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WO2024220895A1 - Antibodies and variant nucleic acid libraries for sirp-alpha - Google Patents

Antibodies and variant nucleic acid libraries for sirp-alpha Download PDF

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Publication number
WO2024220895A1
WO2024220895A1 PCT/US2024/025535 US2024025535W WO2024220895A1 WO 2024220895 A1 WO2024220895 A1 WO 2024220895A1 US 2024025535 W US2024025535 W US 2024025535W WO 2024220895 A1 WO2024220895 A1 WO 2024220895A1
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Prior art keywords
amino acid
seq
acid sequence
respect
chain cdrs
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PCT/US2024/025535
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French (fr)
Inventor
Aaron Sato
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Twist Bioscience Corporation
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Publication of WO2024220895A1 publication Critical patent/WO2024220895A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • Signal-regulatory protein alpha (also known as SIRP-alpha, SIRPa, and SIRPa) is a regulatory membrane glycoprotein that forms the SIRP family which is expressed by cells to negatively control the effector function of innate immune cells. SIRPa diffuses laterally on the macrophage membrane and accumulates at a phagocytic synapse to bind CD47 and signal ‘self, which inhibits the cytoskeleton-invasive process of phagocytosis by a macrophage. Inhibition of the interaction of SIRPa to CD47 enables phagocytosis of tumor cells.
  • SIRPa plays an important role in various diseases and conditions including cancer, and therapeutic antibodies targeting SIRPa have clinical significance.
  • Antibodies possess the capability to bind with high specificity' and affinity to biological targets.
  • the design of therapeutic antibodies is challenging due to balancing of immunological effects with efficacy.
  • antibodies or antibody fragments comprising an amino acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 5-428. In some embodiments, the antibody or antibody fragment comprises an amino acid sequence at least about 95% identical to that set forth in any one of SEQ ID NOs: 5-428. In some embodiments, the antibody or antibody fragment comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 5-428.
  • the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody.
  • scFv single-chain Fvs
  • the antibody or antibody fragment binds to SIRPa with a KD of less than 75 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 50 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 25 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 10 nM.
  • antibodies or antibody fragments that bind SIRPa comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 182-240.
  • the immunoglobulin heavy chain comprises an amino acid sequence at least about 95% identical to that set forth in any one of SEQ ID NOs: 182-240.
  • the immunoglobulin heavy chain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 182-240.
  • the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity' determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti- idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof.
  • scFv single chain antibody
  • Fab fragment a F(ab')2 fragment
  • Fd fragment fragment
  • a single-domain antibody an isolated complementarity' determining region (CDR
  • the antibody or antibody fragment thereof is chimeric or humanized. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 75 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 50 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 25 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 10 nM.
  • antibodies or antibody fragments that bind SIRPa comprising an immunoglobulin light chain comprising an amino acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 382-428.
  • the immunoglobulin heavy chain comprises an amino acid sequence at least about 95% identical to that set forth in any one of SEQ ID NOs: 382-428.
  • the immunoglobulin heavy chain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 382-428.
  • the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idioty pic (anti-Id) antibody, or ab antigen-binding fragments thereof.
  • scFv single chain antibody
  • Fab fragment a F(ab')2 fragment
  • Fd fragment fragment
  • a single-domain antibody an isolated complementarity determining region (CDR)
  • the antibody or antibody fragment thereof is chimeric or humanized. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 75 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 50 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 25 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 10 nM.
  • HCDR1 heavy chain complementary determining region 1
  • HCDR2 heavy chain complementary determining region 2
  • antibodies or antigen-binding fragments thereof that binds SIRPa wherein the antibody or antigen-binding fragment thereof comprises a heavy chain complementary 7 determining region 1 (HCDR1) comprising the amino acid sequence depicted in Table 7 below (for example SEQ ID NO: 58 for SIRPa -54) or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence depicted in Table 7 below (for example SEQ ID NO: 58 for SIRPa -54), a heavy chain complementary 7 determining region 2 (HCDR2) comprising the amino acid sequence depicted in Table 7 below (for example SEQ ID NO: 117 for SIRPa -54) or a functional variant thereof having on or two amino acid substitutions with respect to the amino acid sequence depicted in Table 7 below (for example SEQ ID NO: 1 17 for SIRPa -54) and a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence depicted in Table 7 below (for example SEQ ID NO:
  • antibodies or antigen-binding fragments thereof that binds SIRPa wherein the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 90% or at least 95% identical to the amino acid sequence depicted in Table 8 below (for example SEQ ID NO: 182 for SIRPa -1) and a VL comprising an amino acid sequence at least 90% or at least 95% identical to the amino acid sequence depicted in Table 8 below (for example SEQ ID NO: 382 for SIRPa -1).
  • antibodies or antigen-binding fragments thereof that binds SIRPa, wherein the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 90% or at least 95% identical to the ammo acid sequence of any one of SEQ ID NOs: 229-240 (for example SEQ ID NO: 230 for SIRPa-49).
  • antibodies or antigen-binding fragments thereof that binds SIRPa wherein the antibody is a VHH antibody comprising a variable domain, heavy chain region (VH) wherein VH comprises complementarity determining regions CDRH1, CDRH2. and CDRH3.
  • VHH antibody comprising a variable domain, heavy chain region (VH) wherein VH comprises complementarity determining regions CDRH1, CDRH2. and CDRH3.
  • isolated nucleic acids that encode the antibody or antigen-binding fragment thereof described herein, expression vectors comprising these nucleic acids, isolated host cells comprising these nucleic acids or these expression vectors, and isolated host cells that express the antibody or antigen-binding fragment thereof described herein.
  • libraries comprising nucleic acids, wherein at least one of the nucleic acid encodes for at least one of the antibody or antigen-binding fragment thereof described herein, in particular encoding for a SIRPa VHH antibody.
  • kits for treating a disease or disorder associated with SIRPa dysfunction in a subject in need thereof comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof described herein.
  • the disease is cancer.
  • Figure 1 presents a diagram of steps demonstrating an exemplary process workflow for gene synthesis as disclosed herein.
  • Figure 2 depicts results of phage display panning of three SIRPa libraries.
  • Figures 3A-3C depict ELISA results for the SIRPa in VHH Hi + VHH hShuffle library (Figure 3A), the SIRPa in NAL + SAB + DeepCDR library ( Figure 3B), and the SIRPa in HI NEW library' ( Figure 3C).
  • Figure 3D shows a summary of ELISA results.
  • Figure 4 depicts sequencing and analysis results for all three SIRPa libraries.
  • Figure 5 depicts the distribution of HCDR3 length in SIRPa antibody candidates.
  • Figure 6A depicts an iso-affinity chart of SIRPa binders.
  • Figure 6B-6E depicts kinetic curves of SIRPa IgG candidates.
  • Figure 6F depicts KD values for the purified SIRPa IgG candidates.
  • nucleic acid encompasses double- or triple-stranded nucleic acids, as well as single-stranded molecules.
  • nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands).
  • Nucleic acid sequences, when provided, are listed in the 5’ to 3’ direction, unless stated otherwise. Methods described herein provide for the generation of isolated nucleic acids. Methods described herein additionally provide for the generation of isolated and purified nucleic acids.
  • a “nucleic acid” as referred to herein can comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more bases in length.
  • polypeptide-segments encoding nucleotide sequences, including sequences encoding non-ribosomal peptides (NRPs), sequences encoding non-ribosomal peptidesynthetase (NRPS) modules and synthetic variants, polypeptide segments of other modular proteins, such as antibodies, polypeptide segments from other protein families, including noncoding DNA or RNA.
  • regulatory sequences e.g. promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA, small nucleolar RNA derived from microRNA, or any functional or structural DNA or RNA unit of interest.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification, genomic DNA.
  • loci locus
  • mRNA messenger RNA
  • transfer RNA transfer RNA
  • ribosomal RNA short interfering RNA
  • shRNA short-hairpin RNA
  • miRNA micro-RNA
  • cDNA complementary DNA
  • cDNA complementary DNA
  • cDNA encoding for a gene or gene fragment referred herein may comprise at least one region encoding for exon sequences without an intervening intron sequence in the genomic equivalent sequence.
  • kits for generation of antibodies comprise methods, compositions, and systems for generation of antibodies.
  • Methods, compositions, and systems described herein for the optimization of antibodies comprise a ratio-variant approach that mirror the natural diversity of antibody sequences.
  • libraries of optimized antibodies comprise variant antibody sequences.
  • the variant antibody sequences are designed comprising variant CDR regions.
  • the variant antibody sequences comprising variant CDR regions are generated by shuffling the natural CDR sequences in a llama, humanized, or chimeric framework.
  • such libraries are synthesized, cloned into expression vectors, and translation products (antibodies) evaluated for activity.
  • fragments of sequences are synthesized and subsequently assembled.
  • expression vectors are used to display and enrich desired antibodies, such as phage display.
  • the phage vector is a Fab phagemid vector. Selection pressures used during enrichment in some instances includes binding affinity, toxicity, immunological tolerance, stability, or other factor.
  • Such expression vectors allow antibodies with specific properties to be selected (“panning”), and subsequent propagation or amplification of such sequences enriches the library' with these sequences.
  • Panning rounds can be repeated any number of times, such as 1, 2, 3, 4, 5, 6, 7, or more than 7 rounds.
  • each round of panning involves a number of washes. In some instances, each round of panning involves at least or about 1. 2, 3, 4, 5. 6, 7, 8. 9, 10, 11, 12, 13, 14, 15, 16, or more than 16 washes.
  • Described herein are methods and systems of in-silico library' design. Libraries as described herein, in some instances, are designed based on a database comprising a variety' of antibody sequences.
  • the database comprises a plurality of variant antibody sequences against various targets.
  • the database comprises at least 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more than 5000 antibody sequences.
  • An exemplary database is an iCAN database.
  • the database comprises naive and memory B-cell receptor sequences. In some instances, the naive and memory B-cell receptor sequences are human, mouse, or primate sequences.
  • the naive and memory B- cell receptor sequences are human sequences.
  • the database is analyzed for position specific variation.
  • antibodies described herein comprise position specific variations in CDR regions.
  • the CDR regions comprise multiple sites for variation.
  • the CDR is CDR1, CDR2, or CDR3 of a variable heavy chain. In some instances, the CDR is CDR1, CDR2, or CDR3 of a variable light chain. In some instances, the libraries comprise multiple variants encoding for CDR1, CDR2, or CDR3. In some instances, the libraries as described herein encode for at least 50, 100, 200, 300, 400, 500, 1000, 1200, 1500, 1700, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more than 5000 CDR1 sequences.
  • the libraries as described herein encode for at least 50, 100, 200, 300, 400, 500, 1000, 1200, 1500, 1700, 2000, 2500, 3000. 3500, 4000. 4500. 5000, or more than 5000 CDR2 sequences. In some instances, the libraries as described herein encode for at least 50, 100, 200, 300, 400, 500, 1000, 1200, 1500, 1700, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more than 5000 CDR3 sequences. In-silico antibodies libraries are in some instances synthesized, assembled, and enriched for desired sequences.
  • the CDR1 variants, the CDR2 variants, and the CDR3 variants are shuffled to generate a diverse library.
  • the diversity of the libraries generated by methods described herein have a theoretical diversity of at least or about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 1? , 10 14 , 10 15 , 10 16 , 10 17 , 10 18 , or more than 10 18 sequences.
  • the library has a final library diversity’ of at least or about IO 7 , 10 8 , 10 9 , IO 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , 10 17 , 10 18 , or more than 10 18 sequences.
  • sequences generated by methods described herein comprise at least 1, 2. 3, 4, 5, 6. 7, 8, 9. 10. 11, 12, 13, 14, 15, 16, or more than 16 mutations from the germline sequence.
  • sequences generated comprise no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or no more than 18 mutations from the germline sequence.
  • sequences generated comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15, 16, or about 18 mutations relative to the germline sequence.
  • Antibody Libraries Provided herein are libraries generated from methods described herein. Antibodies described herein result in improved functional activity, structural stability, expression, specificity, or a combination thereof.
  • the antibody is a single domain antibody.
  • the single domain antibody comprises one heavy chain variable domain.
  • the single domain antibody is a VHH antibody.
  • antibody will be understood to include proteins having the characteristic two-armed, Y-shape of a typical antibody molecule as well as one or more fragments of an antibody that retain the abi 1 i ty to specifically bind to an antigen.
  • Exemplary antibodies include, but are not limited to, a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv) (including fragments in which the VL and VH are joined using recombinant methods by a synthetic or natural linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules, including single chain Fab and scFab), a single chain antibody, a Fab fragment (including monovalent fragments comprising the VL, VH.
  • scFv single-chain Fvs
  • a F(ab')2 fragment including bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • a Fd fragment including fragments comprising the VH and CHI fragment
  • a Fv fragment including fragments comprising the VL and VH domains of a single arm of an antibody
  • dAb or sdAb including fragments comprising a VH domain
  • CDR complementarity’ determining region
  • a diabody including fragments comprising bivalent dimers such as two VL and VH domains bound to each other and recognizing two different antigens
  • a fragment comprised of only a single monomeric variable domain disulfide-linked Fvs (sdFv)
  • an intrabody an anti-idioty pic (anti-Id) antibody, or ab antigen-binding fragments thereof.
  • the libraries disclosed herein comprise nucleic acids encoding for an antibody, wherein the antibody is a Fv antibody, including Fv antibodies comprised of the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site.
  • the Fv antibody consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association, and the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer.
  • the six hypervariable regions confer antigen-binding specificity to the antibody.
  • a single variable domain or half of an Fv comprising only three hypervariable regions specific for an antigen, including single domain antibodies isolated from camelid animals comprising one heavy chain variable domain such as VHH antibodies or nanobodies has the ability to recognize and bind antigen.
  • a heavy-chain variable (VHH) antibody is a type of antibody fragment comprising heavy chain variable domains.
  • the libraries disclosed herein comprise nucleic acids encoding for an antibody, wherein the antibody is a single-chain Fv or scFv, including antibody fragments comprising a VH, a VL, or both a VH and VL domain, wherein both domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains allowing the scFv to form the desired structure for antigen binding.
  • a scFv is linked to the Fc fragment or a VHH is linked to the Fc fragment (including minibodies).
  • the antibody comprises immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, e.g., molecules that contain an antigen binding site. Immunoglobulin molecules are of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY). class (e.g., IgG 1, IgG 2, IgG 3, IgG 4, IgA 1 and IgA 2) or subclass.
  • libraries comprise immunoglobulins that are adapted to the species of an intended therapeutic target.
  • these methods include “mammalization” and comprises methods for transferring donor antigen-binding information to a less immunogenic mammal antibody acceptor to generate useful therapeutic treatments.
  • the mammal is mouse, rat, equine, sheep, cow. primate (e.g.. chimpanzee, baboon, gorilla, orangutan, monkey), dog, cat, pig, donkey, rabbit, and human.
  • primate e.g.. chimpanzee, baboon, gorilla, orangutan, monkey
  • dog cat
  • pig donkey
  • rabbit and human.
  • provided herein are libraries and methods for felinization and caninization of antibodies.
  • Humanized forms of non-human antibodies can be chimeric antibodies that contain minimal sequence derived from the non-human antibody.
  • a humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody).
  • the donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect.
  • selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody.
  • Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. In some instances, these modifications are made to further refine antibody performance.
  • Caninization can comprise a method for transferring non-canine antigen-binding information from a donor antibody to a less immunogenic canine antibody acceptor to generate treatments useful as therapeutics in dogs.
  • caninized forms of non-canine antibodies provided herein are chimeric antibodies that contain minimal sequence derived from non-canine antibodies.
  • caninized antibodies are canine antibody sequences ( ⁇ ‘acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-canine species (“donor’” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, nonhuman primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties.
  • donor non-canine species
  • donor such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, nonhuman primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties.
  • donor non-canine species
  • framework region (FR) residues of the canine antibody are replaced by corresponding non-canine FR residues.
  • caninized antibodies include residues that are not found in the recipient
  • the caninized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of a canine antibody.
  • Fc immunoglobulin constant region
  • Felinization can comprise a method for transferring non-feline antigen-binding information from a donor antibody to a less immunogenic feline antibody acceptor to generate treatments useful as therapeutics in cats.
  • felinized forms of non-feline antibodies provided herein are chimeric antibodies that contain minimal sequence derived from non-feline antibodies.
  • felinized antibodies are feline antibody sequences (“acceptor” or “recipient”’ antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-feline species (“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties.
  • donor antibody such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties.
  • donor antibody such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant
  • the felinized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of a felinized antibody.
  • Fc immunoglobulin constant region
  • Exemplary antibody mimetics include, but are not limited to, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, atrimers, DARPins, fynomers, Kunitz domain-based proteins, monobodies, anticalins, knottins, armadillo repeat protein-based proteins, and bicyclic peptides.
  • Libraries described herein comprising nucleic acids encoding for an antibody comprise variations in at least one region of the antibody.
  • Exemplary regions of the antibody for variation include, but are not limited to, a complementarity-determining region (CDR), a variable domain, or a constant domain.
  • the CDR is CDR1, CDR2, or CDR3.
  • the CDR is a heavy domain including, but not limited to. CDRH1, CDRH2, and CDRH3.
  • the CDR is a light domain including, but not limited to, CDRL1, CDRL2. and CDRL3.
  • the variable domain is variable domain, light chain (VL) or variable domain. heavy chain (VH).
  • the CDR1, CDR2, or CDR3 is of a variable domain, light chain (VL).
  • CDR1, CDR2, or CDR3 of a variable domain, light chain (VL) can be referred to as CDRL1, CDRL2, or CDRL3, respectively.
  • CDR1, CDR2, or CDR3 of a variable domain, heavy chain (VH) can be referred to as CDRH1, CDRH2, or CDRH3, respectively.
  • the VL domain comprises kappa or lambda chains.
  • the constant domain is constant domain, light chain (CL) or constant domain, heavy chain (CH).
  • libraries comprising nucleic acids encoding for an antibody comprising variation in at least one region of the antibody, wherein the region is the CDR region.
  • the antibody is a single domain antibody comprising one heavy chain variable domain such as a VHH antibody.
  • the VHH antibody comprises variation in one or more CDR regions.
  • the VHH libraries described herein comprise at least or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400, 2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, or CDR3.
  • the libraries comprise at least 2000 sequences of a CDR1, at least 1200 sequences for CDR2, and at least 1600 sequences for CDR3. In some instances, each sequence is non-identical.
  • Libraries as described herein may comprise varying lengths of a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, or combinations thereof of amino acids when translated.
  • the length of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, or combinations thereof of amino acids when translated is at least or about 5, 6, 7, 8, 9, 10. 11, 12, 13, 14, 15, 16, 17. 18. 19. 20. 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 amino acids.
  • Libraries comprising nucleic acids encoding for antibodies having variant CDR sequences as described herein comprise various lengths of amino acids when translated.
  • the length of each of the amino acid fragments or average length of the amino acid synthesized may be at least or about 15, 20, 25. 30. 35. 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, or more than 150 amino acids.
  • the length of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to 100, or 75 to 95 amino acids. In some instances, the length of the amino acid is about 22 amino acids to about 75 amino acids. In some instances, the antibodies comprise at least or about 100, 200, 300, 400. 500, 600. 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than 5000 amino acids. In some instances, the library is a VHH library. In some instances, the library is an antibody library.
  • Libraries as described herein encoding for a VHH antibody comprise variant CDR sequences that are shuffled to generate a library- with a theoretical diversity of at least or about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , 10 17 , 10 18 , or more than 10 18 sequences.
  • the library' has a final library diversity of at least or about 10 7 , 10 8 , 10 9 , IO 10 , IO 11 , 10 12 , IO 13 , 10 14 , IO 13 , 10 16 , 10 17 , IO 18 . or more than 10 18 sequences.
  • Libraries as described herein encoding for an antibody or immunoglobulin comprise variant CDR sequences that are shuffled to generate a library yvith a theoretical diversity of at least or about 10 7 , 10 8 , 10 9 , IO 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , IO 16 , IO 17 , IO 18 , or more than IO 18 sequences.
  • the library has a final library diversity of at least or about IO 7 , 10 8 , 10 9 , IO 10 , IO 11 , IO 12 . IO 13 . 10 14 , IO 15 , 10 16 , 10 17 . 10 18 , or more than IO 18 sequences.
  • Methods described herein provide for synthesis of libraries comprising nucleic acids encoding an antibody or immunoglobulin, wherein each nucleic acid encodes for a predetermined variant of at least one predetermined reference nucleic acid sequence.
  • the predetermined reference sequence is a nucleic acid sequence encoding for a protein
  • the variant library' comprises sequences encoding for variation of at least a single codon such that a plurality of different variants of a single residue in the subsequent protein encoded by the synthesized nucleic acid are generated by standard translation processes.
  • the antibody library comprises varied nucleic acids collectively encoding variations at multiple positions.
  • the variant library' comprises sequences encoding for variation of at least a single codon of a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, or VH domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons of a CDRH1, CDRH2, CDRH3. CDRL1, CDRL2, CDRL3, VL, or VH domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons of framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4).
  • An exemplary number of codons for variation include, but are not limited to, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons.
  • the at least one region of the antibody for variation is from heavy chain V-gene family, heavy chain D-gene family, heay'y chain J-gene family, light chain V-gene family, or light chain J-gene family.
  • the light chain V-gene family comprises immunoglobulin kappa (IGK) gene or immunoglobulin lambda (IGL).
  • IGK immunoglobulin kappa
  • IGL immunoglobulin lambda
  • Exemplary regions of the antibody for variation include, but are not limited to, IGHV1-18. IGHV1-69, IGHV1-8. IGHV3-21.
  • the gene is IGHV1-69, IGHV3-30, IGHV3-23, IGHV3. IGHV1-46, IGHV3-7. IGHV1, or IGHV1-8. In some instances, the gene is IGHV1-69 and IGHV3-30.
  • the region of the antibody for variation is IGHJ3, IGHJ6, IGHJ, IGHJ4, IGHJ5, IGHJ2. or IGH1. In some instances, the region of the antibody for variation is IGHJ3, IGHJ6, IGHJ. or IGHJ4. In some instances, the at least one region of the antibody for variation is IGHV1-69, IGHV3-23, IGKV3-20, IGKV1-39 or combinations thereof. In some instances, the at least one region of the antibody for variation is IGHV1-69 or IGHV3-23. In some instances, the at least one region of the antibody for variation is IGKV3-20 or IGKV1-39.
