  • the antibody or antigen-binding fragment may be, for example, a human antibody, a monoclonal antibody, a single chain antibody, a Fab, a Fab’, a F(ab’)2, a Fv, or a scFv.
  • the polypeptide to be analyzed is an scFv.
  • the polypeptide to be analyzed is a multi-specific antibody or antigen-binding fragment.
  • the polypeptide to be analyzed is a bispecific antibody or antigen-binding fragment.
  • the polypeptide to be analyzed is a chimeric antigen receptor (CAR).
  • the CAR may be presented on an immune cell, such as a T cell or a NK cell.
  • the sample may be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, cells that are not tissue associated (z.e., free cells), tissues (e.g., surgically resected tumor tissue, biopsies, including fine needle aspiration), histological preparations, and the like.
  • the sample may be contacted with more than one of the linker-specific antibodies or antigen-binding fragments described herein.
  • a sample may be contacted with a first linker-specific antibody, or antigen-binding fragment thereof, and then contacted with a second linker-specific antibody, or antigen-binding fragment thereof, wherein the first antibody or antigen-binding fragment and the second antibody or antigenbinding fragment are not the same antibody or antigen-binding fragment.
  • the first antibody, or antigen-binding fragment thereof may be affixed to a surface, such as a multiwell plate, chip, or similar substrate prior to contacting the sample. In other embodiments the first antibody, or antigen-binding fragment thereof, may not be affixed, or attached, to anything at all prior to contacting the sample.
  • affinity matrices comprising the isolated antibody or antigen-binding fragment described herein are also provided.
  • the described linker-specific antibodies and antigen-binding fragments may be immobilized by attachment to a suitable solid support.
  • solid supports include, but are not limited to, a bead, a membrane, sepharose, a magnetic bead, a plate, a tube, a column.
  • the described linker-specific antibodies and antigen-binding fragments may be attached to an ELISA plate, a magnetic bead, or a surface plasmon resonance biosensor chip.
  • Methods of attaching antibodies and antigen-binding fragments to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g. biotin and streptavidin, or antibodies and antigens, or using covalent linkages.
  • linker-specific antibodies and antigen-binding fragments may be detectably labeled.
  • labeled antibodies and antigen-binding fragments may facilitate the detection of the linker peptide of interest (e.g., SEQ ID NO: 1) via the methods described herein.
  • linker peptide of interest e.g., SEQ ID NO: 1
  • suitable labels include, but should not be considered limited to, radiolabels, fluorescent labels, epitope tags, biotin, chromophore labels, ECL labels, or enzymes.
  • the described labels include ruthenium, U1 ln-DOTA, lu In- diethylenetriaminepentaacetic acid (DTP A), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, poly-histidine (HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazin dyes, phenanthridine dyes, rhodamine dyes, Alexafluor® dyes, and the like.
  • DTP A lu In- diethylenetriaminepentaacetic acid
  • HIS tag poly-histidine
  • acridine dyes cyanine dyes
  • fluorone dyes oxazin dyes
  • phenanthridine dyes phenanthridine dyes
  • rhodamine dyes Alexafluor® dyes, and the like.
  • a detectable label may emit a signal spontaneously, such as when the detectable label is a radioactive isotope. In other cases, the detectable label emits a signal as a result of being stimulated by an external field.
  • Suitable dyes include any commercially available dyes such as, for example, 5(6)-carboxyfluorescein, IRDye 680RD maleimide or IRDye 800CW, ruthenium polypyridyl dyes, and the like.
  • Suitable fluorophores are fluorescein isothiocyanate (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDyes (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594; Alexa647), near infrared (NIR) (700-900 nm) fluorescent dyes, and carbocyanine and aminostyryl dyes.
  • FITC fluorescein isothiocyanate
  • fluorescein thiosemicarbazide e.g., Texas Red
  • CyDyes e.g., Cy3, Cy5, Cy5.5
  • Alexa Fluors e.g., Alexa488, Alexa555, Alexa594; Alexa647
  • NIR near infrared
  • linker-specific antibodies and antigen-binding fragments may be used in a variety of assays to detect the linker peptide of interest (e.g., SEQ ID NO: 1) in a sample.