  • the at least one region of the antibody for variation is IGHV1-69 and IGKV3-20, In some instances, the at least one region of the antibody for variation is IGHV1-69 and IGKV1-39. In some instances, the at least one region of the antibody for variation is IGHV3-23 and IGKV3-20. In some instances, the at least one region of the antibody for variation is IGHV3-23 and IGKV1-39. [0051] Provided herein are libraries comprising nucleic acids encoding for antibodies, wherein the libraries are synthesized with various numbers of fragments. In some instances, the fragments comprise the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, or VH domain.
  • the fragments comprise framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4).
  • the antibody libraries are synthesized with at least or about 2 fragments. 3 fragments, 4 fragments, 5 fragments, or more than 5 fragments.
  • the length of each of the nucleic acid fragments or average length of the nucleic acids synthesized may be at least or about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, or more than 600 base pairs. In some instances, the length is about 50 to 600. 75 to 575, 100 to 550, 125 to 525, 150 to 500. 175 to 475. 200 to 450. 225 to 425, 250 to 400, 275 to 375, or 300 to 350 base pairs.
  • Libraries comprising nucleic acids encoding for antibodies or immunoglobulins as described herein comprise various lengths of amino acids when translated.
  • the length of each of the amino acid fragments or average length of the amino acid synthesized may be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70. 75. 80. 85, 90, 95, 100, 105, 110, 115. 120, 125, 130, 135, 140, 145, 150, or more than 150 amino acids.
  • the length of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to 100, or 75 to 95 amino acids. In some instances, the length of the amino acid is about 22 amino acids to about 75 amino acids. In some instances, the antibodies comprise at least or about 100. 200, 300, 400, 500, 600. 700, 800. 900, 1000. 2000. 3000, 4000, 5000, or more than 5000 amino acids.
  • a number of variant sequences for the at least one region of the antibody for variation are de novo synthesized using methods as described herein. In some instances, a number of variant sequences is de novo synthesized for CDRH1, CDRH2, CDRH3, CDRL1. CDRL2, CDRL3, VL, VH, or combinations thereof. In some instances, a number of variant sequences is de novo synthesized for framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4).
  • the number of variant sequences may be at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more than 500 sequences.
  • the number of variant sequences is at least or about 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000. 6000, 7000, 8000, or more than 8000 sequences.
  • the number of variant sequences is about 10 to 500. 25 to 475, 50 to 450, 75 to 425, 100 to 400. 125 to 375. 150 to 350, 175 to 325, 200 to 300, 225 to 375, 250 to 350, or 275 to 325 sequences.
  • Variant sequences for the at least one region of the antibody vary’ in length or sequence.
  • the at least one region that is de novo synthesized is for CDRH1, CDRH2. CDRH3. CDRL1, CDRL2, CDRL3, VL, VH. or combinations thereof.
  • the at least one region that is de novo synthesized is for framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4).
  • the variant sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40. 45. 50, or more than 50 variant nucleotides or amino acids as compared to wild-type.
  • the variant sequence comprises at least or about 1, 2, 3. 4, 5, 6. 7. 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 additional nucleotides or amino acids as compared to wild-type. In some instances, the variant sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40. 45, or 50 less nucleotides or amino acids as compared to wild-type. In some instances, the libraries comprise at least or about I0 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 . 10 7 , 10 8 , 10 9 . 10 10 , or more than 10 10 variants.
  • antibody libraries may be used for screening and analysis.
  • antibody libraries are assay ed for library 7 displayability and panning.
  • displayability is assayed using a selectable tag.
  • Exemplary 7 tags include, but are not limited to, a radioactive label, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag, an affinity tag or other labels or tags that are known in the art.
  • the tag is histidine, polyhistidine, myc, hemagglutinin (HA), or FLAG.
  • antibody libraries are assayed by sequencing using various methods including, but not limited to, singlemolecule real-time (SMRT) sequencing, Polony sequencing, sequencing by ligation, reversible terminator sequencing, proton detection sequencing, ion semiconductor sequencing, nanopore sequencing, electronic sequencing, pyrosequencing, Maxam-Gilbert sequencing, chain termination (e.g., Sanger) sequencing, +S sequencing, or sequencing by synthesis.
  • SMRT singlemolecule real-time
  • Polony sequencing sequencing by ligation
  • reversible terminator sequencing proton detection sequencing
  • ion semiconductor sequencing nanopore sequencing
  • electronic sequencing pyrosequencing
  • Maxam-Gilbert sequencing Maxam-Gilbert sequencing
  • chain termination e.g., Sanger sequencing
  • +S sequencing e.g., +S sequencing, or sequencing by synthesis.
  • antibody libraries are displayed on the surface of a cell or phage.
  • antibody libraries are enriched for sequences with a desired activity 7 using phage display.
  • the antibody libraries are assayed for functional activity, structural stability (e.g., thermal stable or pH stable), expression, specificity, or a combination thereof.
  • the antibody libraries are assayed for antibody capable of folding.
  • a region of the antibody is assayed for functional activity, structural stability, expression, specificity, folding, or a combination thereof.
  • a VH region or VL region is assayed for functional activity, structural stability, expression, specificity, folding, or a combination thereof.
  • Antibodies or IgGs generated by methods as described herein comprise improved binding affinity.
  • the antibody comprises a binding affinity (e.g., KD) of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 11 nm, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, or less than 30 nM.
  • the antibody comprises a KD of less than 400 nM, less than 350 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nm, less than 100 nM, less than 50 nM, less than 25 nM, less than 15 nM, or less than 10 nM.
  • the antibody comprises a KD of less than 1 nM. In some instances, the antibody comprises a KD of less than 1.2 nM. In some instances, the antibody comprises a KD of less than 2 nM. In some instances, the antibody comprises a KD of less than 5 nM. In some instances, the antibody comprises a KD of less than 10 nM. In some instances, the antibody comprises a KD of less than 13.5 nM. In some instances, the antibody comprises a KD of less than 15 nM. In some instances, the antibody comprises a KD of less than 20 nM. In some instances, the antibody comprises a KD of less than 25 nM. In some instances, the antibody comprises a KD of less than 30 nM.
  • the affinity of antibodies or IgGs generated by methods as described herein is at least or about 1.5x, 2. Ox, 5x, lOx, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx, 200x, or more than 200x improved binding affinity as compared to a comparator antibody. In some instances, the affinity of antibodies or IgGs generated by methods as described herein is at least or about 1.5x, 2. Ox, 5x, lOx, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx, 200x, or more than 200x improved function as compared to a comparator antibody. In some instances, the comparator antibody is an antibody with similar structure, sequence, or antigen target.
  • the variant antibodies or IgGs generated by methods as described herein result in a decreased EC50 in a T-cell cytotoxicity assay as compared to the EC50 in a T-cell cytotoxicity assay of a reference antibody or IgG.
  • the variant antibodies or IgGs have an EC50 in a T-cell cytotoxicity assay that is at least 5X decreased as compared to the EC50 in a T-cell cytotoxicity assay of a reference antibody or IgG.
  • the variant antibodies or IgGs have an EC50 in a T-cell cytotoxicity assay that is at least 8X decreased as compared to the EC50 in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an EC50 in a T-cell cytotoxicity assay that is at least 10X decreased as compared to the EC50 in a T-cell cytotoxicity assay of a reference antibody or IgG.
  • the variant antibodies or IgGs have an EC50 in a T-cell cytotoxicity assay that is at least 20X decreased as compared to the EC50 in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an EC50 in a T-cell cytotoxicity assay that is at least 25X decreased as compared to the EC50 in a T-cell cytotoxicity assay of a reference antibody or IgG.
  • the variant antibodies or IgGs have an EC50 in a T-cell cytotoxicity assay that is at least 30X decreased as compared to the EC50 in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an EC50 in a T-cell cytotoxicity assay that is at least 40X decreased as compared to the EC50 in a T-cell cytotoxicity’ assay of a reference antibody or IgG.
  • the variant antibodies or IgGs have an EC50 in a T-cell cytotoxicity assay that is at least 50X decreased as compared to the EC50 in a T-cell cytotoxicity assay of a reference antibody or IgGs.
  • Methods as described herein result in increased yield of antibodies or IgGs.
  • the yield is at least or about 5, 10, 15, 20, 25, 30, 35. 40. 45. 50. 55. 60. 65, 70, 75, 80, or more than 80 micrograms (ug).
  • the yield is in a range of about 5 to about 80, about 10 to about 75, about 15 to about 60, about 20 to about 50, or about 30 to about 40 micrograms (ug).
  • libraries comprising nucleic acids encoding for antibody comprising binding domains, wherein the libraries have improved specificity, stability, expression, folding, or downstream activity.
  • libraries described herein are used for screening and analysis.
  • libraries comprising nucleic acids encoding for antibody comprising binding domains, w erein the nucleic acid libraries are used for screening and analysis.
  • screening and analysis comprises in vitro, in vivo, or ex vivo assays.
  • Cells for screening include primary cells taken from living subjects or cell lines. Cells may be from prokaryotes (e.g., bacteria and fungi) or eukaryotes (e.g., animals and plants). Exemplary animal cells include, without limitation, those from a mouse, rabbit, primate, and insect.
  • cells for screening include a cell line including, but not limited to, Chinese Hamster Ovary (CHO) cell line, human embryonic kidney (HEK) cell line, or baby hamster kidney (BHK) cell line.
  • CHO Chinese Hamster Ovary
  • HEK human embryonic kidney
  • BHK baby hamster kidney
  • nucleic acid libraries described herein may also be delivered to a multicellular organism.
  • Exemplar ⁇ ' multicellular organisms include, without limitation, a plant, a mouse, rabbit, primate, and insect.
  • Nucleic acid libraries described herein may be screened for various pharmacological or pharmacokinetic properties.
  • the libraries are screened using in vitro assays, in vivo assays, or ex vivo assays.
  • in vitro pharmacological or pharmacokinetic properties that are screened include, but are not limited to, binding affinity, binding specificity, and binding avidity.
  • Exemplary in vivo pharmacological or pharmacokinetic properties of libraries described herein that are screened include, but are not limited to, therapeutic efficacy, activity, preclinical toxicity properties, clinical efficacy properties, clinical toxicity' properties, immunogenicity, potency, and clinical safety properties.
  • nucleic acid libraries wherein the nucleic acid libraries may be expressed in a vector.
  • Expression vectors for inserting nucleic acid libraries disclosed herein may comprise eukaryotic or prokaryotic expression vectors.
  • Exemplar ⁇ ' expression vectors include, without limitation, mammalian expression vectors: pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF- CMV-NEO-COOH-3XFLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV- FLAG(R)-6His.
  • pSF-CMV-Ub-KrYFP are examples of expression vectors: pSF-CMV-Ub-KrYFP.
  • pSF-CMV-FMDV-daGFP pEFla- mCherry-Nl Vector, pEFla-tdTomato Vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), and pSF-CMV-PURO-NH2-CMYC
  • bacterial expression vectors pSF-OXB20- BetaGal,pSF-OXB20-Fluc, pSF-OXB20, and pSF-Tac
  • plant expression vectors pRI 101-AN DNA and pCambia2301
  • yeast expression vectors pTYB21 and pKLAC2
  • insect vectors pAc5.1/V5-His A and pDEST8.
  • the vector is pcDNA3 or pcDNA3.1.
  • nucleic acid libraries that are expressed in a vector to generate a construct comprising an antibody.
  • a size of the construct varies.
  • the construct comprises at least or about 500, 600, 700, 800. 900, 1000, 1100. 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200,4400, 4600, 4800, 5000, 6000, 7000, 8000, 9000, 10000, or more than 10000 bases.
  • a the construct comprises a range of about 300 to 1,000, 300 to 2,000, 300 to 3,000, 300 to 4,000, 300 to 5.000, 300 to 6,000, 300 to 7,000, 300 to 8,000, 300 to 9,000, 300 to 10,000, 1,000 to 2.000, 1,000 to 3.000, 1,000 to 4,000, 1,000 to 5,000, 1,000 to 6,000, 1.000 to 7,000, 1.000 to 8,000, 1,000 to 9,000, 1,000 to 10,000, 2,000 to 3,000, 2,000 to 4,000, 2,000 to 5,000, 2,000 to 6,000, 2,000 to 7,000, 2,000 to 8,000, 2,000 to 9,000, 2,000 to 10,000, 3,000 to 4,000, 3,000 to 5,000, 3,000 to 6,000, 3,000 to 7,000.
  • reporter genes include, but are not limited to, acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), cerulean fluorescent protein, citrine fluorescent protein, orange fluorescent protein , cherry fluorescent protein, turquoise fluorescent protein, blue fluorescent protein, horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), luciferase, and derivatives thereof.
  • AHAS acetohydroxyacid synthase
  • AP alkaline phosphatase
  • LacZ beta galactosidase
  • GUS beta glucoronidase
  • CAT chloramphenicol ace
  • Methods to determine modulation of a reporter gene include, but are not limited to, fluorometric methods (e.g. fluorescence spectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy), and antibiotic resistance determination.
  • fluorometric methods e.g. fluorescence spectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy
  • antibiotic resistance determination e.g. antibiotic resistance determination.
  • libraries comprising nucleic acids encoding for antibodies or immunoglobulins that may have therapeutic effects.
  • the antibodies or immunoglobulin result in protein when translated that is used to treat a disease or disorder in a subject.
  • diseases include, but are not limited to. cancer, inflammatory diseases or disorders, a metabolic disease or disorder, a cardiovascular disease or disorder, an immunodeficiency disease or disorder, a respiratory disease or disorder, pain, a digestive disease or disorder, a reproductive disease or disorder, an endocrine disease or disorder, an immune disease or disorder, an autoimmune disease or disorder, or a neurological disease or disorder.
  • the cancer is a solid cancer or a hematologic cancer.
  • the subject is a mammal. In some instances, the subject is a mouse, rabbit, dog, or human. Subjects treated by methods described herein may be infants, adults, or children. Pharmaceutical compositions comprising antibodies or antibody fragments as described herein may be administered intravenously or subcutaneously. Antibodies or antibody fragments as described herein may be used as a therapeutic agent for a disease.
  • the therapeutic agent comprises the antibodies or antibody fragments as described herein as an active ingredient, and it may further comprise suitable excipient(s).
  • the disease or disorder is associated with SIRPa dysfunction. In some instances, the disease or disorder is associated with aberrant signaling via SIRPa. In some instances, the disease or disorder is cancer.
  • SIRPa can be involved in binding to CD47. The SIRPa/CD47 interaction can lead to bidirectional signaling, resulting in different cell-to-cell responses including inhibition of phagocytosis, stimulation of cell-cell fusion, and T cell activation. Inhibition of the interaction of SIRPa to DC47 can enable phagocytosis of tumor cells. SIRPa can be phosphorylated by tyrosine kinases and can participate in signal transduction mediated by various growth factor receptors. SIRPa has also been identified as a biomarker for various cancers (e g., breast cancer, liver cancer, prostate cancer).
  • antibodies or antigen-binding fragments thereof for use in a method of treating a disease or disorder associated with SIRPa dysfunction in a subject in need thereof, the method comprising administering to the subject an effective amount of such antibodies or antigen-binding fragments thereof.
  • Also provided herein is the use of antibodies or antigen-binding fragments thereof, as described herein, in the manufacture of a therapeutic agent/medi cament for the treatment of a disease or disorder associated with SIRPa dysfunction.
  • libraries comprising nucleic acids encoding for antibodies or immunoglobulins that target SIRPa, also known as signal-regulatory protein alpha, cluster of differentiation 172a (CD172a), and src homology' 2 domain-containing phosphate substrate 1.
  • SIRPa is a membrane protein and is a negative regulator of the phosphatidylinositol 3-kinase signaling and mitogen-activated protein kinase pathways.
  • SIRPa antibodies or immunoglobulins wherein the SIRPa antibody or immunoglobulin comprises a sequence at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity' to any one of SEQ ID NOs: 5-428. In some instances, the antibody or immunoglobulin sequence comprises at least or about 95% sequence identity to any one of SEQ ID NOs: 5-428. In some instances, the antibody or immunoglobulin sequence comprises at least or about 97% sequence identity to any one of SEQ ID NOs: 5-428.
  • the antibody or immunoglobulin sequence comprises at least or about 99% sequence identity to any one of SEQ ID NOs: 5-428. In some instances, the antibody or immunoglobulin sequence comprises at least or about 100% sequence identity to any one SEQ ID NOs: 5-428.
  • the SIRPa antibody or immunoglobulin sequence comprises complementarity' determining regions (CDRs) comprising at least or about 70%, 80%, 85%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 5-181 and 241-381.
  • the antibody or immunoglobulin sequence comprises complementarity determining regions (CDRs) comprising at least or about 95% homology to any one of SEQ ID NOs: 5-181 and 241-381.
  • the antibody or immunoglobulin sequence comprises complementarity determining regions (CDRs) comprising at least or about 97% homology to any one of SEQ ID NOs: 5-181 and 241-381. In some instances, the antibody or immunoglobulin sequence comprises complementarity determining regions (CDRs) comprising at least or about 99% homology to any one of SEQ ID NOs: 5-181 and 241-381. In some instances, the antibody or immunoglobulin sequence comprises complementarity determining regions (CDRs) comprising at least or about 100% homology to any one of SEQ ID NOs: 5-181 and 241-381.
  • the antibody or immunoglobulin sequence comprises complementarity determining regions (CDRs) comprising at least a portion having at least or about 3, 4. 5, 6, 7, 8. 9, 10, 12, 14, 16. 17. 18. 19. 20, 21, 22, or more than 22 amino acids of any one of SEQ ID NOs: 5-181 and 241-381.
  • CDRs complementarity determining regions
  • the SIRPa antibody or immunoglobulin sequence comprises a heavy chain CDR1 (HCDR1) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 5-63.
  • the antibody or immunoglobulin sequence comprises HCDR1 comprising at least or about 95% homology of any one of SEQ ID NOs: 5-63.
  • the antibody or immunoglobulin sequence comprises HCDR1 comprising at least or about 97% homology to any one of SEQ ID NOs: 5-63.
  • the antibody or immunoglobulin sequence comprises HCDR1 comprising at least or about 99% homology to any one of SEQ ID NOs: 5-63. In some instances, the antibody or immunoglobulin sequence comprises HCDR1 comprising at least or about 100% homology to any one of SEQ ID NOs: 5-63. In some instances, the antibody or immunoglobulin sequence comprises HCDR1 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8. or more than 8 amino acids of any one of SEQ ID NOs: 5-63.
  • the SIRPa antibody or immunoglobulin sequence comprises a heavy chain CDR2 (HCDR2) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity 7 to any one of SEQ ID NOs: 64-122.
  • the antibody or immunoglobulin sequence comprises HCDR2 comprising at least or about 95% homology to any one of SEQ ID NOs: 64-122.
  • the antibody or immunoglobulin sequence comprises HCDR2 comprising at least or about 97% homology to any one of SEQ ID NOs: 64-122.
  • the antibody or immunoglobulin sequence comprises HCDR2 comprising at least or about 99% homology to any one of SEQ ID NOs: 64-122. In some instances, the antibody or immunoglobulin sequence comprises HCDR2 comprising at least or about 100% homology to any one of SEQ ID NOs: 64-122. In some instances, the antibody or immunoglobulin sequence comprises HCDR2 comprising at least a portion having at least or about 3, 4, 5, 6, 7. 8, 9, 10, 11, 12 or more than 12 amino acids of any one of SEQ ID NOs: 64-122.
  • the SIRPa antibody or immunoglobulin sequence comprises a heavy chain CDR3 (HCDR3) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity' to any one of SEQ ID NOs: 123-181.
  • the antibody or immunoglobulin sequence comprises HCDR3 comprising at least or about 95% homology to any one of SEQ ID NOs: 123-181.
  • the antibody or immunoglobulin sequence comprises HCDR3 comprising at least or about 97% homology to any one of SEQ ID NOs: 123-181.
  • the antibody or immunoglobulin sequence comprises HCDR3 comprising at least or about 99% homology to any one of SEQ ID NOs: 123-181. In some instances, the antibody or immunoglobulin sequence comprises HCDR3 comprising at least or about 100% homology to any one of SEQ ID NOs: 123-181. In some instances, the antibody or immunoglobulin sequence comprises HCDR3 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22 or more than 22 amino acids of any one of SEQ ID NOs: 123-181.
  • the SIRPa antibody or immunoglobulin sequence comprises a light chain CDR1 (LCDR1) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 241-287.
  • the antibody or immunoglobulin sequence comprises LCDR1 comprising at least or about 95% homology of any one of SEQ ID NOs: 241-287.
  • the antibody or immunoglobulin sequence comprises LCDR1 comprising at least or about 97% homology to any one of SEQ ID NOs: 241-287.
  • the antibody or immunoglobulin sequence comprises HCDR1 comprising at least or about 99% homology to any one of SEQ ID NOs: 241-287. In some instances, the antibody or immunoglobulin sequence comprises LCDR1 comprising at least or about 100% homology to any one of SEQ ID NOs: 241-287. In some instances, the antibody or immunoglobulin sequence comprises LCDR1 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, or more than 8 amino acids of any one of SEQ ID NOs: 241-287.
  • the SIRPa antibody or immunoglobulin sequence comprises a light chain CDR2 (LCDR2) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 288-334.
  • the antibody or immunoglobulin sequence comprises LCDR2 comprising at least or about 95% homology to any one of SEQ ID NOs: 288-334.
  • the antibody or immunoglobulin sequence comprises LCDR2 comprising at least or about 97% homology to any one of SEQ ID NOs: 288-334.
  • the antibody or immunoglobulin sequence comprises LCDR2 comprising at least or about 99% homology to any one of SEQ ID NOs: 288-334. In some instances, the antibody or immunoglobulin sequence comprises LCDR2 comprising at least or about 100% homology to any one of SEQ ID NOs: 288-334. In some instances, the antibody or immunoglobulin sequence comprises LCDR2 comprising at least a portion having at least or about 3. 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 amino acids of any one of SEQ ID NOs: 288-334.
  • the SIRPa antibody or immunoglobulin sequence comprises a light chain CDR3 (LCDR3) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 335-381.