  • suitable assays include, but should not be considered limited to, western blot analysis, radioimmunoassay, surface plasmon resonance, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS) or ELISA assay.
  • the present disclosure provides a method of enriching cells expressing a chimeric antigen receptor (CAR) transgene comprising a BIRD linker peptide, the method comprising contacting a plurality of cells with an isolated antibody or antigen-binding fragment thereof, that specifically binds to the BIRD linker peptide having the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • CAR chimeric antigen receptor
  • the plurality of cells in the methods disclosed herein are derived from a mammal.
  • the mammal is a human.
  • mammals include non-human primates, murines (i.e., mice and rats), canines, felines, equines, bovines, ovines, porcines, and caprines.
  • the plurality of cells of the present disclosure include any T cell of the body that is not a stem cell, a germ cell, or an iPSC.
  • a non-iPSC is a T cell derived from any tissue of the body, including internal organs, skin, bones, blood, nervous tissue, and connective tissue.
  • the isolated population of cells are blood cells.
  • the blood cells are suitably peripheral blood mononuclear cells (PMBCs), and may include all types of blood cells existing on an entire differentiation process from hematopoietic stem cells to final differentiation into peripheral blood.
  • PMBCs peripheral blood mononuclear cells
  • the blood cells include, for example, hematopoietic stem cells, lymphoid stem cells, lymphoid dendritic cell progenitor cells, lymphoid dendritic cells, T lymphocyte progenitor cells, T cells, B lymphocyte progenitor cells, B cells, plasma cells, NK progenitor cells, NK cells, monocytes, and macrophages.
  • the plurality of cells can be peripheral blood mononuclear cells (PBMC), peripheral blood leukocytes (PBL), tumor infiltrating lymphocytes (TIL), or a combination thereof.
  • the isolated population of cells are peripheral blood mononuclear (PBMC) cells.
  • the plurality of cells are T cells.
  • the T cells can be selected from the group consisting of CD4+/CD8+ double positive T cells, cytotoxic T cells, Th3 (Treg) cells, Th9 cells, ThaP helper cells, Tfh cells, stem memory TSCM cells, central memory TCM cells, effector memory TEM cells, effector memory TEMRA cells, gamma delta T cells and any combination thereof.
  • the cells used in the methods disclosed herein are human stem cells.
  • the cells are non-human stem cells.
  • the cells are human non-embryotic stem cells.
  • the enriched cells are iPSCs, primary T cells, CD34 + hematopoietic stem cells, or iPSC-derived T cells.
  • the cells are human iPSCs. Methods for the culture and differentiation of human embryonic and non-embryonic stem cells, including human induced pluripotent stem cells (hiPSC), are known in the art. Methods for producing iPSC-derived T cells are also known in the art.
  • the enriched cells are CAR + human cells. In other embodiments, the enriched cells are CAR + non-human cells. In some embodiments, the enriched cells are CAR + iPSCs. In other embodiments, the enriched cells are CAR + primary T cells, CAR + CD34 + hematopoietic stem cells, or CAR + iPSC-derived T cells.
  • the cells used in the methods disclosed herein are transfected with a vector to express a chimeric antigen receptor (CAR) transgene.
  • CAR chimeric antigen receptor
  • Methods for stably or transiently transfecting a cell line or a primary cell culture are known in the art.
  • the cells are transfected with a CRISPR ribonucleoprotein (RNP) complex and a DNA expression cassette containing the CAR transgene.
  • the CAR binds to a cancer antigen.
  • the cancer antigen is a breast cancer antigen, a lung cancer antigen, a prostate cancer antigen, or a colorectal cancer antigen.
  • the CAR comprises a heavy chain variable region (VH) and a light chain variable region (VL) joined by a spacer region.
  • the spacer region comprises the BIRD linker peptide (SEQ ID NO: 1).
  • the antibody, or antigen binding fragments thereof can be modified to comprise a detectable label, such as, for instance, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and element particles (e.g., gold particles).
  • a detectable label such as, for instance, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and element particles (e.g., gold particles).
  • a detectable label such as, for instance, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FIT
  • Nonlimiting examples of detectable labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorochromes, fluorophores, fluorescent quenching agents, colored molecules, radioactive isotopes, scintillates, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni 2+ , Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donors/acceptors, acridinium esters, and colorimetric substrates.
  • the isolated antibody or antigen-binding fragment thereof comprises a fluorescent tag.
  • Suitable fluorophores are fluorescein isothiocyanate (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDyes (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594; Alexa647), near infrared (NIR) (700-900 nm) fluorescent dyes, and carbocyanine and aminostyryl dyes.
  • FITC fluorescein isothiocyanate
  • fluorescein thiosemicarbazide e.g., Texas Red
  • CyDyes e.g., Cy3, Cy5, Cy5.5
  • Alexa Fluors e.g., Alexa488, Alexa555, Alexa594; Alexa647
  • NIR near infrared
  • the methods disclosed herein further comprise sorting the subpopulation of cells bound to the isolated antibody or antigen-binding fragment thereof, from the remainder of the cell sample, thereby generating the enriched cells.
  • the sorting is by flow cytometry.
  • the sorting is singlecell sorting.
  • the sorting is mechanical cartridge based sorting, microchip based sorting, or bead based sorting
  • the methods disclosed herein further comprise detecting the enriched cells bound to the isolated antibody or antigen-binding fragment thereof. In some embodiments, the detecting comprises detection of the fluorescent tag.
  • the methods disclosed herein further comprise isolating the enriched cells. In some embodiments, the methods disclosed herein further comprise cloning and expanding the enriched cells.
  • less than 1% of the total cells in the plurality of cells express the CAR transgene prior to enrichment. In some embodiments, less than 5% of the total cells in the plurality of cells express the CAR transgene prior to enrichment. In some embodiments, less than 10% of the total cells in the plurality of cells express the CAR transgene prior to enrichment. In some embodiments, less than 20% of the total cells in the plurality of cells express the CAR transgene prior to enrichment. In some embodiments, less than 30% of the total cells in the plurality of cells express the CAR transgene prior to enrichment. In some embodiments, less than 40% of the total cells in the plurality of cells express the CAR transgene prior to enrichment.
  • less than 50% of the total cells in the plurality of cells express the CAR transgene prior to enrichment. In some embodiments, less than 60% of the total cells in the plurality of cells express the CAR transgene prior to enrichment. In some embodiments, less than 70% of the total cells in the plurality of cells express the CAR transgene prior to enrichment. In some embodiments, less than 80% of the total cells in the plurality of cells express the CAR transgene prior to enrichment. In some embodiments, less than 90% of the total cells in the plurality of cells express the CAR transgene prior to enrichment. In some embodiments, less than 95% of the total cells in the plurality of cells express the CAR transgene prior to enrichment.
  • 100% of the cells in the enriched cells express the CAR transgene.
  • greater than 5% of the cells in the enriched cells express the CAR transgene.
  • greater than 10% of the cells in the enriched cells express the CAR transgene.
  • greater than 20% of the cells in the enriched cells express the CAR transgene.
  • greater than 30% of the cells in the enriched cells express the CAR transgene.
  • greater than 40% of the cells in the enriched cells express the CAR transgene.
  • greater than 50% of the cells in the enriched cells express the CAR transgene.
  • greater than 60% of the cells in the enriched cells express the CAR transgene. In some emodiments, greater than 70% of the cells in the enriched cells express the CAR transgene. In some emodiments, greater than 80% of the cells in the enriched cells express the CAR transgene. In some emodiments, greater than 90% of the cells in the enriched cells express the CAR transgene. In some emodiments, greater than 95% of the cells in the enriched cells express the CAR transgene. In some emodiments, greater than 99% of the cells in the enriched cells express the CAR transgene.
  • the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by about 1% to about 99% after the enrichment. In some embodiments, the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 99% after the enrichment.
  • the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% after the enrichment. In some embodiments, the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by at least 1%.
  • the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by at least 5%. In some embodiments, the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by at least 10%. In some embodiments, the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by at least 20%. In some embodiments, the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by at least 30%. In some embodiments, the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by at least 40%.
  • the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by at least 50%. In some embodiments, the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by at least 60%. In some embodiments, the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by at least 70%. In some embodiments, the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by at least 80%. In some embodiments, the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by at least 90%.