  • the antibody or immunoglobulin sequence comprises LCDR3 comprising at least or about 95% homology to any one of SEQ ID NOs: 335-381.
  • the antibody or immunoglobulin sequence comprises LCDR3 comprising at least or about 97% homology to any one of SEQ ID NOs: 335-381.
  • the antibody or immunoglobulin sequence comprises LCDR3 comprising at least or about 99% homology to any one of SEQ ID NOs: 335-381. In some instances, the antibody or immunoglobulin sequence comprises LCDR3 comprising at least or about 100% homology to any one of SEQ ID NOs: 335-381. In some instances, the antibody or immunoglobulin sequence comprises LCDR3 comprising at least a portion having at least or about 3. 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22 or more than 22 amino acids of any one of SEQ ID NOs: 335-381.
  • the SIRPa antibody or immunoglobulin sequence comprises a heavy chain variable domain comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity' to any one of SEQ ID NOs: 182-240.
  • the SIRPa antibody or immunoglobulin sequence comprises a heavy chain variable domain comprising at least or about 95% sequence identity- to any one of SEQ ID NOs: 182-240.
  • the SIRPa antibody or immunoglobulin sequence comprises a heavy chain variable domain comprising at least or about 97% sequence identity to any one of SEQ ID NOs: 182-240.
  • the SIRPa antibody or immunoglobulin sequence comprises a heavy- chain variable domain comprising at least or about 99% sequence identity to any one of SEQ ID NOs: 182-240. In some instances, the SIRPa antibody or immunoglobulin sequence comprises a heavy chain variable domain comprising at least or about 100% sequence identity to any one of SEQ ID NOs: 182-240. In some instances, the SIRPa antibody or immunoglobulin sequence comprises a heavy chain variable domain comprising at least a portion having at least or about 1, 2. 3, 4, 5, 6, 7, 8, 9. 10. 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150. or more than 150 amino acids of any one of SEQ ID NOs: 182-240.
  • the SIRPa antibody or immunoglobulin sequence comprises a light chain variable domain comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity' to any one of SEQ ID NOs: 382-428.
  • the SIRPa antibody or immunoglobulin sequence comprises a light chain variable domain comprising at least or about 95% sequence identity to any one of SEQ ID NOs: 382-428.
  • the SIRPa antibody or immunoglobulin sequence comprises a light chain variable domain comprising at least or about 97% sequence identity to any one of SEQ ID NOs: 382-428.
  • the SIRPa antibody or immunoglobulin sequence comprises a light chain variable domain comprising at least or about 99% sequence identity to any one of SEQ ID NOs: 382-428. In some instances, the SIRPa antibody or immunoglobulin sequence comprises a light chain variable domain comprising at least or about 100% sequence identity to any one of SEQ ID NOs: 382-428. In some instances, the SIRPa antibody or immunoglobulin sequence comprises a light chain variable domain comprising at least a portion having at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9. 10, 12, 14, 16, 18, 20. 30. 40. 50. 60. 70. 80, 90, 100, 110. 120, 130. 140, 150. or more than 150 ammo acids of any one of SEQ ID NOs: 382-428.
  • Variant nucleic acid libraries described herein may comprise a plurality of nucleic acids, wherein each nucleic acid encodes for a variant codon sequence compared to a reference nucleic acid sequence.
  • each nucleic acid of a first nucleic acid population contains a variant at a single variant site.
  • the first nucleic acid population contains a plurality of variants at a single variant site such that the first nucleic acid population contains more than one variant at the same variant site.
  • the first nucleic acid population may comprise nucleic acids collectively encoding multiple codon variants at the same variant site.
  • the first nucleic acid population may comprise nucleic acids collectively encoding up to 19 or more codons at the same position.
  • the first nucleic acid population may comprise nucleic acids collectively encoding up to 60 variant triplets at the same position, or the first nucleic acid population may comprise nucleic acids collectively encoding up to 61 different triplets of codons at the same position.
  • Each variant may encode for a codon that results in a different amino acid during translation.
  • Table 1 provides a listing of each codon possible (and the representative amino acid) for a variant site.
  • a nucleic acid population may comprise varied nucleic acids collectively encoding up to 20 codon variations at multiple positions. In such cases, each nucleic acid in the population comprises variation for codons at more than one position in the same nucleic acid. In some instances, each nucleic acid in the population comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8,
  • each variant long nucleic acid comprises variation for codons at 1, 2, 3, 4. 5, 6, 7, 8, 9,
  • the variant nucleic acid population comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more codons in a single nucleic acid. In some instances, the variant nucleic acid population comprises variation for codons in at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more codons in a single long nucleic acid.
  • a platform approach utilizing miniaturization, parallelization, and vertical integration of the end-to-end process from polynucleotide synthesis to gene assembly within nanowells on silicon to create a revolutionary synthesis platform.
  • Devices described herein provide, with the same footprint as a 96-well plate, a silicon synthesis platform is capable of increasing throughput by a factor of up to 1,000 or more compared to traditional synthesis methods, with production of up to approximately 1,000,000 or more polynucleotides, or 10,000 or more genes in a single highly-parallelized run.
  • Genomic information encoded in the DNA is transcribed into a message that is then translated into the protein that is the active product within a given biological pathway.
  • a library with the desired variants available at the intended frequency in the right position available for testing — in other words, a precision library 7 , enables reduced costs as well as turnaround time for screening.
  • a drug itself can be optimized using methods described herein.
  • a variant polynucleotide library encoding for a portion of the antibody is designed and synthesized.
  • a variant nucleic acid library for the antibody can then be generated by processes described herein (e.g., PCR mutagenesis followed by insertion into a vector).
  • the antibody is then expressed in a production cell line and screened for enhanced activity.
  • Example screens include examining modulation in binding affinity to an antigen, stability 7 , or effector function (e.g., ADCC, complement, or apoptosis).
  • Exemplary regions to optimize the antibody include, without limitation, the Fc region, Fab region, variable region of the Fab region, constant region of the Fab region, variable domain of the heavy chain or light chain (VH or VL), and specific complementarity -determining regions (CDRs) of VH or VL.
  • Nucleic acid libraries synthesized by methods described herein may be expressed in various cells associated with a disease state.
  • Cells associated with a disease state include cell lines, tissue samples, primary cells from a subject, cultured cells expanded from a subject, or cells in a model system.
  • Exemplary model systems include, without limitation, plant and animal models of a disease state.
  • a variant nucleic acid library 7 described herein is expressed in a cell associated with a disease state, or one in which a cell a disease state can be induced.
  • an agent is used to induce a disease state in cells.
  • Exemplary tools for disease state induction include, without limitation, a Cre/Lox recombination system, LPS inflammation induction, and streptozotocin to induce hypoglycemia.
  • the cells associated with a disease state may be cells from a model system or cultured cells, as well as cells from a subject having a particular disease condition.
  • Exemplary disease conditions include a bacterial, fungal, viral, autoimmune, or proliferative disorder (e.g., cancer).
  • the variant nucleic acid library 7 is expressed in the model system, cell line, or primary cells derived from a subject, and screened for changes in at least one cellular activity.
  • Exemplary cellular activities include, without limitation, proliferation, cycle progression, cell death, adhesion, migration, reproduction, cell signaling, energy production, oxygen utilization, metabolic activity 7 , and aging, response to free radical damage, or any combination thereof.
  • Devices used as a surface for polynucleotide synthesis may be in the form of substrates which include, without limitation, homogenous array surfaces, patterned array surfaces, channels, beads, gels, and the like.
  • substrates comprising a plurality of clusters, wherein each cluster comprises a plurality of loci that support the attachment and synthesis of polynucleotides.
  • substrates comprise a homogenous array surface.
  • the homogenous array surface is a homogenous plate.
  • locus 7 refers to a discrete region on a structure which provides support for polynucleotides encoding for a single predetermined sequence to extend from the surface.
  • a locus is on a two dimensional surface, e.g. , a substantially planar surface. In some instances, a locus is on a three- dimensional surface, e.g. . a well, microwell, channel, or post. In some instances, a surface of a locus comprises a material that is actively functionalized to attach to at least one nucleotide for polynucleotide synthesis, or preferably, a population of identical nucleotides for synthesis of a population of polynucleotides. In some instances, polynucleotide refers to a population of polynucleotides encoding for the same nucleic acid sequence.
  • a surface of a substrate is inclusive of one or a plurality of surfaces of a substrate.
  • the average error rates for polynucleotides synthesized within a library described here using the systems and methods provided are often less than 1 in 1000, less than about 1 in 2000, less than about 1 in 3000 or less often without error correction.
  • a substrate provides support for the synthesis of more than 50, 100, 200, 400, 600, 800. 1000, 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200.000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900.000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more non-identical polynucleotides.
  • the surfaces provide support for the synthesis of more than 50. 100, 200, 400, 600, 800, 1000. 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more polynucleotides encoding for distinct sequences.
  • at least a portion of the polynucleotides have an identical sequence or are configured to be synthesized with an identical sequence.
  • the substrate provides a surface environment for the grow th of polynucleotides having at least 80, 90, 100, 120, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400. 425, 450, 475, 500 bases or more.
  • each locus supports the synthesis of a population of polynucleotides.
  • each locus supports the synthesis of a population of polynucleotides having a different sequence than a population of polynucleotides grown on another locus.
  • each polynucleotide sequence is synthesized with 1, 2, 3, 4, 5. 6, 7, 8, 9 or more redundancy across different loci within the same cluster of loci on a surface for polynucleotide synthesis.
  • the loci of a substrate are located within a plurality of clusters.
  • a substrate comprises at least 10, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000. 20000, 30000, 40000, 50000 or more clusters. In some instances, a substrate comprises more than 2,000; 5,000; 10,000; 100,000; 200,000; 300,000; 400.000; 500.000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000; 1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000; 300,000; 400,000; 500,000; 600,000;
  • a substrate comprises about 10,000 distinct loci.
  • the number of loci within a single cluster is varied in different instances.
  • each cluster includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150, 200, 300, 400, 500 or more loci.
  • each cluster includes about 50-500 loci.
  • each cluster includes about 100-200 loci.
  • each cluster includes about 100-150 loci.
  • each cluster includes about 109, 121, 130 or 137 loci. In some instances, each cluster includes about 19, 20, 61, 64 or more loci. Alternatively or in combination, polynucleotide synthesis occurs on a homogenous array surface.
  • the number of distinct polynucleotides synthesized on a substrate is dependent on the number of distinct loci available in the substrate.
  • the density of loci within a cluster or surface of a substrate is at least or about 1, 10, 25, 50, 65, 75, 100, 130, 150, 175, 200, 300. 400, 500, 1,000 or more loci per mm 2 .
  • a substrate comprises 10-500, 25-400, 50-500, 100-500, 150-500, 10-250, 50-250, 10-200, or 50-200 mm 2 .
  • the distance between the centers of two adjacent loci within a cluster or surface is from about 10-500, from about 10-200, or from about 10-100 um.
  • the distance between two centers of adjacent loci is greater than about 10, 20, 30, 40, 50. 60. 70, 80, 90 or 100 um. In some instances, the distance between the centers of two adjacent loci is less than about 200. 150, 100. 80. 70, 60, 50, 40, 30, 20 or 10 um. In some instances, each locus has a width of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In some cases, each locus has a width of about 0.5-100, 0.5-50, 10-75, or 0.5-50 um.
  • the density of clusters within a substrate is at least or about 1 cluster per 100 mm 2 , 1 cluster per 10 mm 2 , 1 cluster per 5 mm 2 , 1 cluster per 4 mm 2 , 1 cluster per 3 mm 2 , 1 cluster per 2 mm 2 , 1 cluster per 1 mm 2 , 2 clusters per 1 mm 2 , 3 clusters per 1 mm 2 , 4 clusters per 1 mm 2 , 5 clusters per 1 mm 2 , 10 clusters per 1 mm 2 , 50 clusters per 1 mm 2 or more.
  • a substrate comprises from about 1 cluster per 10 mm 2 to about 10 clusters per 1 mm 2 .
  • the distance between the centers of two adjacent clusters is at least or about 50. 100, 200, 500, 1000, 2000, or 5000 um. In some cases, the distance between the centers of two adjacent clusters is between about 50-100, 50-200, 50-300, 50-500, and 100-2000 um. In some cases, the distance between the centers of two adjacent clusters is between about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or 0.5-2 mm. In some cases, each cluster has a cross section of about 0.5 to about 2, about 0.5 to about 1, or about 1 to about 2 mm.
  • each cluster has a cross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm. In some cases, each cluster has an interior cross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.15, 1.2, 1.3, 1.4, 1.5, 1.6. 1.7, 1.8, 1.9 or 2 mm.
  • a substrate is about the size of a standard 96 well plate, for example between about 100 and about 200 mm by between about 50 and about 150 mm.
  • a substrate has a diameter less than or equal to about 1000, 500, 450, 400, 300, 250, 200, 150, 100 or 50 mm.
  • the diameter of a substrate is between about 25-1000, 25-800, 25- 600. 25-500, 25-400, 25-300. or 25-200 mm.
  • a substrate has a planar surface area of at least about 100; 200; 500; 1,000; 2,000; 5,000; 10,000; 12,000; 15,000; 20,000; 30,000; 40,000; 50,000 mm 2 or more.
  • the thickness of a substrate is between about 50- 2000, 50- 1000, 100-1000, 200-1000, or 250-1000 mm.
  • Substrates, devices, and reactors provided herein are fabricated from any variety of materials suitable for the methods, compositions, and systems described herein.
  • substrate materials are fabricated to exhibit a low level of nucleotide binding.
  • substrate materials are modified to generate distinct surfaces that exhibit a high level of nucleotide binding.
  • substrate materials are transparent to visible and/or UV light.
  • substrate materials are sufficiently conductive, e.g.. are able to form uniform electric fields across all or a portion of a substrate.
  • conductive materials are connected to an electric ground.
  • the substrate is heat conductive or insulated.
  • a substrate comprises flexible materials.
  • materials can include, without limitation: nylon, both modified and unmodified, nitrocellulose, polypropylene, and the like.
  • a substrate comprises rigid materials.
  • materials can include, without limitation: glass; fuse silica; silicon, plastics (for example polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, and the like); metals (for example, gold, platinum, and the like).
  • the substrate, solid support or reactors can be fabricated from a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, poly dimethylsiloxane (PDMS), and glass.
  • the substrates/solid supports or the microstructures, reactors therein may be manufactured with a combination of materials listed herein or any other suitable material known in the art.
  • a substrate for the methods, compositions, and systems described herein, wherein the substrates have a surface architecture suitable for the methods, compositions, and systems described herein.
  • a substrate comprises raised and/or lowered features.
  • One benefit of having such features is an increase in surface area to support polynucleotide synthesis.
  • a substrate having raised and/or lowered features is referred to as a three-dimensional substrate.
  • a three-dimensional substrate comprises one or more channels.
  • one or more loci comprise a channel.
  • the channels are accessible to reagent deposition via a deposition device such as a material deposition device.
  • reagents and/or fluids collect in a larger well in fluid communication one or more channels.
  • a substrate comprises a plurality of channels corresponding to a plurality of loci with a cluster, and the plurality of channels are in fluid communication with one well of the cluster.
  • a library of polynucleotides is synthesized in a plurality of loci of a cluster.
  • substrates for the methods, compositions, and systems described herein wherein the substrates are configured for polynucleotide synthesis.
  • the structure is configured to allow for controlled flow and mass transfer paths for polynucleotide synthesis on a surface.
  • the configuration of a substrate allows for the controlled and even distribution of mass transfer paths, chemical exposure times, and/or wash efficacy during polynucleotide synthesis.
  • the configuration of a substrate allows for increased sweep efficiency, for example by providing sufficient volume for a growing polynucleotide such that the excluded volume by the growing polynucleotide does not take up more than 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or less of the initially available volume that is available or suitable for growing the polynucleotide.
  • a three-dimensional structure allows for managed flow of fluid to allow for the rapid exchange of chemical exposure.
  • substrates for the methods, compositions, and systems described herein wherein the substrates comprise structures suitable for the methods, compositions, and systems described herein.
  • segregation is achieved by physical structure.
  • segregation is achieved by differential functionalization of the surface generating active and passive regions for polynucleotide synthesis.
  • differential functionalization is achieved by alternating the hydrophobicity across the substrate surface, thereby creating water contact angle effects that cause beading or wetting of the deposited reagents.
  • Employing larger structures can decrease splashing and cross-contamination of distinct polynucleotide synthesis locations with reagents of the neighboring spots.
  • a device such as a material deposition device, is used to deposit reagents to distinct polynucleotide synthesis locations.
  • Substrates having three-dimensional features are configured in a manner that allows for the synthesis of a large number of polynucleotides (e.g. , more than about 10,000) with a low error rate (e.g, less than about 1:500, 1 : 1000. 1: 1500, 1 :2,000, 1:3,000, 1 :5,000, or 1: 10,000).
  • a substrate comprises features with a density of about or greater than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400 or 500 features per mm 2.
  • a well of a substrate may have the same or different width, height, and/or volume as another well of the substrate.
  • a channel of a substrate may have the same or different width, height, and/or volume as another channel of the substrate.
  • the diameter of a cluster or the diameter of a well comprising a cluster, or both is between about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.05-1, 0.05-0.5, 0.05-0.1, 0.1-10. 0.2-10. 0.3-10, 0.4-10, 0.5-10, 0.5-5, or 0.5-2 mm.
  • the diameter of a cluster or well or both is less than or about 5, 4, 3.
  • the diameter of a cluster or well or both is between about 1.0 and 1.3 mm. In some instances, the diameter of a cluster or well, or both is about 1. 150 mm. In some instances, the diameter of a cluster or well, or both is about 0.08 mm.
  • the diameter of a cluster refers to clusters within a two-dimensional or three-dimensional substrate.
  • the height of a well is from about 20-1000, 50-1000, 100- 1000, 200- 1000, 300-1000, 400-1000, or 500-1000 um. In some cases, the height of a well is less than about 1000, 900, 800, 700, or 600 um.
  • a substrate comprises a plurality of channels corresponding to a plurality of loci within a cluster, wherein the height or depth of a channel is 5-500, 5-400, 5-300, 5- 200, 5-100, 5-50, or 10-50 um. In some cases, the height of a channel is less than 100. 80, 60, 40, or 20 um.
  • the diameter of a channel, locus (e.g. , in a substantially planar substrate) or both channel and locus (e.g., in a three-dimensional substrate wherein a locus corresponds to a channel) is from about 1-1000, 1-500, 1-200, 1-100, 5-100, or 10-100 um, for example, about 90, 80, 70, 60, 50, 40, 30. 20 or 10 um. In some instances, the diameter of a channel, locus, or both channel and locus is less than about 100. 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In some instances, the distance between the center of two adjacent channels, loci, or channels and loci is from about 1-500, 1-200, 1-100, 5-200, 5-100, 5-50, or 5-30, for example, about 20 um. [00115] Surface Modifications
  • the surface comprises various surface modifications.
  • the surface modifications are employed for the chemical and/or physical alteration of a surface by an additive or subtractive process to change one or more chemical and/or physical properties of a substrate surface or a selected site or region of a substrate surface.
  • surface modifications include, without limitation, (1) changing the wetting properties of a surface, (2) functionalizing a surface, i.e., providing, modifying or substituting surface functional groups, (3) defunctionalizing a surface, i.e., removing surface functional groups, (4) otherw ise altering the chemical composition of a surface, e.g, through etching, (5) increasing or decreasing surface roughness, (6) providing a coating on a surface, e.g, a coating that exhibits wetting properties that are different from the wetting properties of the surface, and/or (7) depositing particulates on a surface.
  • adhesion promoter facilitates structured patterning of loci on a surface of a substrate.
  • exemplary surfaces for application of adhesion promotion include, without limitation, glass, silicon, silicon dioxide and silicon nitride.
  • the adhesion promoter is a chemical with a high surface energy .
  • a second chemical layer is deposited on a surface of a substrate.
  • the second chemical layer has a low surface energy.
  • surface energy of a chemical layer coated on a surface supports localization of droplets on the surface. Depending on the patterning arrangement selected, the proximity of loci and/or area of fluid contact at the loci are alterable.
  • a substrate surface, or resolved loci, onto which nucleic acids or other moieties are deposited, e.g.. for polynucleotide synthesis are smooth or substantially planar (e.g., two-dimensional) or have irregularities, such as raised or lowered features (e.g., three- dimensional features).
  • a substrate surface is modified with one or more different layers of compounds.
  • modification layers of interest include, without limitation, inorganic and organic layers such as metals, metal oxides, polymers, small organic molecules and the like.
  • resolved loci of a substrate are functionalized with one or more moieties that increase and/or decrease surface energy'.
  • a moiety is chemically inert.
  • a moiety is configured to support a desired chemical reaction, for example, one or more processes in a polynucleotide synthesis reaction.
  • the surface energy, or hydrophobicity, of a surface is a factor for determining the affinity of a nucleotide to attach onto the surface.
  • a method for substrate functionalization comprises: (a) providing a substrate having a surface that comprises silicon dioxide; and (b) silanizing the surface using, a suitable silanizing agent described herein or otherwise known in the art. for example, an organofunctional alkoxysilane molecule. Methods and functionalizing agents are described in U.S. Patent No. 5474796, which is herein incorporated by reference in its entirety.
  • a substrate surface is functionalized by contact with a derivatizing composition that contains a mixture of silanes, under reaction conditions effective to couple the silanes to the substrate surface, typically via reactive hydrophilic moieties present on the substrate surface.
  • Silanization generally covers a surface through self-assembly with organofunctional alkoxysilane molecules.
  • a variety' of siloxane functionalizing reagents can further be used as currently known in the art, e.g. , for lowering or increasing surface energy.
  • the organofunctional alkoxysilanes are classified according to their organic functions.
  • Methods of the current disclosure for polynucleotide synthesis may include processes involving phosphoramidite chemistry.
  • polynucleotide synthesis comprises coupling a base with phosphoramidite.
  • Polynucleotide synthesis may comprise coupling a base by deposition of phosphoramidite under coupling conditions, wherein the same base is optionally deposited with phosphoramidite more than once, i.e., double coupling.
  • Polynucleotide synthesis may comprise capping of unreacted sites. In some instances, capping is optional.
  • Polynucleotide synthesis may also comprise oxidation or an oxidation step or oxidation steps.
  • Polynucleotide synthesis may comprise deblocking, detritylation, and sulfurization. In some instances, polynucleotide synthesis comprises either oxidation or sulfurization. In some instances, between one or each step during a polynucleotide synthesis reaction, the device is washed, for example, using tetrazole or acetonitrile. Time frames for any one step in a phosphoramidite synthesis method may be less than about 2 min. 1 min, 50 sec, 40 sec, 30 sec, 20 sec and 10 sec.
  • Polynucleotide synthesis using a phosphoramidite method may comprise a subsequent addition of a phosphoramidite building block (e.g., nucleoside phosphoramidite) to a growing polynucleotide chain for the formation of a phosphite triester linkage.
  • a phosphoramidite building block e.g., nucleoside phosphoramidite
  • Phosphoramidite polynucleotide synthesis proceeds in the 3’ to 5’ direction.
  • Phosphoramidite polynucleotide synthesis allows for the controlled addition of one nucleotide to a growing nucleic acid chain per synthesis cycle. In some instances, each synthesis cycle comprises a coupling step.
  • Phosphoramidite coupling involves the formation of a phosphite triester linkage between an activated nucleoside phosphoramidite and a nucleoside bound to the substrate, for example, via a linker.
  • the nucleoside phosphoramidite is provided to the device activated.
  • the nucleoside phosphoramidite is provided to the device with an activator.
  • nucleoside phosphoramidites are provided to the device in a 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90. 100-fold excess or more over the substrate-bound nucleosides.
  • nucleoside phosphoramidite is performed in an anhydrous environment, for example, in anhydrous acetonitrile.
  • the device is optionally washed.
  • the coupling step is repeated one or more additional times, optionally with a wash step between nucleoside phosphoramidite additions to the substrate.
  • a polynucleotide synthesis method used herein comprises 1, 2, 3 or more sequential coupling steps.
  • the nucleoside bound to the device is de-protected by removal of a protecting group, where the protecting group functions to prevent polymerization.
  • a common protecting group is 4,4’-dimethoxytrityl (DMT).
  • phosphoramidite polynucleotide synthesis methods optionally comprise a capping step.
  • a capping step the growing polynucleotide is treated with a capping agent.
  • a capping step is useful to block unreacted substrate-bound 5 ’-OH groups after coupling from further chain elongation, preventing the formation of polynucleotides with internal base deletions.
  • phosphoramidites activated with IH-tetrazole may react, to a small extent, with the 06 position of guanosine. Without being bound by theory, upon oxidation with h /water, this side product, possibly via O6-N7 migration, may undergo depurination.
  • the apurinic sites may end up being cleaved in the course of the final deprotection of the polynucleotide thus reducing the yield of the full-length product.
  • the 06 modifications may be removed by treatment with the capping reagent prior to oxidation with b/water.
  • inclusion of a capping step during polynucleotide synthesis decreases the error rate as compared to synthesis without capping.
  • the capping step comprises treating the substrate-bound polynucleotide with a mixture of acetic anhydride and 1 -methylimidazole. Following a capping step, the device is optionally washed.
  • the device bound growing nucleic acid is oxidized.
  • the oxidation step comprises the phosphite triester is oxidized into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester intemucleoside linkage.
  • oxidation of the growing polynucleotide is achieved by treatment with iodine and water, optionally in the presence of a weak base (e g., pyridine, lutidine, collidine). Oxidation may be carried out under anhydrous conditions using, e.g.
  • a capping step is performed following oxidation.
  • a second capping step allows for device drying, as residual water from oxidation that may persist can inhibit subsequent coupling.
  • the device and growing polynucleotide is optionally washed.
  • the step of oxidation is substituted with a sulfurization step to obtain polynucleotide phosphorothioates, wherein any capping steps can be performed after the sulfurization.
  • reagents are capable of the efficient sulfur transfer, including but not limited to 3-(Dimethylaminomethylidene)amino)-3H-l,2,4-dithiazole-3-thione, DDTT, 3H-l,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent, and N,N,N'N'- Tetraethylthiuram disulfide (TETD).
  • DDTT 3-(Dimethylaminomethylidene)amino)-3H-l,2,4-dithiazole-3-thione
  • DDTT 3H-l,2-benzodithiol-3-one 1,1-dioxide
  • Beaucage reagent also known as Beaucage reagent
  • TETD N,N,N'N'- Tetraethylthiuram disulfide
  • the protected 5’ end of the device bound growing polynucleotide is removed so that the primary hydroxyl group is reactive with a next nucleoside phosphoramidite.
  • the protecting group is DMT and deblocking occurs with trichloroacetic acid in dichloromethane. Conducting detritylation for an extended time or with stronger than recommended solutions of acids may lead to increased depurination of solid support-bound polynucleotide and thus reduces the yield of the desired full-length product.
  • Methods and compositions of the disclosure described herein provide for controlled deblocking conditions limiting undesired depurination reactions.
  • the device bound polynucleotide is washed after deblocking. In some instances, efficient washing after deblocking contributes to synthesized polynucleotides having a low error rate.
  • Methods for the synthesis of polynucleotides typically involve an iterating sequence of the following steps: application of a protected monomer to an actively functionalized surface (e.g, locus) to link with either the activated surface, a linker or with a previously deprotected monomer; deprotection of the applied monomer so that it is reactive with a subsequently applied protected monomer; and application of another protected monomer for linking.
  • One or more intermediate steps include oxidation or sulfurization.
  • one or more wash steps precede or follow one or all of the steps.
  • Methods for phosphoramidite-based polynucleotide synthesis comprise a series of chemical steps.
  • one or more steps of a synthesis method involve reagent cycling, where one or more steps of the method comprise application to the device of a reagent useful for the step.
  • reagents are cycled by a series of liquid deposition and vacuum drying steps.
  • substrates comprising three-dimensional features such as wells, microwells, channels and the like, reagents are optionally passed through one or more regions of the device via the wells and/or channels.
  • Methods and systems described herein relate to polynucleotide synthesis devices for the synthesis of polynucleotides.
  • the synthesis may be in parallel.
  • at least or about at least 2, 3, 4, 5, 6, 7, 8. 9, 10, 11, 12, 13. 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40. 45, 50, 100. 150, 200. 250, 300, 350, 400, 450. 500, 550. 600, 650, 700, 750, 800. 850, 900, 1000, 10000, 50000, 75000, 100000 or more polynucleotides can be synthesized in parallel.
  • the total number polynucleotides that may be synthesized in parallel may be from 2-100000, 3-50000, 4- 10000, 5-1000, 6-900, 7-850, 8-800, 9-750, 10-700, 11-650, 12-600, 13-550, 14-500, 15-450, 16- 400, 17-350, 18-300, 19-250, 20-200, 21-150,22-100, 23-50. 24-45, 25-40, 30-35.
  • the total number of polynucleotides synthesized in parallel may fall within any range bound by any of these values, for example 25-100.
  • the total number of polynucleotides synthesized in parallel may fall within any range defined by any of the values serving as endpoints of the range.
  • Total molar mass of polynucleotides synthesized within the device or the molar mass of each of the polynucleotides may be at least or at least about 10, 20, 30, 40, 50, 100. 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 25000, 50000, 75000, 100000 picomoles, or more.
  • the length of each of the polynucleotides or average length of the polynucleotides within the device may be at least or about at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 100. 150, 200. 300, 400, 500 nucleotides, or more.
  • the length of each of the polynucleotides or average length of the polynucleotides within the device may be at most or about at most 500, 400, 300, 200, 150. 100, 50, 45, 35, 30, 25, 20, 19, 18. 17. 16, 15, 14, 13, 12, 11, 10 nucleotides, or less.
  • the length of each of the polynucleotides or average length of the polynucleotides within the device may fall from 10-500, 9-400, 11-300, 12-200, 13-150, 14-100, 15-50, 16-45, 17-40, 18-35, 19-25.
  • each of the polynucleotides or average length of the polynucleotides within the device may fall within any range bound by any of these values, for example 100-300.
  • the length of each of the polynucleotides or average length of the polynucleotides within the device may fall within any range defined by any of the values serving as endpoints of the range.
  • Methods for polynucleotide synthesis on a surface allow for synthesis at a fast rate.
  • at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. 26. 27. 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, 200 nucleotides per hour, or more are synthesized.
  • Nucleotides include adenine, guanine, thymine, cytosine, uridine building blocks, or analogs/modified versions thereof.
  • libraries of polynucleotides are synthesized in parallel on substrate. For example, a device comprising about or at least about 100; 1,000; 10.000; 30,000; 75,000; 100,000; 1,000,000; 2,000.000; 3,000,000;
  • 4,000.000; or 5.000.000 resolved loci is able to support the synthesis of at least the same number of distinct polynucleotides, wherein polynucleotide encoding a distinct sequence is synthesized on a resolved locus.
  • a library of polynucleotides is synthesized on a device with low error rates described herein in less than about three months, two months, one month, three weeks, 15, 14, 13, 12. 11. 10. 9, 8, 7, 6. 5, 4, 3. 2 days. 24 hours or less.
  • nucleic acids assembled from a polynucleotide library synthesized with low error rate using the substrates and methods described herein are prepared in less than about three months, two months, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours or less.
  • methods described herein provide for generation of a library’ of nucleic acids comprising variant nucleic acids differing at a plurality of codon sites.
  • a nucleic acid may have 1 site, 2 sites, 3 sites, 4 sites, 5 sites, 6 sites, 7 sites, 8 sites, 9 sites, 10 sites, 11 sites, 12 sites, 13 sites, 14 sites, 15 sites, 16 sites, 17 sites 18 sites, 19 sites, 20 sites, 30 sites, 40 sites, 50 sites, or more of variant codon sites.
  • the one or more sites of variant codon sites may be adjacent. In some instances, the one or more sites of variant codon sites may not be adjacent and separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more codons.
  • a nucleic acid may comprise multiple sites of variant codon sites, wherein all the variant codon sites are adjacent to one another, forming a stretch of variant codon sites. In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein none the variant codon sites are adjacent to one another. In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein some the variant codon sites are adjacent to one another, forming a stretch of variant codon sites, and some of the variant codon sites are not adjacent to one another.
  • FIG. 1 illustrates an exemplary 7 process workflow for synthesis of nucleic acids (e.g., genes) from shorter nucleic acids.
  • the workflow is divided generally into phases: (1) de novo synthesis of a single stranded nucleic acid library, (2) joining nucleic acids to form larger fragments, (3) error correction, (4) quality control, and (5) shipment.
  • an intended nucleic acid sequence or group of nucleic acid sequences is preselected. For example, a group of genes is preselected for generation.
  • a predetermined library of nucleic acids is designed for de novo synthesis.
  • Various suitable methods are known for generating high density polynucleotide arrays.
  • a device surface layer is provided.
  • chemistry of the surface is altered in order to improve the polynucleotide synthesis process. Areas of low surface energy are generated to repel liquid while areas of high surface energy- are generated to attract liquids.
  • the surface itself may be in the form of a planar surface or contain variations in shape, such as protrusions or microwells which increase surface area.
  • high surface energy molecules selected serve a dual function of supporting DNA chemistry, as disclosed in International Patent Application Publication WO/2015/021080. which is herein incorporated by reference in its entirety 7 .
  • a deposition device such as a material deposition device, is designed to release reagents in a step wise fashion such that multiple polynucleotides extend, in parallel, one residue at a time to generate oligomers with a predetermined nucleic acid sequence 102.
  • polynucleotides are cleaved from the surface at this stage.
  • Cleavage includes gas cleavage, e.g., with ammonia or methylamine.
  • the generated polynucleotide libraries are placed in a reaction chamber.
  • the reaction chamber also referred to as “nanoreactor” is a silicon coated well, containing PCR reagents and lowered onto the polynucleotide library 103.
  • a reagent is added to release the polynucleotides from the substrate.
  • the polynucleotides are released subsequent to sealing of the nanoreactor 105. Once released, fragments of single stranded polynucleotides hybridize in order to span an entire long range sequence of DNA. Partial hybridization 105 is possible because each synthesized polynucleotide is designed to have a small portion overlapping with at least one other polynucleotide in the pool.
  • a PCA reaction is commenced.
  • the polynucleotides anneal to complementary fragments and gaps are fdled in by a polymerase.
  • Each cycle increases the length of various fragments randomly depending on which polynucleotides find each other. Complementarity amongst the fragments allows for forming a complete large span of double stranded DNA 106.
  • the nanoreactor is separated from the device 107 and positioned for interaction with a device having primers for PCR 108. After sealing, the nanoreactor is subject to PCR 109 and the larger nucleic acids are amplified. After PCR 110, the nanochamber is opened 111. error correction reagents are added 112, the chamber is sealed 113 and an error correction reaction occurs to remove mismatched base pairs and/or strands with poor complementarity from the double stranded PCR amplification products 114. The nanoreactor is opened and separated 115. Error corrected product is next subject to additional processing steps, such as PCR and molecular bar coding, and then packaged 122 for shipment 123.
  • additional processing steps such as PCR and molecular bar coding
  • quality control measures are taken. After error correction, quality control steps include for example interaction with a wafer having sequencing primers for amplification of the error corrected product 116, sealing the wafer to a chamber containing error corrected amplification product 117, and performing an additional round of amplification 118. The nanoreactor is opened 119 and the products are pooled 120 and sequenced 121. After an acceptable quality control determination is made, the packaged product 122 is approved for shipment 123.
  • a nucleic acid generated by a workflow such as that in FIG. 1 is subject to mutagenesis using overlapping primers disclosed herein.
  • a library 7 of primers are generated by in situ preparation on a solid support and utilize single nucleotide extension process to extend multiple oligomers in parallel.
  • a deposition device such as a material deposition device, is designed to release reagents in a step wise fashion such that multiple polynucleotides extend, in parallel, one residue at a time to generate oligomers with a predetermined nucleic acid sequence 102.
  • Example 1 Functionalization of a device surface
  • a device was functionalized to support the attachment and synthesis of a library of polynucleotides.
  • the device surface w as first w et cleaned using a piranha solution comprising 90% H2SO4 and 10% H2O2 for 20 minutes.
  • the device was rinsed in several beakers with DI water, held under a DI water gooseneck faucet for 5 min. and dried with N2.
  • the device was subsequently soaked in NH4OH (1 : 100; 3 mL:300 mL) for 5 min, rinsed with DI water using a handgun, soaked in three successive beakers with DI w ater for 1 min each, and then rinsed again with DI w ater using the handgun.
  • the device was then plasma cleaned by exposing the device surface to O2.
  • a SAMCO PC-300 instrument was used to plasma etch O2 at 250 watts for 1 min in downstream mode.
  • the cleaned device surface was actively functionalized with a solution comprising N-(3- triethoxysilylpropyl)-4-hydroxybutyramide using a YES-1224P vapor deposition oven system with the following parameters: 0.5 to 1 torr, 60 min, 70 °C, 135 °C vaporizer.
  • the device surface was resist coated using a Brew er Science 200X spin coater. SPRTM 3612 photoresist was spin coated on the device at 2500 rpm for 40 sec. The device was pre-baked for 30 min at 90 °C on a Brewer hot plate. The device was subjected to photolithography using a Karl Suss MA6 mask aligner instrument.
  • the device was exposed for 2.2 sec and developed for 1 min in MSF 26A. Remaining developer was rinsed with the handgun and the device soaked in water for 5 min. The device was baked for 30 min at 100 °C in the oven, followed by visual inspection for lithography defects using a Nikon L200. A descum process was used to remove residual resist using the SAMCO PC-300 instrument to O2 plasma etch at 250 watts for 1 min.
  • the device surface w as passively functionalized with a 100 pL solution of perfluorooctyltri chlorosilane mixed with 10 pL light mineral oil.
  • the device w as placed in a chamber, pumped for 10 min. and then the valve was closed to the pump and left to stand for 10 min. The chamber was vented to air.
  • the device was resist stripped by performing two soaks for 5 min in 500 mL NMP at 70 °C with ultrasonication at maximum power (9 on Crest system).
  • the device w as then soaked for 5 min in 500 mL isopropanol at room temperature with ultrasonication at maximum power.
  • the device was dipped in 300 mL of 200 proof ethanol and blown dry with N2.
  • Example 2 Synthesis of a 50-mer sequence on an oligonucleotide synthesis device [00149] A two-dimensional oligonucleotide synthesis device was assembled into a flowcell, which was connected to a flowcell (Applied Biosystems (ABI394 DNA Synthesizer").
  • the two- dimensional oligonucleotide synthesis device was uniformly functionalized with N-(3- TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest) was used to sy nthesize an exemplary polynucleotide of 50 bp ("50-mer polynucleotide”) using polynucleotide synthesis methods described herein.
  • sequence of the 50-mer was as described below: 5'AGACAATCAACCATTTGGGGTGGACAGCCTTGACCTCTAGACTTCGGCAT##TTTTTTT TTT3' (SEQ ID NO: 1), where # denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP -2244 from ChemGenes), which is a cleavable linker enabling the release of oligos from the surface during deprotection.
  • CLP -2244 Thymidine-succinyl hexamide CED phosphoramidite
  • the synthesis was done using standard DNA synthesis chemistry (coupling, capping, oxidation, and deblocking) according to the protocol in Table 2 and an ABI synthesizer.
  • the phosphorami dite/activator combination was delivered similar to the delivery' of bulk reagents through the flowcell. No drying steps were performed as the environment stays "wet” with reagent the entire time.
  • the flow restrictor was removed from the ABI 394 synthesizer to enable faster flow. Without flow restrictor, flow rates for amidites (0. IM in ACN), Activator.
  • Example 3 Synthesis of a 100-mer sequence on an oligonucleotide synthesis device
  • 100-mer polynucleotide (“100-mer polynucleotide”; 5' CGGGATCCTTATCGTCATCGTCGTACAGATCCCGACCCATTTGCTGTCCACCAGTCATG CTAGCCATACCATGATGATGATGATGATGAGAACCCCGCAT##TTTTTTTTTT3' (SEQ ID NO: 2) , where # denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 from ChemGenes) on two different silicon chips, the first one uniformly functionalized with N-(3- TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE and the second one functionalized with 5/95 mix of 11 -acetoxyundecy
  • Table 4 summarizes error characteristics for the sequences obtained from the polynucleotide samples from spots 1-10.
  • the invention may be according to the following aspects.
  • Aspect 1 An antibody or antibody fragment comprising an amino acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 5-428.
  • Aspect 2 The antibody or antibody fragment of aspect 1, wherein the antibody or antibody fragment comprises an amino acid sequence at least about 95% identical to that set forth in any one of SEQ ID NOs: 5-428.
  • Aspect 3 The antibody or antibody fragment of aspect 1, wherein the antibody or antibody fragment comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 5- 428.
  • Aspect 4 The antibody or antibody fragment of any one of aspects 1-Error! Reference source not found., wherein the antibody is a monoclonal antibody, a polyclonal antibody, a bispecific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody. a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof.
  • scFv single chain antibody
  • Fab fragment a F(ab')2 fragment
  • Aspect 5 The antibody or antibody fragment of any one of aspects 1 -Error! Reference source not found., wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 75 nM.
  • Aspect 6 The antibody or antibody fragment of any one of aspects 1 -Error! Reference source not found., wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 50 nM.
  • Aspect 7 The antibody or antibody fragment of any one of aspects 1 -Error! Reference source not found., wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 25 nM.
  • Aspect 8 The antibody or antibody fragment of any one of aspects 1 -Error! Reference source not found., wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 10 nM.
  • Aspect 9 An antibody or antibody fragment that binds SIRPa, comprising an immunoglobulin heavy chain compnsing an ammo acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 182-240.
  • Aspect 10 The antibody or antibody fragment of aspect Error! Reference source not found., wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 95% identical to that set forth in any one of SEQ ID NOs: 182-240.
  • Aspect 1 The antibody or antibody fragment of aspect Error! Reference source not found., wherein the immunoglobulin heavy chain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 182-240.
  • Aspect 12 The antibody or antibody fragment of any one of claims Error! Reference source not found.-l l, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a bispecific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof.
  • scFv single chain antibody
  • Fab fragment a F(ab')2
  • Aspect 13 The antibody or antibody fragment of any one of aspects Error! Reference source not found.- 12, wherein the antibody or antibody fragment thereof is chimeric or humanized.
  • Aspect 14 The antibody or antibody fragment of any one of aspects Error! Reference source not found.-13, wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 75 nM.
  • Aspect 15 The antibody or antibody fragment of any one of aspects Error! Reference source not found.-14, wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 50 nM.
  • Aspect 16 The antibody or antibody fragment of any one of aspects Error! Reference source not found.-15, wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 25 nM.
  • Aspect 17 The antibody or antibody fragment of any one of aspects Error! Reference source not found.- 16, wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 10 nM.
  • Aspect 18 An antibody or antibody fragment that binds SIRPa, comprising an immunoglobulin light chain comprising an amino acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 382-428.
  • Aspect 19 The antibody or antibody fragment of aspect 18, wherein the immunoglobulin light chain comprises an amino acid sequence at least about 95% identical to that set forth in any one of SEQ ID NOs: 382-428.
  • Aspect 20 The antibody or antibody fragment of aspect 18, wherein the immunoglobulin light chain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 382-428.
  • Aspect 21 The antibody or antibody fragment of any one of aspects 18-20, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity 7 determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof.
  • scFv single chain antibody
  • Fab fragment a F(ab')2 fragment
  • Fd fragment a F
  • Aspect 22 The antibody or antibody fragment of any one of aspects 18-21, wherein the antibody or antibody fragment thereof is chimeric or humanized.
  • Aspect 23 The antibody or antibody fragment of any one of aspects 18-22, wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 75 nM.
  • Aspect 24 The antibody or antibody fragment of any one of aspects 18-23, wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 50 nM.
  • Aspect 25 The antibody or antibody fragment of any one of aspects 18-24, wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 25 nM.
  • Aspect 26 The antibody or antibody fragment of any one of aspects 18-25, wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 10 nM.
  • Aspect 27 A method of treating a disease comprising administering the antibody or antibody fragment of any one of aspects 1-26.
  • Aspect 28 The method of aspect 27, wherein the disease is cancer.