  • the percentage of the total cells in the plurality of cells expressing the CAR transgene is increased by at least 95%.
  • the enriched iPSCs are measured for the expression level of one or more pluripotency markers and/or one or more non-iPSC surface marker.
  • greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, or greater than 95% of the iPSCs in the enriched cells express a pluripotency marker.
  • greater than 80% of the iPSCs in the enriched cells express a pluripotency marker.
  • greater than 85% of the iPSCs in the enriched cells express a pluripotency marker.
  • the pluripotency marker is stage-specific embryonic antigen-3 (SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), OCT3/4, SOX2, or any combination thereof.
  • the pluripotency marker is SSEA-3, and greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, or greater than 95% of the iPSCs in the enriched cells express SSEA-3.
  • the pluripotency marker is SSEA-4, and greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, or greater than 95% of the iPSCs in the enriched cells express SSEA-4.
  • the pluripotency marker is SOX2, greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, or greater than 95% of the iPSCs in the enriched cells express SOX2.
  • the pluripotency marker is OCT3/4, and greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, or greater than 95% of the iPSCs in the enriched cells express OCT3/4.
  • less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 1.5%, less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than 4%, less than 4.5%, or less than 5% of the iPSCs in the enriched cells express a non-iPSC surface marker. In some embodiments, less than 0.1% of the iPSCs in the enriched cells express a non-iPSC surface marker. In some embodiments, less than 0.5% of the iPSCs in the enriched cells express a non-iPSC surface marker.
  • the iPSCs in the enriched cells express a non-iPSC surface marker. In some embodiments, less than 5% of the iPSCs in the enriched cells express a non-iPSC surface marker. In some embodiments, the non-iPSC surface marker is stage-specific embryonic antigen-1 (SSEA-1), CD34, or both.
  • SSEA-1 stage-specific embryonic antigen-1
  • the non-iPSC surface marker is SSEA-1, and less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 1.5%, less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than 4%, less than 4.5%, or less than 5% of the iPSCs in the enriched cells express SSEA-1.
  • the surface marker is CD34, and less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 1.5%, less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than 4%, less than 4.5%, or less than 5% of the iPSCs in the enriched cells express CD34.
  • the methods disclosed herein further comprise forming a subpopulation of cells bound to the isolated antibody or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain (HC) comprising a heavy chain complementarity determining region 1 (HCCDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3), a light chain (LC) comprising a light chain complementarity determining region 1 (CDR1), a light chain complementarity determining region 2 (CDR2), and a light chain complementarity determining region 3 (CDR3), where the HCDR1, HCDR2, and HCDR3 comprise amino acid sequences of GYTFTRYW (SEQ ID NO: 2), IHPSDSDT (SEQ ID NO: 3), and LTMPVEGDY (SEQ ID NO: 4), respectively, and the LCDR1, LCDR2, and LCDR3 comprise amino acid sequences of QSLLDSDGETY (SEQ ID NO: 5), QVS (SEQ ID NO: 6), and WQGTFFPRT
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain (HC) comprising a heavy chain complementarity determining region 1 (HCCDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3), a light chain (LC) comprising a light chain complementarity determining region 1 (CDR1), a light chain complementarity determining region 2 (CDR2), and a light chain complementarity determining region 3 (CDR3), where the HCDR1, HCDR2, and HCDR3 comprise amino acid sequences of GYPFTRYW (SEQ ID NO: 8), IHPSDSDT (SEQ ID NO: 9), and LTMPVEGDY (SEQ ID NO: 10), respectively, and the LCDR1, LCDR2, and LCDR3 comprise amino acid sequences of QSLLDSDGETY (SEQ ID NO: 11), QVS (SEQ ID NO: 12), and WQGTFFPRT
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and a LCDR1, a LCDR2, and a LCDR3 comprise amino acid sequences of SEQ ID NO: 5, 6, and 7, respectively.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18 and/or a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain (HC) comprising an amino acid sequence of
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a light chain (LC) comprising an amino acid sequence of DIKMTQTPLTLSVSLGQPASFSCKSSQSLLDSDGETYLNWLFQGPGQSPKRLIYQVSK LDSRVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTFFPRTFGGGTKLEIERAD AAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQD SKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 15).