  • Aspect 29 An antibody or antigen-binding fragment thereof that binds SIRPa, wherein the antibody or antigen-binding fragment thereof comprises the following combinations of complementary determining regions (CDR) sequences (or a functional variant thereof having one, two or three amino acid substitutions with respect to each amino acid sequence):
  • CDR complementary determining regions
  • Aspect 30 An antibody or antigen-binding fragment thereof that binds SIRPa, wherein the antibody or antigen-binding fragment thereof comprises the following combinations of complementary determining regions (CDR) sequences (or a functional variant thereof having one, two or three amino acid substitutions with respect to each amino acid sequence):
  • CDR complementary determining regions
  • Aspect 31 The antibody or antigen-binding fragment thereof of aspect 30, wherein the antibody or antigen-binding fragment thereof is a VHH antibody.
  • Aspect 32 An antibody or antigen-binding fragment thereof that binds SIRPa, wherein the antibody or antigen-binding fragment thereof comprises the following combinations of variable domain, heavy chain (VH) and variable domain, light chain (VL) sequences (or a functional variant thereof having one, two or three amino acid substitutions with respect to each amino acid sequence):
  • Aspect 33 An antibody or antigen-binding fragment thereof that binds SIRPa. wherein the antibody or antigen-binding fragment thereof is a VHH antibody comprising the variable domain, heavy chain region (VH) sequences (or a functional variant thereof having one, two or three amino acid substitutions with respect to each amino acid sequence) of any one of SEQ ID NOs: 229-240.
  • VHH antibody comprising the variable domain, heavy chain region (VH) sequences (or a functional variant thereof having one, two or three amino acid substitutions with respect to each amino acid sequence) of any one of SEQ ID NOs: 229-240.

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Abstract

Provided herein are methods and compositions relating to SIRPα antibodies and libraries having nucleic acids encoding for a scaffold comprising a SIRPα domain. SIRPα libraries described herein encode for immunoglobulins such as antibodies.

Description

ANTIBODIES AND VARIANT NUCLEIC ACID LIBRARIES FOR SIRP-ALPHA
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/497,385 filed on April 20, 2023, which is incorporated by reference in its entirety'.
BACKGROUND
[0002] Signal-regulatory protein alpha (also known as SIRP-alpha, SIRPa, and SIRPa) is a regulatory membrane glycoprotein that forms the SIRP family which is expressed by cells to negatively control the effector function of innate immune cells. SIRPa diffuses laterally on the macrophage membrane and accumulates at a phagocytic synapse to bind CD47 and signal ‘self, which inhibits the cytoskeleton-invasive process of phagocytosis by a macrophage. Inhibition of the interaction of SIRPa to CD47 enables phagocytosis of tumor cells.
[0003] SIRPa plays an important role in various diseases and conditions including cancer, and therapeutic antibodies targeting SIRPa have clinical significance. Antibodies possess the capability to bind with high specificity' and affinity to biological targets. However, the design of therapeutic antibodies is challenging due to balancing of immunological effects with efficacy. Thus, there is a need to develop compositions and methods for generation of antibodies for use in therapeutics.
INCORPORATION BY REFERENCE
[0004] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF SUMMARY
[0005] Provided herein are antibodies or antibody fragments comprising an amino acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 5-428. In some embodiments, the antibody or antibody fragment comprises an amino acid sequence at least about 95% identical to that set forth in any one of SEQ ID NOs: 5-428. In some embodiments, the antibody or antibody fragment comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 5-428. In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody. a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 75 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 50 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 25 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 10 nM.
[0006] Provided herein are antibodies or antibody fragments that bind SIRPa, comprising an immunoglobulin heavy chain comprising an amino acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 182-240. In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence at least about 95% identical to that set forth in any one of SEQ ID NOs: 182-240. In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 182-240. In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity' determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti- idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof. In some embodiments, the antibody or antibody fragment thereof is chimeric or humanized. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 75 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 50 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 25 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 10 nM.
[0007] Provided herein are antibodies or antibody fragments that bind SIRPa, comprising an immunoglobulin light chain comprising an amino acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 382-428. In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence at least about 95% identical to that set forth in any one of SEQ ID NOs: 382-428. In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 382-428. In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idioty pic (anti-Id) antibody, or ab antigen-binding fragments thereof. In some embodiments, the antibody or antibody fragment thereof is chimeric or humanized. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 75 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 50 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 25 nM. In some embodiments, the antibody or antibody fragment binds to SIRPa with a KD of less than 10 nM.
[0008] Provided herein are methods of treating a disease comprising administering any one of the antibody or antibody fragments described herein. In some embodiments, the disease is cancer. [0009] Provided herein are antibodies or antigen-binding fragments thereof that binds SIRPa, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence depicted in Table 6 below (for example SEQ ID NO: 12 for SIRPa-8) or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence depicted in Table 6 below (for example SEQ ID NO: 12 for SIRPa-8), a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence depicted in Table 6 below (for example SEQ ID NO: 71 for SIRPa -8) or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence depicted in Table 6 below (for example SEQ ID NO: 71 for SIRPa -8), a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence depicted in Table 6 below (for example SEQ ID NO: 130 for SIRPa -8) or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence depicted in Table 6 below (for example SEQ ID NO: 130 for SIRPa -8), a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence depicted in Table 6 below (for example SEQ ID NO: 248 for SIRPa -8) or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence depicted in Table 6 below (for example SEQ ID NO: 248 for SIRPa -8), a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence depicted in Table 6 below (for example SEQ ID NO: 295 for SIRPa -8) or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence depicted in Table 6 below (for example SEQ ID NO: 295 for SIRPa -8), and a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence depicted in Table 6 below (for example SEQ ID NO: 342 for SIRPa -8) or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence depicted in Table 6 below (for example SEQ ID NO: 342 for SIRPa -8). [0010] Provided herein are antibodies or antigen-binding fragments thereof that binds SIRPa, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain complementary7 determining region 1 (HCDR1) comprising the amino acid sequence depicted in Table 7 below (for example SEQ ID NO: 58 for SIRPa -54) or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence depicted in Table 7 below (for example SEQ ID NO: 58 for SIRPa -54), a heavy chain complementary7 determining region 2 (HCDR2) comprising the amino acid sequence depicted in Table 7 below (for example SEQ ID NO: 117 for SIRPa -54) or a functional variant thereof having on or two amino acid substitutions with respect to the amino acid sequence depicted in Table 7 below (for example SEQ ID NO: 1 17 for SIRPa -54) and a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence depicted in Table 7 below (for example SEQ ID NO: 176 for SIRPa -54) or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence depicted in Table 7 below (for example SEQ ID NO: 176 for SIRPa -54).
[0011] Provided herein are antibodies or antigen-binding fragments thereof that binds SIRPa, wherein the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 90% or at least 95% identical to the amino acid sequence depicted in Table 8 below (for example SEQ ID NO: 182 for SIRPa -1) and a VL comprising an amino acid sequence at least 90% or at least 95% identical to the amino acid sequence depicted in Table 8 below (for example SEQ ID NO: 382 for SIRPa -1).
[0012] Provided herein are antibodies or antigen-binding fragments thereof that binds SIRPa, wherein the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 90% or at least 95% identical to the ammo acid sequence of any one of SEQ ID NOs: 229-240 (for example SEQ ID NO: 230 for SIRPa-49).
[0013] Provided herein are antibodies or antigen-binding fragments thereof that binds SIRPa, wherein the antibody is a VHH antibody comprising a variable domain, heavy chain region (VH) wherein VH comprises complementarity determining regions CDRH1, CDRH2. and CDRH3. [0014] Provided herein are isolated nucleic acids that encode the antibody or antigen-binding fragment thereof described herein, expression vectors comprising these nucleic acids, isolated host cells comprising these nucleic acids or these expression vectors, and isolated host cells that express the antibody or antigen-binding fragment thereof described herein. [0015] Provided herein are libraries comprising nucleic acids, wherein at least one of the nucleic acid encodes for at least one of the antibody or antigen-binding fragment thereof described herein, in particular encoding for a SIRPa VHH antibody.
[0016] Provided herein are methods of treating a disease or disorder associated with SIRPa dysfunction in a subject in need thereof, the methods comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof described herein. In some embodiments, the disease is cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 presents a diagram of steps demonstrating an exemplary process workflow for gene synthesis as disclosed herein.
[0018] Figure 2 depicts results of phage display panning of three SIRPa libraries.
[0019] Figures 3A-3C depict ELISA results for the SIRPa in VHH Hi + VHH hShuffle library (Figure 3A), the SIRPa in NAL + SAB + DeepCDR library (Figure 3B), and the SIRPa in HI NEW library' (Figure 3C). Figure 3D shows a summary of ELISA results.
[0020] Figure 4 depicts sequencing and analysis results for all three SIRPa libraries.
[0021] Figure 5 depicts the distribution of HCDR3 length in SIRPa antibody candidates.
[0022] Figure 6A depicts an iso-affinity chart of SIRPa binders. Figure 6B-6E depicts kinetic curves of SIRPa IgG candidates. Figure 6F depicts KD values for the purified SIRPa IgG candidates.
DETAILED DESCRIPTION
[0023] The present disclosure employs, unless otherwise indicated, conventional molecular biology techniques, which are within the skill of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art.
[0024] Definitions
[0025] Throughout this disclosure, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range to the tenth of the unit of the lower limit unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.
[0026] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms "a." “an’' and “the” are intended to include the plural forms as well, unless the context clearly indicates otherw ise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.
[0027] Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers +/- 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.
[0028] Unless specifically stated, as used herein, the term “nucleic acid” encompasses double- or triple-stranded nucleic acids, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands). Nucleic acid sequences, when provided, are listed in the 5’ to 3’ direction, unless stated otherwise. Methods described herein provide for the generation of isolated nucleic acids. Methods described herein additionally provide for the generation of isolated and purified nucleic acids. A “nucleic acid” as referred to herein can comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more bases in length. Moreover, provided herein are methods for the synthesis of any number of polypeptide-segments encoding nucleotide sequences, including sequences encoding non-ribosomal peptides (NRPs), sequences encoding non-ribosomal peptidesynthetase (NRPS) modules and synthetic variants, polypeptide segments of other modular proteins, such as antibodies, polypeptide segments from other protein families, including noncoding DNA or RNA. such as regulatory sequences e.g. promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA, small nucleolar RNA derived from microRNA, or any functional or structural DNA or RNA unit of interest. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification, genomic DNA. recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. cDNA encoding for a gene or gene fragment referred herein may comprise at least one region encoding for exon sequences without an intervening intron sequence in the genomic equivalent sequence.
[0029] Antibody Libraries
[0030] Provided herein are methods, compositions, and systems for generation of antibodies. Methods, compositions, and systems described herein for the optimization of antibodies comprise a ratio-variant approach that mirror the natural diversity of antibody sequences. In some instances, libraries of optimized antibodies comprise variant antibody sequences. In some instances, the variant antibody sequences are designed comprising variant CDR regions. In some instances, the variant antibody sequences comprising variant CDR regions are generated by shuffling the natural CDR sequences in a llama, humanized, or chimeric framework. In some instances, such libraries are synthesized, cloned into expression vectors, and translation products (antibodies) evaluated for activity. In some instances, fragments of sequences are synthesized and subsequently assembled. In some instances, expression vectors are used to display and enrich desired antibodies, such as phage display. In some instances, the phage vector is a Fab phagemid vector. Selection pressures used during enrichment in some instances includes binding affinity, toxicity, immunological tolerance, stability, or other factor. Such expression vectors allow antibodies with specific properties to be selected (“panning”), and subsequent propagation or amplification of such sequences enriches the library' with these sequences. Panning rounds can be repeated any number of times, such as 1, 2, 3, 4, 5, 6, 7, or more than 7 rounds. In some instances, each round of panning involves a number of washes. In some instances, each round of panning involves at least or about 1. 2, 3, 4, 5. 6, 7, 8. 9, 10, 11, 12, 13, 14, 15, 16, or more than 16 washes.
[0031] Described herein are methods and systems of in-silico library' design. Libraries as described herein, in some instances, are designed based on a database comprising a variety' of antibody sequences. In some instances, the database comprises a plurality of variant antibody sequences against various targets. In some instances, the database comprises at least 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more than 5000 antibody sequences. An exemplary database is an iCAN database. In some instances, the database comprises naive and memory B-cell receptor sequences. In some instances, the naive and memory B-cell receptor sequences are human, mouse, or primate sequences. In some instances, the naive and memory B- cell receptor sequences are human sequences. In some instances, the database is analyzed for position specific variation. In some instances, antibodies described herein comprise position specific variations in CDR regions. In some instances, the CDR regions comprise multiple sites for variation.
[0032] Described herein are libraries comprising variation in a CDR region. In some instances, the CDR is CDR1, CDR2, or CDR3 of a variable heavy chain. In some instances, the CDR is CDR1, CDR2, or CDR3 of a variable light chain. In some instances, the libraries comprise multiple variants encoding for CDR1, CDR2, or CDR3. In some instances, the libraries as described herein encode for at least 50, 100, 200, 300, 400, 500, 1000, 1200, 1500, 1700, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more than 5000 CDR1 sequences. In some instances, the libraries as described herein encode for at least 50, 100, 200, 300, 400, 500, 1000, 1200, 1500, 1700, 2000, 2500, 3000. 3500, 4000. 4500. 5000, or more than 5000 CDR2 sequences. In some instances, the libraries as described herein encode for at least 50, 100, 200, 300, 400, 500, 1000, 1200, 1500, 1700, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more than 5000 CDR3 sequences. In-silico antibodies libraries are in some instances synthesized, assembled, and enriched for desired sequences.
[0033] Following synthesis of CDR1 variants, CDR2 variants, and CDR3 variants, in some instances, the CDR1 variants, the CDR2 variants, and the CDR3 variants are shuffled to generate a diverse library. In some instances, the diversity of the libraries generated by methods described herein have a theoretical diversity of at least or about 107, 108, 109, 1010, 1011, 1012, 101?, 1014, 1015, 1016, 1017, 1018, or more than 1018 sequences. In some instances, the library has a final library diversity’ of at least or about IO7, 108, 109, IO10, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, or more than 1018 sequences.
[0034] The germline sequences corresponding to a variant sequence may also be modified to generate sequences in a library. For example, sequences generated by methods described herein comprise at least 1, 2. 3, 4, 5, 6. 7, 8, 9. 10. 11, 12, 13, 14, 15, 16, or more than 16 mutations from the germline sequence. In some instances, sequences generated comprise no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or no more than 18 mutations from the germline sequence. In some instances, sequences generated comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15, 16, or about 18 mutations relative to the germline sequence.
[0035] Antibody Libraries [0036] Provided herein are libraries generated from methods described herein. Antibodies described herein result in improved functional activity, structural stability, expression, specificity, or a combination thereof. In some instances, the antibody is a single domain antibody. In some instances, the single domain antibody comprises one heavy chain variable domain. In some instances, the single domain antibody is a VHH antibody.
[0037] As used herein, the term “antibody” will be understood to include proteins having the characteristic two-armed, Y-shape of a typical antibody molecule as well as one or more fragments of an antibody that retain the abi 1 i ty to specifically bind to an antigen. Exemplary antibodies include, but are not limited to, a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv) (including fragments in which the VL and VH are joined using recombinant methods by a synthetic or natural linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules, including single chain Fab and scFab), a single chain antibody, a Fab fragment (including monovalent fragments comprising the VL, VH. CL, and CHI domains), a F(ab')2 fragment (including bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region), a Fd fragment (including fragments comprising the VH and CHI fragment), a Fv fragment (including fragments comprising the VL and VH domains of a single arm of an antibody), a single-domain antibody (dAb or sdAb) (including fragments comprising a VH domain), an isolated complementarity’ determining region (CDR), a diabody (including fragments comprising bivalent dimers such as two VL and VH domains bound to each other and recognizing two different antigens), a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idioty pic (anti-Id) antibody, or ab antigen-binding fragments thereof. In some instances, the libraries disclosed herein comprise nucleic acids encoding for an antibody, wherein the antibody is a Fv antibody, including Fv antibodies comprised of the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. In some embodiments, the Fv antibody consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association, and the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. In some embodiments, the six hypervariable regions confer antigen-binding specificity to the antibody. In some embodiments, a single variable domain or half of an Fv comprising only three hypervariable regions specific for an antigen, including single domain antibodies isolated from camelid animals comprising one heavy chain variable domain such as VHH antibodies or nanobodies has the ability to recognize and bind antigen. A heavy-chain variable (VHH) antibody is a type of antibody fragment comprising heavy chain variable domains. In some instances, the libraries disclosed herein comprise nucleic acids encoding for an antibody, wherein the antibody is a single-chain Fv or scFv, including antibody fragments comprising a VH, a VL, or both a VH and VL domain, wherein both domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains allowing the scFv to form the desired structure for antigen binding. In some instances, a scFv is linked to the Fc fragment or a VHH is linked to the Fc fragment (including minibodies). In some instances, the antibody comprises immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, e.g., molecules that contain an antigen binding site. Immunoglobulin molecules are of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY). class (e.g., IgG 1, IgG 2, IgG 3, IgG 4, IgA 1 and IgA 2) or subclass.
[0038] In some embodiments, libraries comprise immunoglobulins that are adapted to the species of an intended therapeutic target. Generally, these methods include “mammalization” and comprises methods for transferring donor antigen-binding information to a less immunogenic mammal antibody acceptor to generate useful therapeutic treatments. In some instances, the mammal is mouse, rat, equine, sheep, cow. primate (e.g.. chimpanzee, baboon, gorilla, orangutan, monkey), dog, cat, pig, donkey, rabbit, and human. In some instances, provided herein are libraries and methods for felinization and caninization of antibodies.
[0039] "Humanized" forms of non-human antibodies can be chimeric antibodies that contain minimal sequence derived from the non-human antibody. A humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. In some instances, these modifications are made to further refine antibody performance.
[0040] "Caninization" can comprise a method for transferring non-canine antigen-binding information from a donor antibody to a less immunogenic canine antibody acceptor to generate treatments useful as therapeutics in dogs. In some instances, caninized forms of non-canine antibodies provided herein are chimeric antibodies that contain minimal sequence derived from non-canine antibodies. In some instances, caninized antibodies are canine antibody sequences (■‘acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-canine species (“donor’" antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, nonhuman primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the canine antibody are replaced by corresponding non-canine FR residues. In some instances, caninized antibodies include residues that are not found in the recipient antibody or in the donor antibody. In some instances, these modifications are made to further refine antibody performance. The caninized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of a canine antibody. [0041] ‘Felinization” can comprise a method for transferring non-feline antigen-binding information from a donor antibody to a less immunogenic feline antibody acceptor to generate treatments useful as therapeutics in cats. In some instances, felinized forms of non-feline antibodies provided herein are chimeric antibodies that contain minimal sequence derived from non-feline antibodies. In some instances, felinized antibodies are feline antibody sequences (“acceptor” or “recipient"’ antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-feline species (“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the feline antibody are replaced by corresponding non-feline FR residues. In some instances, felinized antibodies include residues that are not found in the recipient antibody or in the donor antibody. In some instances, these modifications are made to further refine antibody performance. The felinized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of a felinized antibody. [0042] Methods as described herein may be used for generation of libraries encoding a nonimmunoglobulin. In some instances, the libraries comprise antibody mimetics. Exemplary antibody mimetics include, but are not limited to, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, atrimers, DARPins, fynomers, Kunitz domain-based proteins, monobodies, anticalins, knottins, armadillo repeat protein-based proteins, and bicyclic peptides.
[0043] Libraries described herein comprising nucleic acids encoding for an antibody comprise variations in at least one region of the antibody. Exemplary regions of the antibody for variation include, but are not limited to, a complementarity-determining region (CDR), a variable domain, or a constant domain. In some instances, the CDR is CDR1, CDR2, or CDR3. In some instances, the CDR is a heavy domain including, but not limited to. CDRH1, CDRH2, and CDRH3. In some instances, the CDR is a light domain including, but not limited to, CDRL1, CDRL2. and CDRL3. In some instances, the variable domain is variable domain, light chain (VL) or variable domain. heavy chain (VH). In some instances, the CDR1, CDR2, or CDR3 is of a variable domain, light chain (VL). CDR1, CDR2, or CDR3 of a variable domain, light chain (VL) can be referred to as CDRL1, CDRL2, or CDRL3, respectively. CDR1, CDR2, or CDR3 of a variable domain, heavy chain (VH) can be referred to as CDRH1, CDRH2, or CDRH3, respectively. In some instances, the VL domain comprises kappa or lambda chains. In some instances, the constant domain is constant domain, light chain (CL) or constant domain, heavy chain (CH).
[0044] Provided herein are libraries comprising nucleic acids encoding for an antibody comprising variation in at least one region of the antibody, wherein the region is the CDR region. In some instances, the antibody is a single domain antibody comprising one heavy chain variable domain such as a VHH antibody. In some instances, the VHH antibody comprises variation in one or more CDR regions. In some instances, the VHH libraries described herein comprise at least or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400, 2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, or CDR3. For example, the libraries comprise at least 2000 sequences of a CDR1, at least 1200 sequences for CDR2, and at least 1600 sequences for CDR3. In some instances, each sequence is non-identical.
[0045] Libraries as described herein may comprise varying lengths of a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, or combinations thereof of amino acids when translated. In some instances, the length of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, or combinations thereof of amino acids when translated is at least or about 5, 6, 7, 8, 9, 10. 11, 12, 13, 14, 15, 16, 17. 18. 19. 20. 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 amino acids.
[0046] Libraries comprising nucleic acids encoding for antibodies having variant CDR sequences as described herein comprise various lengths of amino acids when translated. In some instances, the length of each of the amino acid fragments or average length of the amino acid synthesized may be at least or about 15, 20, 25. 30. 35. 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, or more than 150 amino acids. In some instances, the length of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to 100, or 75 to 95 amino acids. In some instances, the length of the amino acid is about 22 amino acids to about 75 amino acids. In some instances, the antibodies comprise at least or about 100, 200, 300, 400. 500, 600. 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than 5000 amino acids. In some instances, the library is a VHH library. In some instances, the library is an antibody library.
[0047] Libraries as described herein encoding for a VHH antibody comprise variant CDR sequences that are shuffled to generate a library- with a theoretical diversity of at least or about 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, or more than 1018 sequences. In some instances, the library' has a final library diversity of at least or about 107, 108, 109, IO10, IO11, 1012, IO13, 1014, IO13, 1016, 1017, IO18. or more than 1018 sequences.