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain (HC) comprising an amino acid sequence of SEQ ID NO: 14, and a light chain comprising an amino acid sequence of SEQ ID NO: 15.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively, and a LCDR1, a LCDR2, and a LCDR3 comprising amino acid sequences of SEQ ID NO: 11, 12, and 13, respectively.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 20 and/or a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 21.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain (HC) comprising an amino acid sequence of EVQLQESGAELVKPGASVKVSCKASGYPFTRYWMHWVKHRPGQGLEWIGRIHPSDS DTNYNQNFKGKATLTVDRSSNTAYMQLSSLTSEDSAVYYCLTMPVEGDYWGQGTT LTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTF PAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKC PAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTA QTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVR APQVYVLPPPEEEMTKKQ
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a light chain (LC) comprising an amino acid sequence of DIKMTQSPLTLSVTLGQPASFSCKSSQSLLDSDGETYLNWLFQGPGQSPKRLIYQVSK LDSRVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTFFPRTFGGGTKLEIKRAD
  • LC light chain
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain (HC) comprising an amino acid sequence of SEQ ID NO: 16, and a light chain comprising an amino acid sequence of SEQ ID NO: 17.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences of SEQ ID NOs: 23-25, respectively, and a LCDR1, a LCDR2, and a LCDR3 comprise amino acid sequences of SEQ ID NO: 26-28, respectively.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 41 and/or a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 42.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences of SEQ ID NOs: 29-31, respectively, and a LCDR1, a LCDR2, and a LCDR3 comprise amino acid sequences of SEQ ID NO: 32-34, respectively.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 43 and/or a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 44.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences of SEQ ID NOs: 35-37, respectively, and a LCDR1, a LCDR2, and a LCDR3 comprise amino acid sequences of SEQ ID NO: 38-40, respectively.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 45 and/or a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 46.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • VH heavy chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 90% identity thereto.
  • the isolated antibody or antigenbinding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 95% identity thereto. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45.
  • the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof.
  • the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 90% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 95% identity thereto. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46.
  • the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof.
  • the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45,, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto; and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • VH heavy chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 90% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 95% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45, and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45, and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45, and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46.
  • the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof.
  • the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45,; and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto; and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 18, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 19, or a sequence having at least 90% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 18, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 19, or a sequence having at least 95% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 18 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 19. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 18 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 19. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 18 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 19. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 20, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto; and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 21, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 20, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 21, or a sequence having at least 90% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 20, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 21, or a sequence having at least 95% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 20 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 21. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 20 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 21. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 20 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 21. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 41, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto; and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 42, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 41, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 42, or a sequence having at least 90% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 41, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 42, or a sequence having at least 95% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 41 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 42. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 41 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 42. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 41 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 42. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 43, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto; and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 44, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 43, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 44, or a sequence having at least 90% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 43, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 44, or a sequence having at least 95% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 43 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 44. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 43 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 44. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 43 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 44.
  • the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 45, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto; and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 46, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 45, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 46, or a sequence having at least 90% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 45, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 46, or a sequence having at least 95% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 45 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 46. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 45 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 46. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 45 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 46. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18 and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 20 and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 21.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody, or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 41 and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 42.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody, or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 43 and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 44.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody, or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 45 and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 46.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 4; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10; or.
  • a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 31; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 37.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain CDR1 of SEQ ID NO: 2, 8, 23, 29, or 35, and conservative modifications thereof, wherein the anti-BIRD linker antibody or antigenbinding fragment binds the linker peptide comprising the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain CDR2 of SEQ ID NO: 3, 9, 24, 30, or 36, and conservative modifications thereof, wherein the anti-BIRD linker antibody or antigenbinding fragment binds the linker peptide comprising the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain CDR3 of SEQ ID NO: 4, 10, 25, 31, or 37, and conservative modifications thereof, wherein the anti-BIRD linker antibody or antigenbinding fragment binds the linker peptide comprising the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7; or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11 , a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13; or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 28; or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a light chain CDR1 of SEQ ID NO: 5, 11, 26, 32, or 38, and conservative modifications thereof, wherein the anti-BIRD linker antibody or antigenbinding fragment binds the linker peptide comprising the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a light chain CDR2 of SEQ ID NO: 6, 12, 27, 33, or 39, and conservative modifications thereof, wherein the anti-BIRD linker antibody or antigenbinding fragment binds the linker peptide comprising the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a light chain CDR3 of SEQ ID NO: 7, 13, 28, 34, or 40, and conservative modifications thereof, wherein the anti-BIRD linker antibody or antigenbinding fragment binds the linker peptide comprising the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 1).
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2 and the light chain CDR3 comprising the amino acid sequence of a) SEQ ID NOs: 2, 3, 4, 8, 9, and 10, respectively; b) SEQ ID NOs: 5, 6, 7, 11, 12, and 13, respectively; c) SEQ ID NOs: 23-28, respectively; d) SEQ ID NOs: 29-34, respectively; or e) SEQ ID NOs: 35-40, respectively.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto; and/or a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 90% identity thereto. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 95% identity thereto. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 90% identity thereto. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 95% identity thereto. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 90% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46, or a sequence having at least 95% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45, and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45, and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 18, 20, 41, 43, or 45, and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 19, 21, 42, 44, or 46.
  • the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof.
  • the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 18, 20, 41, 43, or 45; and/or a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19, 21, 42, 44, or 46.
  • VH heavy chain variable region
  • VL light chain variable region
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 18, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 19, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 18, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 19, or a sequence having at least 90% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 18, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 19, or a sequence having at least 95% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 18 and an amino acid sequence
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 18 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 19.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 18 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 19.
  • the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof.
  • the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 20, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 21, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 20, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 21, or a sequence having at least 90% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 20, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 21, or a sequence having at least 95% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 20 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 21. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 20 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 21. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 20 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 21. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 41, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 42, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 41, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 42, or a sequence having at least 90% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 41, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 42, or a sequence having at least 95% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 41 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 42. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 41 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 42. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 41 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 42.
  • the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 43, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 44, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 43, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 44, or a sequence having at least 90% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 43, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 44, or a sequence having at least 95% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 43 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 44. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 43 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 44. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 43 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 44. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 45, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 46, or a sequence having at least 80%, 85%, 90%, preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 45, or a sequence having at least 90% identity thereto; and an amino acid sequence of SEQ ID NO: 46, or a sequence having at least 90% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence of SEQ ID NO: 45, or a sequence having at least 95% identity thereto; and an amino acid sequence of SEQ ID NO: 46, or a sequence having at least 95% identity thereto.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having one amino acid difference compared to SEQ ID NO: 45 and an amino acid sequence having one amino acid difference compared to SEQ ID NO: 46. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having two amino acid difference compared to SEQ ID NO: 45 and an amino acid sequence having two amino acid difference compared to SEQ ID NO: 46. In some embodiments, the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 45 and an amino acid sequence having at least three amino acid difference compared to SEQ ID NO: 46. In some embodiments, the amino acid difference is limited to the framework regions of the antibody, or antigen-binding fragment thereof. In some embodiments, the amino acid changes does not affect the binding affinity of the antibody, or antigen-binding fragment thereof.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 19.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 21.
  • the isolated antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 41 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 42.
  • the isolated antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 43 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 44.
  • the isolated antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 45 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 46.
  • the isolated antibody or antigen-binding fragment thereof used in the methods disclosed herein does not bind to the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22).
  • Vectors for expressing a transgene in a host cell are know in the art.
  • the vectors can be expression vectors.
  • Recombinant expression vectors containing a sequence encoding a polypeptide of interest are thus contemplated as within the scope of this disclosure.
  • the expression vector may contain one or more additional sequences such as but not limited to regulatory sequences (e.g., promoter, enhancer), a selection marker, and a polyadenylation signal.
  • Vectors for transforming a wide variety of host cells include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), as well as other bacterial, yeast and viral vectors.
  • Recombinant expression vectors within the scope of the description include synthetic, genomic, or cDNA-derived nucleic acid fragments that encode at least one recombinant protein which may be operably linked to suitable regulatory elements.