[0048] Libraries as described herein encoding for an antibody or immunoglobulin comprise variant CDR sequences that are shuffled to generate a library yvith a theoretical diversity of at least or about 107, 108, 109, IO10, 1011, 1012, 1013, 1014, 1015, IO16, IO17, IO18, or more than IO18 sequences. In some instances, the library has a final library diversity of at least or about IO7, 108, 109, IO10, IO11, IO12. IO13. 1014, IO15, 1016, 1017. 1018, or more than IO18 sequences.
[0049] Methods described herein provide for synthesis of libraries comprising nucleic acids encoding an antibody or immunoglobulin, wherein each nucleic acid encodes for a predetermined variant of at least one predetermined reference nucleic acid sequence. In some cases, the predetermined reference sequence is a nucleic acid sequence encoding for a protein, and the variant library' comprises sequences encoding for variation of at least a single codon such that a plurality of different variants of a single residue in the subsequent protein encoded by the synthesized nucleic acid are generated by standard translation processes. In some instances, the antibody library comprises varied nucleic acids collectively encoding variations at multiple positions. In some instances, the variant library' comprises sequences encoding for variation of at least a single codon of a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, or VH domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons of a CDRH1, CDRH2, CDRH3. CDRL1, CDRL2, CDRL3, VL, or VH domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons of framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). An exemplary number of codons for variation include, but are not limited to, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons.
[0050] In some instances, the at least one region of the antibody for variation is from heavy chain V-gene family, heavy chain D-gene family, heay'y chain J-gene family, light chain V-gene family, or light chain J-gene family. In some instances, the light chain V-gene family comprises immunoglobulin kappa (IGK) gene or immunoglobulin lambda (IGL). Exemplary regions of the antibody for variation include, but are not limited to, IGHV1-18. IGHV1-69, IGHV1-8. IGHV3-21. IGHV3-23, IGHV3-30/33m, IGHV3-28, IGHV1-69, IGHV3-74, IGHV4-39, IGHV4-59/61, IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, IGLV2-14, IGLV1-40, and IGLV3-1. In some instances, the gene is IGHV1-69, IGHV3-30, IGHV3-23, IGHV3. IGHV1-46, IGHV3-7. IGHV1, or IGHV1-8. In some instances, the gene is IGHV1-69 and IGHV3-30. In some instances, the region of the antibody for variation is IGHJ3, IGHJ6, IGHJ, IGHJ4, IGHJ5, IGHJ2. or IGH1. In some instances, the region of the antibody for variation is IGHJ3, IGHJ6, IGHJ. or IGHJ4. In some instances, the at least one region of the antibody for variation is IGHV1-69, IGHV3-23, IGKV3-20, IGKV1-39 or combinations thereof. In some instances, the at least one region of the antibody for variation is IGHV1-69 or IGHV3-23. In some instances, the at least one region of the antibody for variation is IGKV3-20 or IGKV1-39. In some instances, the at least one region of the antibody for variation is IGHV1-69 and IGKV3-20, In some instances, the at least one region of the antibody for variation is IGHV1-69 and IGKV1-39. In some instances, the at least one region of the antibody for variation is IGHV3-23 and IGKV3-20. In some instances, the at least one region of the antibody for variation is IGHV3-23 and IGKV1-39. [0051] Provided herein are libraries comprising nucleic acids encoding for antibodies, wherein the libraries are synthesized with various numbers of fragments. In some instances, the fragments comprise the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, or VH domain. In some instances, the fragments comprise framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). In some instances, the antibody libraries are synthesized with at least or about 2 fragments. 3 fragments, 4 fragments, 5 fragments, or more than 5 fragments. The length of each of the nucleic acid fragments or average length of the nucleic acids synthesized may be at least or about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, or more than 600 base pairs. In some instances, the length is about 50 to 600. 75 to 575, 100 to 550, 125 to 525, 150 to 500. 175 to 475. 200 to 450. 225 to 425, 250 to 400, 275 to 375, or 300 to 350 base pairs.
[0052] Libraries comprising nucleic acids encoding for antibodies or immunoglobulins as described herein comprise various lengths of amino acids when translated. In some instances, the length of each of the amino acid fragments or average length of the amino acid synthesized may be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70. 75. 80. 85, 90, 95, 100, 105, 110, 115. 120, 125, 130, 135, 140, 145, 150, or more than 150 amino acids. In some instances, the length of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to 100, or 75 to 95 amino acids. In some instances, the length of the amino acid is about 22 amino acids to about 75 amino acids. In some instances, the antibodies comprise at least or about 100. 200, 300, 400, 500, 600. 700, 800. 900, 1000. 2000. 3000, 4000, 5000, or more than 5000 amino acids.
[0053] A number of variant sequences for the at least one region of the antibody for variation are de novo synthesized using methods as described herein. In some instances, a number of variant sequences is de novo synthesized for CDRH1, CDRH2, CDRH3, CDRL1. CDRL2, CDRL3, VL, VH, or combinations thereof. In some instances, a number of variant sequences is de novo synthesized for framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). The number of variant sequences may be at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more than 500 sequences. In some instances, the number of variant sequences is at least or about 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000. 6000, 7000, 8000, or more than 8000 sequences. In some instances, the number of variant sequences is about 10 to 500. 25 to 475, 50 to 450, 75 to 425, 100 to 400. 125 to 375. 150 to 350, 175 to 325, 200 to 300, 225 to 375, 250 to 350, or 275 to 325 sequences.
[0054] Variant sequences for the at least one region of the antibody, in some instances, vary’ in length or sequence. In some instances, the at least one region that is de novo synthesized is for CDRH1, CDRH2. CDRH3. CDRL1, CDRL2, CDRL3, VL, VH. or combinations thereof. In some instances, the at least one region that is de novo synthesized is for framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). In some instances, the variant sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40. 45. 50, or more than 50 variant nucleotides or amino acids as compared to wild-type. In some instances, the variant sequence comprises at least or about 1, 2, 3. 4, 5, 6. 7. 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 additional nucleotides or amino acids as compared to wild-type. In some instances, the variant sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40. 45, or 50 less nucleotides or amino acids as compared to wild-type. In some instances, the libraries comprise at least or about I01, 102, 103, 104, 105, 106. 107, 108, 109. 1010, or more than 1010 variants.
[0055] Following synthesis of antibody libraries, antibody libraries may be used for screening and analysis. For example, antibody libraries are assay ed for library7 displayability and panning. In some instances, displayability is assayed using a selectable tag. Exemplary7 tags include, but are not limited to, a radioactive label, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag, an affinity tag or other labels or tags that are known in the art. In some instances, the tag is histidine, polyhistidine, myc, hemagglutinin (HA), or FLAG. In some instances, antibody libraries are assayed by sequencing using various methods including, but not limited to, singlemolecule real-time (SMRT) sequencing, Polony sequencing, sequencing by ligation, reversible terminator sequencing, proton detection sequencing, ion semiconductor sequencing, nanopore sequencing, electronic sequencing, pyrosequencing, Maxam-Gilbert sequencing, chain termination (e.g., Sanger) sequencing, +S sequencing, or sequencing by synthesis. In some instances, antibody libraries are displayed on the surface of a cell or phage. In some instances, antibody libraries are enriched for sequences with a desired activity7 using phage display. [0056] In some instances, the antibody libraries are assayed for functional activity, structural stability (e.g., thermal stable or pH stable), expression, specificity, or a combination thereof. In some instances, the antibody libraries are assayed for antibody capable of folding. In some instances, a region of the antibody is assayed for functional activity, structural stability, expression, specificity, folding, or a combination thereof. For example, a VH region or VL region is assayed for functional activity, structural stability, expression, specificity, folding, or a combination thereof. [0057] Antibodies or IgGs generated by methods as described herein comprise improved binding affinity. In some instances, the antibody comprises a binding affinity (e.g., KD) of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 11 nm, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, or less than 30 nM. In some instances, the antibody comprises a KD of less than 400 nM, less than 350 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nm, less than 100 nM, less than 50 nM, less than 25 nM, less than 15 nM, or less than 10 nM. In some instances, the antibody comprises a KD of less than 1 nM. In some instances, the antibody comprises a KD of less than 1.2 nM. In some instances, the antibody comprises a KD of less than 2 nM. In some instances, the antibody comprises a KD of less than 5 nM. In some instances, the antibody comprises a KD of less than 10 nM. In some instances, the antibody comprises a KD of less than 13.5 nM. In some instances, the antibody comprises a KD of less than 15 nM. In some instances, the antibody comprises a KD of less than 20 nM. In some instances, the antibody comprises a KD of less than 25 nM. In some instances, the antibody comprises a KD of less than 30 nM.
[0058] In some instances, the affinity of antibodies or IgGs generated by methods as described herein is at least or about 1.5x, 2. Ox, 5x, lOx, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx, 200x, or more than 200x improved binding affinity as compared to a comparator antibody. In some instances, the affinity of antibodies or IgGs generated by methods as described herein is at least or about 1.5x, 2. Ox, 5x, lOx, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx, 200x, or more than 200x improved function as compared to a comparator antibody. In some instances, the comparator antibody is an antibody with similar structure, sequence, or antigen target.
[0059] In some embodiments, the variant antibodies or IgGs generated by methods as described herein result in a decreased EC50 in a T-cell cytotoxicity assay as compared to the EC50 in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an EC50 in a T-cell cytotoxicity assay that is at least 5X decreased as compared to the EC50 in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an EC50 in a T-cell cytotoxicity assay that is at least 8X decreased as compared to the EC50 in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an EC50 in a T-cell cytotoxicity assay that is at least 10X decreased as compared to the EC50 in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an EC50 in a T-cell cytotoxicity assay that is at least 20X decreased as compared to the EC50 in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an EC50 in a T-cell cytotoxicity assay that is at least 25X decreased as compared to the EC50 in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an EC50 in a T-cell cytotoxicity assay that is at least 30X decreased as compared to the EC50 in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an EC50 in a T-cell cytotoxicity assay that is at least 40X decreased as compared to the EC50 in a T-cell cytotoxicity’ assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an EC50 in a T-cell cytotoxicity assay that is at least 50X decreased as compared to the EC50 in a T-cell cytotoxicity assay of a reference antibody or IgGs.
[0060] Methods as described herein, in some instances, result in increased yield of antibodies or IgGs. In some instances, the yield is at least or about 5, 10, 15, 20, 25, 30, 35. 40. 45. 50. 55. 60. 65, 70, 75, 80, or more than 80 micrograms (ug). In some instances, the yield is in a range of about 5 to about 80, about 10 to about 75, about 15 to about 60, about 20 to about 50, or about 30 to about 40 micrograms (ug).
[0061] Expression Systems
[0062] Provided herein are libraries comprising nucleic acids encoding for antibody comprising binding domains, wherein the libraries have improved specificity, stability, expression, folding, or downstream activity. In some instances, libraries described herein are used for screening and analysis.
[0063] Provided herein are libraries comprising nucleic acids encoding for antibody comprising binding domains, w erein the nucleic acid libraries are used for screening and analysis. In some instances, screening and analysis comprises in vitro, in vivo, or ex vivo assays. Cells for screening include primary cells taken from living subjects or cell lines. Cells may be from prokaryotes (e.g., bacteria and fungi) or eukaryotes (e.g., animals and plants). Exemplary animal cells include, without limitation, those from a mouse, rabbit, primate, and insect. In some instances, cells for screening include a cell line including, but not limited to, Chinese Hamster Ovary (CHO) cell line, human embryonic kidney (HEK) cell line, or baby hamster kidney (BHK) cell line. In some instances, nucleic acid libraries described herein may also be delivered to a multicellular organism. Exemplar^' multicellular organisms include, without limitation, a plant, a mouse, rabbit, primate, and insect.
[0064] Nucleic acid libraries described herein may be screened for various pharmacological or pharmacokinetic properties. In some instances, the libraries are screened using in vitro assays, in vivo assays, or ex vivo assays. For example, in vitro pharmacological or pharmacokinetic properties that are screened include, but are not limited to, binding affinity, binding specificity, and binding avidity. Exemplary in vivo pharmacological or pharmacokinetic properties of libraries described herein that are screened include, but are not limited to, therapeutic efficacy, activity, preclinical toxicity properties, clinical efficacy properties, clinical toxicity' properties, immunogenicity, potency, and clinical safety properties.
[0065] Provided herein are nucleic acid libraries, wherein the nucleic acid libraries may be expressed in a vector. Expression vectors for inserting nucleic acid libraries disclosed herein may comprise eukaryotic or prokaryotic expression vectors. Exemplar}' expression vectors include, without limitation, mammalian expression vectors: pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF- CMV-NEO-COOH-3XFLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV- FLAG(R)-6His. pCEP4 pDEST27. pSF-CMV-Ub-KrYFP. pSF-CMV-FMDV-daGFP, pEFla- mCherry-Nl Vector, pEFla-tdTomato Vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), and pSF-CMV-PURO-NH2-CMYC; bacterial expression vectors: pSF-OXB20- BetaGal,pSF-OXB20-Fluc, pSF-OXB20, and pSF-Tac; plant expression vectors: pRI 101-AN DNA and pCambia2301; and yeast expression vectors: pTYB21 and pKLAC2, and insect vectors: pAc5.1/V5-His A and pDEST8. In some instances, the vector is pcDNA3 or pcDNA3.1.
[0066] Described herein are nucleic acid libraries that are expressed in a vector to generate a construct comprising an antibody. In some instances, a size of the construct varies. In some instances, the construct comprises at least or about 500, 600, 700, 800. 900, 1000, 1100. 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200,4400, 4600, 4800, 5000, 6000, 7000, 8000, 9000, 10000, or more than 10000 bases. In some instances, a the construct comprises a range of about 300 to 1,000, 300 to 2,000, 300 to 3,000, 300 to 4,000, 300 to 5.000, 300 to 6,000, 300 to 7,000, 300 to 8,000, 300 to 9,000, 300 to 10,000, 1,000 to 2.000, 1,000 to 3.000, 1,000 to 4,000, 1,000 to 5,000, 1,000 to 6,000, 1.000 to 7,000, 1.000 to 8,000, 1,000 to 9,000, 1,000 to 10,000, 2,000 to 3,000, 2,000 to 4,000, 2,000 to 5,000, 2,000 to 6,000, 2,000 to 7,000, 2,000 to 8,000, 2,000 to 9,000, 2,000 to 10,000, 3,000 to 4,000, 3,000 to 5,000, 3,000 to 6,000, 3,000 to 7,000. 3,000 to 8,000, 3,000 to 9,000, 3,000 to 10,000, 4,000 to 5,000, 4,000 to 6,000. 4,000 to 7,000. 4,000 to 8.000, 4,000 to 9.000, 4,000 to 10,000. 5,000 to 6,000, 5,000 to 7,000, 5,000 to 8,000, 5,000 to 9,000, 5,000 to 10,000, 6,000 to 7,000, 6,000 to 8,000, 6,000 to 9,000, 6,000 to 10,000, 7,000 to 8,000, 7.000 to 9,000, 7,000 to 10,000, 8,000 to 9,000, 8,000 to 10,000, or 9,000 to 10,000 bases.
[0067] Provided herein are libraries comprising nucleic acids encoding for antibodies, wherein the nucleic acid libraries are expressed in a cell. In some instances, the libraries are synthesized to express a reporter gene. Exemplary reporter genes include, but are not limited to, acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), cerulean fluorescent protein, citrine fluorescent protein, orange fluorescent protein , cherry fluorescent protein, turquoise fluorescent protein, blue fluorescent protein, horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), luciferase, and derivatives thereof. Methods to determine modulation of a reporter gene are well known in the art, and include, but are not limited to, fluorometric methods (e.g. fluorescence spectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy), and antibiotic resistance determination. [0068] Diseases and Disorders
[0069] Provided herein are libraries comprising nucleic acids encoding for antibodies or immunoglobulins that may have therapeutic effects. In some instances, the antibodies or immunoglobulin result in protein when translated that is used to treat a disease or disorder in a subject. Exemplary diseases include, but are not limited to. cancer, inflammatory diseases or disorders, a metabolic disease or disorder, a cardiovascular disease or disorder, an immunodeficiency disease or disorder, a respiratory disease or disorder, pain, a digestive disease or disorder, a reproductive disease or disorder, an endocrine disease or disorder, an immune disease or disorder, an autoimmune disease or disorder, or a neurological disease or disorder. In some instances, the cancer is a solid cancer or a hematologic cancer. In some instances, the subject is a mammal. In some instances, the subject is a mouse, rabbit, dog, or human. Subjects treated by methods described herein may be infants, adults, or children. Pharmaceutical compositions comprising antibodies or antibody fragments as described herein may be administered intravenously or subcutaneously. Antibodies or antibody fragments as described herein may be used as a therapeutic agent for a disease. The therapeutic agent comprises the antibodies or antibody fragments as described herein as an active ingredient, and it may further comprise suitable excipient(s).
[0070] In some instances, the disease or disorder is associated with SIRPa dysfunction. In some instances, the disease or disorder is associated with aberrant signaling via SIRPa. In some instances, the disease or disorder is cancer. [0071] For example, SIRPa can be involved in binding to CD47. The SIRPa/CD47 interaction can lead to bidirectional signaling, resulting in different cell-to-cell responses including inhibition of phagocytosis, stimulation of cell-cell fusion, and T cell activation. Inhibition of the interaction of SIRPa to DC47 can enable phagocytosis of tumor cells. SIRPa can be phosphorylated by tyrosine kinases and can participate in signal transduction mediated by various growth factor receptors. SIRPa has also been identified as a biomarker for various cancers (e g., breast cancer, liver cancer, prostate cancer).
[0072] Provided herein are antibodies or antigen-binding fragments thereof, as described herein, for use in a method of treating a disease or disorder associated with SIRPa dysfunction in a subject in need thereof, the method comprising administering to the subject an effective amount of such antibodies or antigen-binding fragments thereof.
[0073] Also provided herein is the use of antibodies or antigen-binding fragments thereof, as described herein, in the manufacture of a therapeutic agent/medi cament for the treatment of a disease or disorder associated with SIRPa dysfunction.
[0074] Protein Targets
[0075] Provided herein are libraries comprising nucleic acids encoding for antibodies or immunoglobulins that target SIRPa, also known as signal-regulatory protein alpha, cluster of differentiation 172a (CD172a), and src homology' 2 domain-containing phosphate substrate 1. SIRPa is a membrane protein and is a negative regulator of the phosphatidylinositol 3-kinase signaling and mitogen-activated protein kinase pathways.
[0076] Provided herein are SIRPa antibodies or immunoglobulins, wherein the SIRPa antibody or immunoglobulin comprises a sequence at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity' to any one of SEQ ID NOs: 5-428. In some instances, the antibody or immunoglobulin sequence comprises at least or about 95% sequence identity to any one of SEQ ID NOs: 5-428. In some instances, the antibody or immunoglobulin sequence comprises at least or about 97% sequence identity to any one of SEQ ID NOs: 5-428. In some instances, the antibody or immunoglobulin sequence comprises at least or about 99% sequence identity to any one of SEQ ID NOs: 5-428. In some instances, the antibody or immunoglobulin sequence comprises at least or about 100% sequence identity to any one SEQ ID NOs: 5-428.
[0077] In some embodiments, the SIRPa antibody or immunoglobulin sequence comprises complementarity' determining regions (CDRs) comprising at least or about 70%, 80%, 85%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 5-181 and 241-381. In some instances, the antibody or immunoglobulin sequence comprises complementarity determining regions (CDRs) comprising at least or about 95% homology to any one of SEQ ID NOs: 5-181 and 241-381. In some instances, the antibody or immunoglobulin sequence comprises complementarity determining regions (CDRs) comprising at least or about 97% homology to any one of SEQ ID NOs: 5-181 and 241-381. In some instances, the antibody or immunoglobulin sequence comprises complementarity determining regions (CDRs) comprising at least or about 99% homology to any one of SEQ ID NOs: 5-181 and 241-381. In some instances, the antibody or immunoglobulin sequence comprises complementarity determining regions (CDRs) comprising at least or about 100% homology to any one of SEQ ID NOs: 5-181 and 241-381. In some instances, the antibody or immunoglobulin sequence comprises complementarity determining regions (CDRs) comprising at least a portion having at least or about 3, 4. 5, 6, 7, 8. 9, 10, 12, 14, 16. 17. 18. 19. 20, 21, 22, or more than 22 amino acids of any one of SEQ ID NOs: 5-181 and 241-381.
[0078] In some embodiments, the SIRPa antibody or immunoglobulin sequence comprises a heavy chain CDR1 (HCDR1) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 5-63. In some instances, the antibody or immunoglobulin sequence comprises HCDR1 comprising at least or about 95% homology of any one of SEQ ID NOs: 5-63. In some instances, the antibody or immunoglobulin sequence comprises HCDR1 comprising at least or about 97% homology to any one of SEQ ID NOs: 5-63. In some instances, the antibody or immunoglobulin sequence comprises HCDR1 comprising at least or about 99% homology to any one of SEQ ID NOs: 5-63. In some instances, the antibody or immunoglobulin sequence comprises HCDR1 comprising at least or about 100% homology to any one of SEQ ID NOs: 5-63. In some instances, the antibody or immunoglobulin sequence comprises HCDR1 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8. or more than 8 amino acids of any one of SEQ ID NOs: 5-63.