  • suitable regulatory elements may include a transcriptional promoter, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation.
  • Expression vectors may also include one or more nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5' or 3' flanking nontranscribed sequences, 5' or 3' nontranslated sequences (such as necessary ribosome binding sites), a polyadenylation site, splice donor and acceptor sites, or transcriptional termination sequences.
  • an origin of replication that confers the ability to replicate in a host may also be incorporated.
  • transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells may be provided by viral sources.
  • Exemplary vectors may be constructed as described by Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).
  • the vectors may comprise selection markers, which are well known in the art.
  • Selection markers include positive and negative selection markers, for example, antibiotic resistance genes (e.g., neomycin resistance gene, a hygromycin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene, a penicillin resistance gene, a puromycin resistance gene, a blasticidin resistance gene), glutamate synthase genes, HSV-TK, HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)).
  • a nucleic acid sequence encoding a selection marker or the cloning site may be upstream or downstream of a nucleic acid sequence encoding a polypeptide of interest or cloning site.
  • chromosome transfer e.g., cell fusion, chromosome mediated gene transfer, micro cell mediated gene transfer
  • physical methods e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome carrier
  • viral vector transfer e.g., recombinant DNA viruses, recombinant RNA viruses
  • Calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells may also be used to transform cells.
  • Cells transformed with expression vectors described herein may be selected or screened for recombinant expression of the antibodies or antigen-binding fragments described herein.
  • Recombinant-positive cells are expanded and screened for subclones exhibiting a desired phenotype, such as high level expression, enhanced growth properties, or the ability to yield proteins with desired biochemical characteristics, for example, due to protein modification or altered post-translational modifications. These phenotypes may be due to inherent properties of a given subclone or to mutation. Mutations may be effected through the use of chemicals, UV-wavelength light, radiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or a combination of such methods.
  • the methods disclosed herein further comprises delivery of a donor template comprising a knock-in cassette comprising a cargo sequence for insertion within the target gene.
  • endonucleases are introduced to edit and insert knock-in genes into the target gene.
  • endonucleases comprise zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), or endonuclease of the CRISPR/CAS systems.
  • the target gene is a CAR gene.
  • transposases, recombinases, or integrases are introduced to insert knock-in genes into a target gene.
  • transposases, recombinases, or integrases are conjugated to a targeting moiety such as a zinc finger, transcription activatorlike effector (TALE), or nuclease-deficient CRISPR/CAS molecule.
  • introduction of a cargo encoding construct includes the introduction of a vector DNA and a transposase.
  • a zinc finger nuclease is used as a gene editing agent in any one of the embodiments described herein.
  • Particular embodiments may use a transcription activator like nuclease (TALEN) as a gene editing agent.
  • TALEN transcription activator like nuclease
  • targeted genetic engineering approaches can be used, such as the CRISPR nuclease system.
  • Example 1 General methods iPSC Cell Culture iPSCs were cultured on 96-well, 24-well, or 6-well plates precoated with 0.5pg/cm2 of iMatrix-511 according to manufacturer’s protocol (Takara #T304). At time of passaging, media was supplemented with the CEPT cocktail (chroman 1 (MedChem Express #HY - 15392, emricasan (SelleckChem S7775), polyamine supplement (Sigma- Alrdich P8483), and trans-ISRIB (R&D Systems 5284)).
  • CEPT cocktail chroman 1 (MedChem Express #HY - 15392, emricasan (SelleckChem S7775), polyamine supplement (Sigma- Alrdich P8483), and trans-ISRIB (R&D Systems 5284)
  • the final concentrations of the CEPT cocktail 50nM chroman 1, 5pM emricasan, polyamine supplement 1:1,000, and 0.7pM trans-ISRIB were diluted in Stemfit Basic 04 Complete Type Medium (Ajinomoto BasicO4CT).
  • IxlO 6 CAR + Clone 1 iPSCs were collected and centrifuged at 300g for 5 minutes. The supernatant was aspirated and the cell pellet resuspended in lOOpL of Stain Buffer (BD #554656) with final concentration of l.Opg/mL of BIRDB123.AF488.001. The cells were stained for 30 minutes at 4°C away from light. The cells were then washed once with Stain Buffer and resuspended in ImL of MACSQuant Tyto Running Buffer (Miltenyi #130-107- 206) and run on the MACSQuant Tyto Cell Sorter according to Miltenyi’ s protocol.