[0079] In some embodiments, the SIRPa antibody or immunoglobulin sequence comprises a heavy chain CDR2 (HCDR2) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity7 to any one of SEQ ID NOs: 64-122. In some instances, the antibody or immunoglobulin sequence comprises HCDR2 comprising at least or about 95% homology to any one of SEQ ID NOs: 64-122. In some instances, the antibody or immunoglobulin sequence comprises HCDR2 comprising at least or about 97% homology to any one of SEQ ID NOs: 64-122. In some instances, the antibody or immunoglobulin sequence comprises HCDR2 comprising at least or about 99% homology to any one of SEQ ID NOs: 64-122. In some instances, the antibody or immunoglobulin sequence comprises HCDR2 comprising at least or about 100% homology to any one of SEQ ID NOs: 64-122. In some instances, the antibody or immunoglobulin sequence comprises HCDR2 comprising at least a portion having at least or about 3, 4, 5, 6, 7. 8, 9, 10, 11, 12 or more than 12 amino acids of any one of SEQ ID NOs: 64-122. [0080] In some embodiments, the SIRPa antibody or immunoglobulin sequence comprises a heavy chain CDR3 (HCDR3) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity' to any one of SEQ ID NOs: 123-181. In some instances, the antibody or immunoglobulin sequence comprises HCDR3 comprising at least or about 95% homology to any one of SEQ ID NOs: 123-181. In some instances, the antibody or immunoglobulin sequence comprises HCDR3 comprising at least or about 97% homology to any one of SEQ ID NOs: 123-181. In some instances, the antibody or immunoglobulin sequence comprises HCDR3 comprising at least or about 99% homology to any one of SEQ ID NOs: 123-181. In some instances, the antibody or immunoglobulin sequence comprises HCDR3 comprising at least or about 100% homology to any one of SEQ ID NOs: 123-181. In some instances, the antibody or immunoglobulin sequence comprises HCDR3 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22 or more than 22 amino acids of any one of SEQ ID NOs: 123-181.
[0081] In some embodiments, the SIRPa antibody or immunoglobulin sequence comprises a light chain CDR1 (LCDR1) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 241-287. In some instances, the antibody or immunoglobulin sequence comprises LCDR1 comprising at least or about 95% homology of any one of SEQ ID NOs: 241-287. In some instances, the antibody or immunoglobulin sequence comprises LCDR1 comprising at least or about 97% homology to any one of SEQ ID NOs: 241-287. In some instances, the antibody or immunoglobulin sequence comprises HCDR1 comprising at least or about 99% homology to any one of SEQ ID NOs: 241-287. In some instances, the antibody or immunoglobulin sequence comprises LCDR1 comprising at least or about 100% homology to any one of SEQ ID NOs: 241-287. In some instances, the antibody or immunoglobulin sequence comprises LCDR1 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, or more than 8 amino acids of any one of SEQ ID NOs: 241-287.
[0082] In some embodiments, the SIRPa antibody or immunoglobulin sequence comprises a light chain CDR2 (LCDR2) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 288-334. In some instances, the antibody or immunoglobulin sequence comprises LCDR2 comprising at least or about 95% homology to any one of SEQ ID NOs: 288-334. In some instances, the antibody or immunoglobulin sequence comprises LCDR2 comprising at least or about 97% homology to any one of SEQ ID NOs: 288-334. In some instances, the antibody or immunoglobulin sequence comprises LCDR2 comprising at least or about 99% homology to any one of SEQ ID NOs: 288-334. In some instances, the antibody or immunoglobulin sequence comprises LCDR2 comprising at least or about 100% homology to any one of SEQ ID NOs: 288-334. In some instances, the antibody or immunoglobulin sequence comprises LCDR2 comprising at least a portion having at least or about 3. 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 amino acids of any one of SEQ ID NOs: 288-334.
[0083] In some embodiments, the SIRPa antibody or immunoglobulin sequence comprises a light chain CDR3 (LCDR3) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 335-381. In some instances, the antibody or immunoglobulin sequence comprises LCDR3 comprising at least or about 95% homology to any one of SEQ ID NOs: 335-381. In some instances, the antibody or immunoglobulin sequence comprises LCDR3 comprising at least or about 97% homology to any one of SEQ ID NOs: 335-381. In some instances, the antibody or immunoglobulin sequence comprises LCDR3 comprising at least or about 99% homology to any one of SEQ ID NOs: 335-381. In some instances, the antibody or immunoglobulin sequence comprises LCDR3 comprising at least or about 100% homology to any one of SEQ ID NOs: 335-381. In some instances, the antibody or immunoglobulin sequence comprises LCDR3 comprising at least a portion having at least or about 3. 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22 or more than 22 amino acids of any one of SEQ ID NOs: 335-381.
[0084] In some instances, the SIRPa antibody or immunoglobulin sequence comprises a heavy chain variable domain comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity' to any one of SEQ ID NOs: 182-240. In some instances, the SIRPa antibody or immunoglobulin sequence comprises a heavy chain variable domain comprising at least or about 95% sequence identity- to any one of SEQ ID NOs: 182-240. In some instances, the SIRPa antibody or immunoglobulin sequence comprises a heavy chain variable domain comprising at least or about 97% sequence identity to any one of SEQ ID NOs: 182-240. In some instances, the SIRPa antibody or immunoglobulin sequence comprises a heavy- chain variable domain comprising at least or about 99% sequence identity to any one of SEQ ID NOs: 182-240. In some instances, the SIRPa antibody or immunoglobulin sequence comprises a heavy chain variable domain comprising at least or about 100% sequence identity to any one of SEQ ID NOs: 182-240. In some instances, the SIRPa antibody or immunoglobulin sequence comprises a heavy chain variable domain comprising at least a portion having at least or about 1, 2. 3, 4, 5, 6, 7, 8, 9. 10. 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150. or more than 150 amino acids of any one of SEQ ID NOs: 182-240.
[0085] In some instances, the SIRPa antibody or immunoglobulin sequence comprises a light chain variable domain comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity' to any one of SEQ ID NOs: 382-428. In some instances, the SIRPa antibody or immunoglobulin sequence comprises a light chain variable domain comprising at least or about 95% sequence identity to any one of SEQ ID NOs: 382-428. In some instances, the SIRPa antibody or immunoglobulin sequence comprises a light chain variable domain comprising at least or about 97% sequence identity to any one of SEQ ID NOs: 382-428. In some instances, the SIRPa antibody or immunoglobulin sequence comprises a light chain variable domain comprising at least or about 99% sequence identity to any one of SEQ ID NOs: 382-428. In some instances, the SIRPa antibody or immunoglobulin sequence comprises a light chain variable domain comprising at least or about 100% sequence identity to any one of SEQ ID NOs: 382-428. In some instances, the SIRPa antibody or immunoglobulin sequence comprises a light chain variable domain comprising at least a portion having at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9. 10, 12, 14, 16, 18, 20. 30. 40. 50. 60. 70. 80, 90, 100, 110. 120, 130. 140, 150. or more than 150 ammo acids of any one of SEQ ID NOs: 382-428.
[0086] Variant Libraries
[0087] Codon variation
[0088] Variant nucleic acid libraries described herein may comprise a plurality of nucleic acids, wherein each nucleic acid encodes for a variant codon sequence compared to a reference nucleic acid sequence. In some instances, each nucleic acid of a first nucleic acid population contains a variant at a single variant site. In some instances, the first nucleic acid population contains a plurality of variants at a single variant site such that the first nucleic acid population contains more than one variant at the same variant site. The first nucleic acid population may comprise nucleic acids collectively encoding multiple codon variants at the same variant site. The first nucleic acid population may comprise nucleic acids collectively encoding up to 19 or more codons at the same position. The first nucleic acid population may comprise nucleic acids collectively encoding up to 60 variant triplets at the same position, or the first nucleic acid population may comprise nucleic acids collectively encoding up to 61 different triplets of codons at the same position. Each variant may encode for a codon that results in a different amino acid during translation. Table 1 provides a listing of each codon possible (and the representative amino acid) for a variant site.
Table 1. List of codons and amino acids
Figure imgf000027_0001
[0089] A nucleic acid population may comprise varied nucleic acids collectively encoding up to 20 codon variations at multiple positions. In such cases, each nucleic acid in the population comprises variation for codons at more than one position in the same nucleic acid. In some instances, each nucleic acid in the population comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more codons in a single nucleic acid. In some instances, each variant long nucleic acid comprises variation for codons at 1, 2, 3, 4. 5, 6, 7, 8, 9,
10, 11, 12, 13. 14. 15. 16. 17. 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29. 30 or more codons in a single long nucleic acid. In some instances, the variant nucleic acid population comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more codons in a single nucleic acid. In some instances, the variant nucleic acid population comprises variation for codons in at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more codons in a single long nucleic acid.
[0090] Highly Parallel Nucleic Acid Synthesis
[0091] Provided herein is a platform approach utilizing miniaturization, parallelization, and vertical integration of the end-to-end process from polynucleotide synthesis to gene assembly within nanowells on silicon to create a revolutionary synthesis platform. Devices described herein provide, with the same footprint as a 96-well plate, a silicon synthesis platform is capable of increasing throughput by a factor of up to 1,000 or more compared to traditional synthesis methods, with production of up to approximately 1,000,000 or more polynucleotides, or 10,000 or more genes in a single highly-parallelized run.
[0092] With the advent of next-generation sequencing, high resolution genomic data has become an important factor for studies that delve into the biological roles of various genes in both normal biology and disease pathogenesis. At the core of this research is the central dogma of molecular biology and the concept of “residue-by-residue transfer of sequential information A Genomic information encoded in the DNA is transcribed into a message that is then translated into the protein that is the active product within a given biological pathway.
[0093] Another exciting area of study is on the discovery, development and manufacturing of therapeutic molecules focused on a highly-specific cellular target. High diversity' DNA sequence libraries are at the core of development pipelines for targeted therapeutics. Gene mutants are used to express proteins in a design, build, and test protein engineering cycle that ideally culminates in an optimized gene for high expression of a protein with high affinity7 for its therapeutic target. As an example, consider the binding pocket of a receptor. The ability to test all sequence permutations of all residues within the binding pocket simultaneously will allow for a thorough exploration, increasing chances of success. Saturation mutagenesis, in which a researcher attempts to generate all possible mutations at a specific site within the receptor, represents one approach to this development challenge. Though costly and time and labor-intensive, it enables each variant to be introduced into each position. In contrast, combinatorial mutagenesis, where a few selected positions or short stretch of DNA may be modified extensively, generates an incomplete repertoire of variants with biased representation.
[0094] To accelerate the drug development pipeline, a library with the desired variants available at the intended frequency in the right position available for testing — in other words, a precision library7, enables reduced costs as well as turnaround time for screening. Provided herein are methods for synthesizing nucleic acid synthetic variant libraries which provide for precise introduction of each intended variant at the desired frequency. To the end user, this translates to the ability to not only thoroughly sample sequence space but also be able to query these hypotheses in an efficient manner, reducing cost and screening time. Genome-wide editing can elucidate important pathways, libraries where each variant and sequence permutation can be tested for optimal functionality7, and thousands of genes can be used to reconstruct entire pathways and genomes to re-engineer biological systems for drug discovery.
[0095] In a first example, a drug itself can be optimized using methods described herein. For example, to improve a specified function of an antibody, a variant polynucleotide library encoding for a portion of the antibody is designed and synthesized. A variant nucleic acid library for the antibody can then be generated by processes described herein (e.g., PCR mutagenesis followed by insertion into a vector). The antibody is then expressed in a production cell line and screened for enhanced activity. Example screens include examining modulation in binding affinity to an antigen, stability7, or effector function (e.g., ADCC, complement, or apoptosis). Exemplary regions to optimize the antibody include, without limitation, the Fc region, Fab region, variable region of the Fab region, constant region of the Fab region, variable domain of the heavy chain or light chain (VH or VL), and specific complementarity -determining regions (CDRs) of VH or VL.
[0096] Nucleic acid libraries synthesized by methods described herein may be expressed in various cells associated with a disease state. Cells associated with a disease state include cell lines, tissue samples, primary cells from a subject, cultured cells expanded from a subject, or cells in a model system. Exemplary model systems include, without limitation, plant and animal models of a disease state.
[0097] To identify a variant molecule associated with prevention, reduction or treatment of a disease state, a variant nucleic acid library7 described herein is expressed in a cell associated with a disease state, or one in which a cell a disease state can be induced. In some instances, an agent is used to induce a disease state in cells. Exemplary tools for disease state induction include, without limitation, a Cre/Lox recombination system, LPS inflammation induction, and streptozotocin to induce hypoglycemia. The cells associated with a disease state may be cells from a model system or cultured cells, as well as cells from a subject having a particular disease condition. Exemplary disease conditions include a bacterial, fungal, viral, autoimmune, or proliferative disorder (e.g., cancer). In some instances, the variant nucleic acid library7 is expressed in the model system, cell line, or primary cells derived from a subject, and screened for changes in at least one cellular activity. Exemplary cellular activities include, without limitation, proliferation, cycle progression, cell death, adhesion, migration, reproduction, cell signaling, energy production, oxygen utilization, metabolic activity7, and aging, response to free radical damage, or any combination thereof.
[0098] Substrates
[0099] Devices used as a surface for polynucleotide synthesis may be in the form of substrates which include, without limitation, homogenous array surfaces, patterned array surfaces, channels, beads, gels, and the like. Provided herein are substrates comprising a plurality of clusters, wherein each cluster comprises a plurality of loci that support the attachment and synthesis of polynucleotides. In some instances, substrates comprise a homogenous array surface. For example, the homogenous array surface is a homogenous plate. The term “locus7’ as used herein refers to a discrete region on a structure which provides support for polynucleotides encoding for a single predetermined sequence to extend from the surface. In some instances, a locus is on a two dimensional surface, e.g. , a substantially planar surface. In some instances, a locus is on a three- dimensional surface, e.g. . a well, microwell, channel, or post. In some instances, a surface of a locus comprises a material that is actively functionalized to attach to at least one nucleotide for polynucleotide synthesis, or preferably, a population of identical nucleotides for synthesis of a population of polynucleotides. In some instances, polynucleotide refers to a population of polynucleotides encoding for the same nucleic acid sequence. In some cases, a surface of a substrate is inclusive of one or a plurality of surfaces of a substrate. The average error rates for polynucleotides synthesized within a library described here using the systems and methods provided are often less than 1 in 1000, less than about 1 in 2000, less than about 1 in 3000 or less often without error correction.
[00100] Provided herein are surfaces that support the parallel synthesis of a plurality of polynucleotides having different predetermined sequences at addressable locations on a common support. In some instances, a substrate provides support for the synthesis of more than 50, 100, 200, 400, 600, 800. 1000, 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200.000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900.000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more non-identical polynucleotides. In some cases, the surfaces provide support for the synthesis of more than 50. 100, 200, 400, 600, 800, 1000. 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more polynucleotides encoding for distinct sequences. In some instances, at least a portion of the polynucleotides have an identical sequence or are configured to be synthesized with an identical sequence. In some instances, the substrate provides a surface environment for the grow th of polynucleotides having at least 80, 90, 100, 120, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400. 425, 450, 475, 500 bases or more.
[00101] Provided herein are methods for polynucleotide synthesis on distinct loci of a substrate, wherein each locus supports the synthesis of a population of polynucleotides. In some cases, each locus supports the synthesis of a population of polynucleotides having a different sequence than a population of polynucleotides grown on another locus. In some instances, each polynucleotide sequence is synthesized with 1, 2, 3, 4, 5. 6, 7, 8, 9 or more redundancy across different loci within the same cluster of loci on a surface for polynucleotide synthesis. In some instances, the loci of a substrate are located within a plurality of clusters. In some instances, a substrate comprises at least 10, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000. 20000, 30000, 40000, 50000 or more clusters. In some instances, a substrate comprises more than 2,000; 5,000; 10,000; 100,000; 200,000; 300,000; 400.000; 500.000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000; 1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000; 300,000; 400,000; 500,000; 600,000;
700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4.500,000; 5,000.000; or 10,000.000 or more distinct loci. In some instances, a substrate comprises about 10,000 distinct loci. The number of loci within a single cluster is varied in different instances. In some cases, each cluster includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150, 200, 300, 400, 500 or more loci. In some instances, each cluster includes about 50-500 loci. In some instances, each cluster includes about 100-200 loci. In some instances, each cluster includes about 100-150 loci. In some instances, each cluster includes about 109, 121, 130 or 137 loci. In some instances, each cluster includes about 19, 20, 61, 64 or more loci. Alternatively or in combination, polynucleotide synthesis occurs on a homogenous array surface.
[00102] In some instances, the number of distinct polynucleotides synthesized on a substrate is dependent on the number of distinct loci available in the substrate. In some instances, the density of loci within a cluster or surface of a substrate is at least or about 1, 10, 25, 50, 65, 75, 100, 130, 150, 175, 200, 300. 400, 500, 1,000 or more loci per mm2. In some cases, a substrate comprises 10-500, 25-400, 50-500, 100-500, 150-500, 10-250, 50-250, 10-200, or 50-200 mm2. In some instances, the distance between the centers of two adjacent loci within a cluster or surface is from about 10-500, from about 10-200, or from about 10-100 um. In some instances, the distance between two centers of adjacent loci is greater than about 10, 20, 30, 40, 50. 60. 70, 80, 90 or 100 um. In some instances, the distance between the centers of two adjacent loci is less than about 200. 150, 100. 80. 70, 60, 50, 40, 30, 20 or 10 um. In some instances, each locus has a width of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In some cases, each locus has a width of about 0.5-100, 0.5-50, 10-75, or 0.5-50 um.
[00103] In some instances, the density of clusters within a substrate is at least or about 1 cluster per 100 mm2, 1 cluster per 10 mm2, 1 cluster per 5 mm2, 1 cluster per 4 mm2, 1 cluster per 3 mm2, 1 cluster per 2 mm2, 1 cluster per 1 mm2, 2 clusters per 1 mm2, 3 clusters per 1 mm2, 4 clusters per 1 mm2, 5 clusters per 1 mm2, 10 clusters per 1 mm2, 50 clusters per 1 mm2 or more. In some instances, a substrate comprises from about 1 cluster per 10 mm2 to about 10 clusters per 1 mm2. In some instances, the distance between the centers of two adjacent clusters is at least or about 50. 100, 200, 500, 1000, 2000, or 5000 um. In some cases, the distance between the centers of two adjacent clusters is between about 50-100, 50-200, 50-300, 50-500, and 100-2000 um. In some cases, the distance between the centers of two adjacent clusters is between about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or 0.5-2 mm. In some cases, each cluster has a cross section of about 0.5 to about 2, about 0.5 to about 1, or about 1 to about 2 mm. In some cases, each cluster has a cross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm. In some cases, each cluster has an interior cross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.15, 1.2, 1.3, 1.4, 1.5, 1.6. 1.7, 1.8, 1.9 or 2 mm.
[00104] In some instances, a substrate is about the size of a standard 96 well plate, for example between about 100 and about 200 mm by between about 50 and about 150 mm. In some instances, a substrate has a diameter less than or equal to about 1000, 500, 450, 400, 300, 250, 200, 150, 100 or 50 mm. In some instances, the diameter of a substrate is between about 25-1000, 25-800, 25- 600. 25-500, 25-400, 25-300. or 25-200 mm. In some instances, a substrate has a planar surface area of at least about 100; 200; 500; 1,000; 2,000; 5,000; 10,000; 12,000; 15,000; 20,000; 30,000; 40,000; 50,000 mm2 or more. In some instances, the thickness of a substrate is between about 50- 2000, 50- 1000, 100-1000, 200-1000, or 250-1000 mm.
[00105] Surface materials [00106] Substrates, devices, and reactors provided herein are fabricated from any variety of materials suitable for the methods, compositions, and systems described herein. In certain instances, substrate materials are fabricated to exhibit a low level of nucleotide binding. In some instances, substrate materials are modified to generate distinct surfaces that exhibit a high level of nucleotide binding. In some instances, substrate materials are transparent to visible and/or UV light. In some instances, substrate materials are sufficiently conductive, e.g.. are able to form uniform electric fields across all or a portion of a substrate. In some instances, conductive materials are connected to an electric ground. In some instances, the substrate is heat conductive or insulated. In some instances, the materials are chemical resistant and heat resistant to support chemical or biochemical reactions, for example polynucleotide synthesis reaction processes. In some instances, a substrate comprises flexible materials. For flexible materials, materials can include, without limitation: nylon, both modified and unmodified, nitrocellulose, polypropylene, and the like. In some instances, a substrate comprises rigid materials. For rigid materials, materials can include, without limitation: glass; fuse silica; silicon, plastics (for example polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, and the like); metals (for example, gold, platinum, and the like). The substrate, solid support or reactors can be fabricated from a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, poly dimethylsiloxane (PDMS), and glass. The substrates/solid supports or the microstructures, reactors therein may be manufactured with a combination of materials listed herein or any other suitable material known in the art.
[00107] Surface Architecture
[00108] Provided herein are substrates for the methods, compositions, and systems described herein, wherein the substrates have a surface architecture suitable for the methods, compositions, and systems described herein. In some instances, a substrate comprises raised and/or lowered features. One benefit of having such features is an increase in surface area to support polynucleotide synthesis. In some instances, a substrate having raised and/or lowered features is referred to as a three-dimensional substrate. In some cases, a three-dimensional substrate comprises one or more channels. In some cases, one or more loci comprise a channel. In some cases, the channels are accessible to reagent deposition via a deposition device such as a material deposition device. In some cases, reagents and/or fluids collect in a larger well in fluid communication one or more channels. For example, a substrate comprises a plurality of channels corresponding to a plurality of loci with a cluster, and the plurality of channels are in fluid communication with one well of the cluster. In some methods, a library of polynucleotides is synthesized in a plurality of loci of a cluster. [00109] Provided herein are substrates for the methods, compositions, and systems described herein, wherein the substrates are configured for polynucleotide synthesis. In some instances, the structure is configured to allow for controlled flow and mass transfer paths for polynucleotide synthesis on a surface. In some instances, the configuration of a substrate allows for the controlled and even distribution of mass transfer paths, chemical exposure times, and/or wash efficacy during polynucleotide synthesis. In some instances, the configuration of a substrate allows for increased sweep efficiency, for example by providing sufficient volume for a growing polynucleotide such that the excluded volume by the growing polynucleotide does not take up more than 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or less of the initially available volume that is available or suitable for growing the polynucleotide. In some instances, a three-dimensional structure allows for managed flow of fluid to allow for the rapid exchange of chemical exposure. [00110] Provided herein are substrates for the methods, compositions, and systems described herein, wherein the substrates comprise structures suitable for the methods, compositions, and systems described herein. In some instances, segregation is achieved by physical structure. In some instances, segregation is achieved by differential functionalization of the surface generating active and passive regions for polynucleotide synthesis. In some instances, differential functionalization is achieved by alternating the hydrophobicity across the substrate surface, thereby creating water contact angle effects that cause beading or wetting of the deposited reagents. Employing larger structures can decrease splashing and cross-contamination of distinct polynucleotide synthesis locations with reagents of the neighboring spots. In some cases, a device, such as a material deposition device, is used to deposit reagents to distinct polynucleotide synthesis locations. Substrates having three-dimensional features are configured in a manner that allows for the synthesis of a large number of polynucleotides (e.g. , more than about 10,000) with a low error rate (e.g, less than about 1:500, 1 : 1000. 1: 1500, 1 :2,000, 1:3,000, 1 :5,000, or 1: 10,000). In some cases, a substrate comprises features with a density of about or greater than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400 or 500 features per mm 2.