  • MACSQuant Tyto Running Buffer MACSQuant Tyto Running Buffer
  • the cells were gated and sorted based on the positive BIRDB123 AF488 fluorescent signal.
  • the enriched cells (2xl0 3 cells) were then transferred to one well of 6 well plate coated with 0.5pg/cm2 of iMatrix-511 (Takara #T304) containing Stemfit Basic 04 Complete Type Medium with CEPT cocktail.
  • IxlO 6 CAR + iPSCs were collected and centrifuged at 300g for 5 minutes. The supernatant was aspirated and the cell pellet resuspended in lOOpE of DPBS (Gibco #14190144) with final concentration of EOpg/mL of BIRDB123.AF488.001. The cells were stained for 30 minutes at 4°C away from light. The cells were then washed once with DPBS and resuspended in ImE DPBS. The cells were then washed once with Stain Buffer and resuspended in ImE of DPBS and run on the f.sight 2.0 according to Cytena’s protocol.
  • the cells were selected to be single-cell sorted based on the positive BIRDB123 AF488 fluorescent signal.
  • a single-cell with a positive BIRDB123 AF488 signal was deposited into one-well of a 96-well plate coated with 0.5pg/cm2 of iMatrix-511 (Takara #T304) containing Stemfit Basic 04 Complete Type Medium with CEPT cocktail.
  • the clones were cultured, expanded, and evaluated for CAR expression and pluripotency via flow cytometry 2 weeks later.
  • Table 1 presents non-limiting examples of pluripotency markers used in the experiment.
  • CRISPR-edited knock-in iPSCs (CAR + Clone 1) were generated (Signals ELN H.Sun E175345) by nucleofecting the CRISPR ribonucleoprotein (RNP) complex and DNA expression cassette containing the CAR transgene.
  • the iPSCs were then cultured for 8 days, stained with BIRDB123 mAh Alexa Fluor (AF) 488 conjugate, and determined to be 0.3% CAR + .
  • This sample was then enriched to 80% CAR+ with the Tyto Cell Sorter by gating on the positive BIRDB123 AF488 fluorescent population (Signals ELN J.McCaffrey E177509) and the flow plot of CAR expression post enrichment is shown in Figure la.
  • the enriched sample was then cultured for one week, stained with BIRDB123 mAh AF488 conjugate, and single-cell sorted based on the positive BIRDB123 AF488 fluorescent signal with the f.sight 2.0.
  • Out of a 96-well plate 62 CAR + clones were generated and expanded for approximately 2 weeks.
  • a representative clone, Clone Al has nearly 100% CAR expression determined by flow cytometry shown in Figure lb.
  • the pluripotency markers (SSEA-3 and SSEA-4) have acceptable expression and the surface markers (SSEA-1 and CD34) that are not expressed in iPSCs also meet the acceptance criteria.
  • CRISPR-edited knock-in iPSCs (CAR + Clone 2) were generated (Signals ELN P.Diviney E167765) by nucleofecting the CRISPR ribonucleoprotein (RNP) complex and DNA expression cassette containing the CAR transgene.
  • the iPSCs were then cultured for 8 days, stained with BIRDB123 mAh Alexa Fluor (AF) 488 conjugate, and determined to be 8.57% CAR + ( Figure 2a).
  • the sample was then directly single-cell sorted based on the positive BIRDB123 AF488 fluorescent signal with the f.sight 2.0 (Signals ELN J.McCaffrey E169071).

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Abstract

La divulgation concerne des procédés d'enrichissement de cellules exprimant un transgène de récepteur antigénique chimérique (CAR) comprenant un peptide lieur BIRD. En particulier, l'invention concerne des procédés d'utilisation d'anticorps qui se lient au peptide lieur BIRD pour enrichir des cellules CAR+.
PCT/IB2025/054718 2024-05-06 2025-05-06 Enrichissement de cellules exprimant un lieur bird Pending WO2025233825A1 (fr)

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