[00111] A well of a substrate may have the same or different width, height, and/or volume as another well of the substrate. A channel of a substrate may have the same or different width, height, and/or volume as another channel of the substrate. In some instances, the diameter of a cluster or the diameter of a well comprising a cluster, or both, is between about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.05-1, 0.05-0.5, 0.05-0.1, 0.1-10. 0.2-10. 0.3-10, 0.4-10, 0.5-10, 0.5-5, or 0.5-2 mm. In some instances, the diameter of a cluster or well or both is less than or about 5, 4, 3.
2, 1, 0.5, 0.1, 0.09, 0.08, 0.07, 0.06, or 0.05 mm. In some instances, the diameter of a cluster or well or both is between about 1.0 and 1.3 mm. In some instances, the diameter of a cluster or well, or both is about 1. 150 mm. In some instances, the diameter of a cluster or well, or both is about 0.08 mm. The diameter of a cluster refers to clusters within a two-dimensional or three-dimensional substrate.
[00112] In some instances, the height of a well is from about 20-1000, 50-1000, 100- 1000, 200- 1000, 300-1000, 400-1000, or 500-1000 um. In some cases, the height of a well is less than about 1000, 900, 800, 700, or 600 um.
[00113] In some instances, a substrate comprises a plurality of channels corresponding to a plurality of loci within a cluster, wherein the height or depth of a channel is 5-500, 5-400, 5-300, 5- 200, 5-100, 5-50, or 10-50 um. In some cases, the height of a channel is less than 100. 80, 60, 40, or 20 um.
[00114] In some instances, the diameter of a channel, locus (e.g. , in a substantially planar substrate) or both channel and locus (e.g., in a three-dimensional substrate wherein a locus corresponds to a channel) is from about 1-1000, 1-500, 1-200, 1-100, 5-100, or 10-100 um, for example, about 90, 80, 70, 60, 50, 40, 30. 20 or 10 um. In some instances, the diameter of a channel, locus, or both channel and locus is less than about 100. 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In some instances, the distance between the center of two adjacent channels, loci, or channels and loci is from about 1-500, 1-200, 1-100, 5-200, 5-100, 5-50, or 5-30, for example, about 20 um. [00115] Surface Modifications
[00116] Provided herein are methods for polynucleotide synthesis on a surface, wherein the surface comprises various surface modifications. In some instances, the surface modifications are employed for the chemical and/or physical alteration of a surface by an additive or subtractive process to change one or more chemical and/or physical properties of a substrate surface or a selected site or region of a substrate surface. For example, surface modifications include, without limitation, (1) changing the wetting properties of a surface, (2) functionalizing a surface, i.e., providing, modifying or substituting surface functional groups, (3) defunctionalizing a surface, i.e., removing surface functional groups, (4) otherw ise altering the chemical composition of a surface, e.g, through etching, (5) increasing or decreasing surface roughness, (6) providing a coating on a surface, e.g, a coating that exhibits wetting properties that are different from the wetting properties of the surface, and/or (7) depositing particulates on a surface.
[00117] In some cases, the addition of a chemical layer on top of a surface (referred to as adhesion promoter) facilitates structured patterning of loci on a surface of a substrate. Exemplary surfaces for application of adhesion promotion include, without limitation, glass, silicon, silicon dioxide and silicon nitride. In some cases, the adhesion promoter is a chemical with a high surface energy . In some instances, a second chemical layer is deposited on a surface of a substrate. In some cases, the second chemical layer has a low surface energy. In some cases, surface energy of a chemical layer coated on a surface supports localization of droplets on the surface. Depending on the patterning arrangement selected, the proximity of loci and/or area of fluid contact at the loci are alterable.
[00118] In some instances, a substrate surface, or resolved loci, onto which nucleic acids or other moieties are deposited, e.g.. for polynucleotide synthesis, are smooth or substantially planar (e.g., two-dimensional) or have irregularities, such as raised or lowered features (e.g., three- dimensional features). In some instances, a substrate surface is modified with one or more different layers of compounds. Such modification layers of interest include, without limitation, inorganic and organic layers such as metals, metal oxides, polymers, small organic molecules and the like.
[00119] In some instances, resolved loci of a substrate are functionalized with one or more moieties that increase and/or decrease surface energy'. In some cases, a moiety is chemically inert. In some cases, a moiety is configured to support a desired chemical reaction, for example, one or more processes in a polynucleotide synthesis reaction. The surface energy, or hydrophobicity, of a surface is a factor for determining the affinity of a nucleotide to attach onto the surface. In some instances, a method for substrate functionalization comprises: (a) providing a substrate having a surface that comprises silicon dioxide; and (b) silanizing the surface using, a suitable silanizing agent described herein or otherwise known in the art. for example, an organofunctional alkoxysilane molecule. Methods and functionalizing agents are described in U.S. Patent No. 5474796, which is herein incorporated by reference in its entirety.
[00120] In some instances, a substrate surface is functionalized by contact with a derivatizing composition that contains a mixture of silanes, under reaction conditions effective to couple the silanes to the substrate surface, typically via reactive hydrophilic moieties present on the substrate surface. Silanization generally covers a surface through self-assembly with organofunctional alkoxysilane molecules. A variety' of siloxane functionalizing reagents can further be used as currently known in the art, e.g. , for lowering or increasing surface energy. The organofunctional alkoxysilanes are classified according to their organic functions.
[00121] Polynucleotide Synthesis
[00122] Methods of the current disclosure for polynucleotide synthesis may include processes involving phosphoramidite chemistry. In some instances, polynucleotide synthesis comprises coupling a base with phosphoramidite. Polynucleotide synthesis may comprise coupling a base by deposition of phosphoramidite under coupling conditions, wherein the same base is optionally deposited with phosphoramidite more than once, i.e., double coupling. Polynucleotide synthesis may comprise capping of unreacted sites. In some instances, capping is optional. Polynucleotide synthesis may also comprise oxidation or an oxidation step or oxidation steps. Polynucleotide synthesis may comprise deblocking, detritylation, and sulfurization. In some instances, polynucleotide synthesis comprises either oxidation or sulfurization. In some instances, between one or each step during a polynucleotide synthesis reaction, the device is washed, for example, using tetrazole or acetonitrile. Time frames for any one step in a phosphoramidite synthesis method may be less than about 2 min. 1 min, 50 sec, 40 sec, 30 sec, 20 sec and 10 sec.
[00123] Polynucleotide synthesis using a phosphoramidite method may comprise a subsequent addition of a phosphoramidite building block (e.g., nucleoside phosphoramidite) to a growing polynucleotide chain for the formation of a phosphite triester linkage. Phosphoramidite polynucleotide synthesis proceeds in the 3’ to 5’ direction. Phosphoramidite polynucleotide synthesis allows for the controlled addition of one nucleotide to a growing nucleic acid chain per synthesis cycle. In some instances, each synthesis cycle comprises a coupling step.
Phosphoramidite coupling involves the formation of a phosphite triester linkage between an activated nucleoside phosphoramidite and a nucleoside bound to the substrate, for example, via a linker. In some instances, the nucleoside phosphoramidite is provided to the device activated. In some instances, the nucleoside phosphoramidite is provided to the device with an activator. In some instances, nucleoside phosphoramidites are provided to the device in a 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90. 100-fold excess or more over the substrate-bound nucleosides. In some instances, the addition of nucleoside phosphoramidite is performed in an anhydrous environment, for example, in anhydrous acetonitrile. Following addition of a nucleoside phosphoramidite, the device is optionally washed. In some instances, the coupling step is repeated one or more additional times, optionally with a wash step between nucleoside phosphoramidite additions to the substrate. In some instances, a polynucleotide synthesis method used herein comprises 1, 2, 3 or more sequential coupling steps. Prior to coupling, in many cases, the nucleoside bound to the device is de-protected by removal of a protecting group, where the protecting group functions to prevent polymerization. A common protecting group is 4,4’-dimethoxytrityl (DMT).
[00124] Following coupling, phosphoramidite polynucleotide synthesis methods optionally comprise a capping step. In a capping step, the growing polynucleotide is treated with a capping agent. A capping step is useful to block unreacted substrate-bound 5 ’-OH groups after coupling from further chain elongation, preventing the formation of polynucleotides with internal base deletions. Further, phosphoramidites activated with IH-tetrazole may react, to a small extent, with the 06 position of guanosine. Without being bound by theory, upon oxidation with h /water, this side product, possibly via O6-N7 migration, may undergo depurination. The apurinic sites may end up being cleaved in the course of the final deprotection of the polynucleotide thus reducing the yield of the full-length product. The 06 modifications may be removed by treatment with the capping reagent prior to oxidation with b/water. In some instances, inclusion of a capping step during polynucleotide synthesis decreases the error rate as compared to synthesis without capping. As an example, the capping step comprises treating the substrate-bound polynucleotide with a mixture of acetic anhydride and 1 -methylimidazole. Following a capping step, the device is optionally washed.
[00125] In some instances, following addition of a nucleoside phosphoramidite, and optionally after capping and one or more wash steps, the device bound growing nucleic acid is oxidized. The oxidation step comprises the phosphite triester is oxidized into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester intemucleoside linkage. In some instances, oxidation of the growing polynucleotide is achieved by treatment with iodine and water, optionally in the presence of a weak base (e g., pyridine, lutidine, collidine). Oxidation may be carried out under anhydrous conditions using, e.g. tert-Butyl hydroperoxide or (1 S)-(+)- (lO-camphorsulfonyl)-oxaziridine (CSO). In some methods, a capping step is performed following oxidation. A second capping step allows for device drying, as residual water from oxidation that may persist can inhibit subsequent coupling. Following oxidation, the device and growing polynucleotide is optionally washed. In some instances, the step of oxidation is substituted with a sulfurization step to obtain polynucleotide phosphorothioates, wherein any capping steps can be performed after the sulfurization. Many reagents are capable of the efficient sulfur transfer, including but not limited to 3-(Dimethylaminomethylidene)amino)-3H-l,2,4-dithiazole-3-thione, DDTT, 3H-l,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent, and N,N,N'N'- Tetraethylthiuram disulfide (TETD).
[00126] In order for a subsequent cycle of nucleoside incorporation to occur through coupling, the protected 5’ end of the device bound growing polynucleotide is removed so that the primary hydroxyl group is reactive with a next nucleoside phosphoramidite. In some instances, the protecting group is DMT and deblocking occurs with trichloroacetic acid in dichloromethane. Conducting detritylation for an extended time or with stronger than recommended solutions of acids may lead to increased depurination of solid support-bound polynucleotide and thus reduces the yield of the desired full-length product. Methods and compositions of the disclosure described herein provide for controlled deblocking conditions limiting undesired depurination reactions. In some instances, the device bound polynucleotide is washed after deblocking. In some instances, efficient washing after deblocking contributes to synthesized polynucleotides having a low error rate.
[00127] Methods for the synthesis of polynucleotides typically involve an iterating sequence of the following steps: application of a protected monomer to an actively functionalized surface (e.g, locus) to link with either the activated surface, a linker or with a previously deprotected monomer; deprotection of the applied monomer so that it is reactive with a subsequently applied protected monomer; and application of another protected monomer for linking. One or more intermediate steps include oxidation or sulfurization. In some instances, one or more wash steps precede or follow one or all of the steps.
[00128] Methods for phosphoramidite-based polynucleotide synthesis comprise a series of chemical steps. In some instances, one or more steps of a synthesis method involve reagent cycling, where one or more steps of the method comprise application to the device of a reagent useful for the step. For example, reagents are cycled by a series of liquid deposition and vacuum drying steps. For substrates comprising three-dimensional features such as wells, microwells, channels and the like, reagents are optionally passed through one or more regions of the device via the wells and/or channels.
[00129] Methods and systems described herein relate to polynucleotide synthesis devices for the synthesis of polynucleotides. The synthesis may be in parallel. For example, at least or about at least 2, 3, 4, 5, 6, 7, 8. 9, 10, 11, 12, 13. 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40. 45, 50, 100. 150, 200. 250, 300, 350, 400, 450. 500, 550. 600, 650, 700, 750, 800. 850, 900, 1000, 10000, 50000, 75000, 100000 or more polynucleotides can be synthesized in parallel. The total number polynucleotides that may be synthesized in parallel may be from 2-100000, 3-50000, 4- 10000, 5-1000, 6-900, 7-850, 8-800, 9-750, 10-700, 11-650, 12-600, 13-550, 14-500, 15-450, 16- 400, 17-350, 18-300, 19-250, 20-200, 21-150,22-100, 23-50. 24-45, 25-40, 30-35. Those of skill in the art appreciate that the total number of polynucleotides synthesized in parallel may fall within any range bound by any of these values, for example 25-100. The total number of polynucleotides synthesized in parallel may fall within any range defined by any of the values serving as endpoints of the range. Total molar mass of polynucleotides synthesized within the device or the molar mass of each of the polynucleotides may be at least or at least about 10, 20, 30, 40, 50, 100. 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 25000, 50000, 75000, 100000 picomoles, or more. The length of each of the polynucleotides or average length of the polynucleotides within the device may be at least or about at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 100. 150, 200. 300, 400, 500 nucleotides, or more. The length of each of the polynucleotides or average length of the polynucleotides within the device may be at most or about at most 500, 400, 300, 200, 150. 100, 50, 45, 35, 30, 25, 20, 19, 18. 17. 16, 15, 14, 13, 12, 11, 10 nucleotides, or less. The length of each of the polynucleotides or average length of the polynucleotides within the device may fall from 10-500, 9-400, 11-300, 12-200, 13-150, 14-100, 15-50, 16-45, 17-40, 18-35, 19-25. Those of skill in the art appreciate that the length of each of the polynucleotides or average length of the polynucleotides within the device may fall within any range bound by any of these values, for example 100-300. The length of each of the polynucleotides or average length of the polynucleotides within the device may fall within any range defined by any of the values serving as endpoints of the range.
[00130] Methods for polynucleotide synthesis on a surface provided herein allow for synthesis at a fast rate. As an example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. 26. 27. 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, 200 nucleotides per hour, or more are synthesized. Nucleotides include adenine, guanine, thymine, cytosine, uridine building blocks, or analogs/modified versions thereof. In some instances, libraries of polynucleotides are synthesized in parallel on substrate. For example, a device comprising about or at least about 100; 1,000; 10.000; 30,000; 75,000; 100,000; 1,000,000; 2,000.000; 3,000,000;
4,000.000; or 5.000.000 resolved loci is able to support the synthesis of at least the same number of distinct polynucleotides, wherein polynucleotide encoding a distinct sequence is synthesized on a resolved locus. In some instances, a library of polynucleotides is synthesized on a device with low error rates described herein in less than about three months, two months, one month, three weeks, 15, 14, 13, 12. 11. 10. 9, 8, 7, 6. 5, 4, 3. 2 days. 24 hours or less. In some instances, larger nucleic acids assembled from a polynucleotide library synthesized with low error rate using the substrates and methods described herein are prepared in less than about three months, two months, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours or less.
[00131] In some instances, methods described herein provide for generation of a library’ of nucleic acids comprising variant nucleic acids differing at a plurality of codon sites. In some instances, a nucleic acid may have 1 site, 2 sites, 3 sites, 4 sites, 5 sites, 6 sites, 7 sites, 8 sites, 9 sites, 10 sites, 11 sites, 12 sites, 13 sites, 14 sites, 15 sites, 16 sites, 17 sites 18 sites, 19 sites, 20 sites, 30 sites, 40 sites, 50 sites, or more of variant codon sites.
[00132] In some instances, the one or more sites of variant codon sites may be adjacent. In some instances, the one or more sites of variant codon sites may not be adjacent and separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more codons.
[00133] In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein all the variant codon sites are adjacent to one another, forming a stretch of variant codon sites. In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein none the variant codon sites are adjacent to one another. In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein some the variant codon sites are adjacent to one another, forming a stretch of variant codon sites, and some of the variant codon sites are not adjacent to one another.
[00134] Referring to the Figures, FIG. 1 illustrates an exemplary7 process workflow for synthesis of nucleic acids (e.g., genes) from shorter nucleic acids. The workflow is divided generally into phases: (1) de novo synthesis of a single stranded nucleic acid library, (2) joining nucleic acids to form larger fragments, (3) error correction, (4) quality control, and (5) shipment. Prior to de novo synthesis, an intended nucleic acid sequence or group of nucleic acid sequences is preselected. For example, a group of genes is preselected for generation.
[00135] Once large nucleic acids for generation are selected, a predetermined library of nucleic acids is designed for de novo synthesis. Various suitable methods are known for generating high density polynucleotide arrays. In the workflow example, a device surface layer is provided. In the example, chemistry of the surface is altered in order to improve the polynucleotide synthesis process. Areas of low surface energy are generated to repel liquid while areas of high surface energy- are generated to attract liquids. The surface itself may be in the form of a planar surface or contain variations in shape, such as protrusions or microwells which increase surface area. In the workflow example, high surface energy molecules selected serve a dual function of supporting DNA chemistry, as disclosed in International Patent Application Publication WO/2015/021080. which is herein incorporated by reference in its entirety7.
[00136] In situ preparation of polynucleotide arrays is generated on a solid support and utilizes single nucleotide extension process to extend multiple oligomers in parallel. A deposition device, such as a material deposition device, is designed to release reagents in a step wise fashion such that multiple polynucleotides extend, in parallel, one residue at a time to generate oligomers with a predetermined nucleic acid sequence 102. In some instances, polynucleotides are cleaved from the surface at this stage. Cleavage includes gas cleavage, e.g., with ammonia or methylamine.
[00137] The generated polynucleotide libraries are placed in a reaction chamber. In this exemplary workflow, the reaction chamber (also referred to as “nanoreactor”) is a silicon coated well, containing PCR reagents and lowered onto the polynucleotide library 103. Prior to or after the sealing 104 of the polynucleotides, a reagent is added to release the polynucleotides from the substrate. In the exemplary workflow, the polynucleotides are released subsequent to sealing of the nanoreactor 105. Once released, fragments of single stranded polynucleotides hybridize in order to span an entire long range sequence of DNA. Partial hybridization 105 is possible because each synthesized polynucleotide is designed to have a small portion overlapping with at least one other polynucleotide in the pool.
[00138] After hybridization, a PCA reaction is commenced. During the polymerase cycles, the polynucleotides anneal to complementary fragments and gaps are fdled in by a polymerase. Each cycle increases the length of various fragments randomly depending on which polynucleotides find each other. Complementarity amongst the fragments allows for forming a complete large span of double stranded DNA 106.
[00139] After PCA is complete, the nanoreactor is separated from the device 107 and positioned for interaction with a device having primers for PCR 108. After sealing, the nanoreactor is subject to PCR 109 and the larger nucleic acids are amplified. After PCR 110, the nanochamber is opened 111. error correction reagents are added 112, the chamber is sealed 113 and an error correction reaction occurs to remove mismatched base pairs and/or strands with poor complementarity from the double stranded PCR amplification products 114. The nanoreactor is opened and separated 115. Error corrected product is next subject to additional processing steps, such as PCR and molecular bar coding, and then packaged 122 for shipment 123.
[00140] In some instances, quality control measures are taken. After error correction, quality control steps include for example interaction with a wafer having sequencing primers for amplification of the error corrected product 116, sealing the wafer to a chamber containing error corrected amplification product 117, and performing an additional round of amplification 118. The nanoreactor is opened 119 and the products are pooled 120 and sequenced 121. After an acceptable quality control determination is made, the packaged product 122 is approved for shipment 123. [00141] In some instances, a nucleic acid generated by a workflow such as that in FIG. 1 is subject to mutagenesis using overlapping primers disclosed herein. In some instances, a library7 of primers are generated by in situ preparation on a solid support and utilize single nucleotide extension process to extend multiple oligomers in parallel. A deposition device, such as a material deposition device, is designed to release reagents in a step wise fashion such that multiple polynucleotides extend, in parallel, one residue at a time to generate oligomers with a predetermined nucleic acid sequence 102.
[00142] The following examples are set forth to illustrate more clearly the principle and practice of embodiments disclosed herein to those skilled in the art and are not to be construed as limiting the scope of any claimed embodiments. Unless otherwise stated, all parts and percentages are on a weight basis.
EXAMPLES [00143] The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.
[00144] Example 1: Functionalization of a device surface
[00145] A device was functionalized to support the attachment and synthesis of a library of polynucleotides. The device surface w as first w et cleaned using a piranha solution comprising 90% H2SO4 and 10% H2O2 for 20 minutes. The device was rinsed in several beakers with DI water, held under a DI water gooseneck faucet for 5 min. and dried with N2. The device was subsequently soaked in NH4OH (1 : 100; 3 mL:300 mL) for 5 min, rinsed with DI water using a handgun, soaked in three successive beakers with DI w ater for 1 min each, and then rinsed again with DI w ater using the handgun. The device was then plasma cleaned by exposing the device surface to O2. A SAMCO PC-300 instrument was used to plasma etch O2 at 250 watts for 1 min in downstream mode.
[00146] The cleaned device surface was actively functionalized with a solution comprising N-(3- triethoxysilylpropyl)-4-hydroxybutyramide using a YES-1224P vapor deposition oven system with the following parameters: 0.5 to 1 torr, 60 min, 70 °C, 135 °C vaporizer. The device surface was resist coated using a Brew er Science 200X spin coater. SPR™ 3612 photoresist was spin coated on the device at 2500 rpm for 40 sec. The device was pre-baked for 30 min at 90 °C on a Brewer hot plate. The device was subjected to photolithography using a Karl Suss MA6 mask aligner instrument. The device was exposed for 2.2 sec and developed for 1 min in MSF 26A. Remaining developer was rinsed with the handgun and the device soaked in water for 5 min. The device was baked for 30 min at 100 °C in the oven, followed by visual inspection for lithography defects using a Nikon L200. A descum process was used to remove residual resist using the SAMCO PC-300 instrument to O2 plasma etch at 250 watts for 1 min.
[00147] The device surface w as passively functionalized with a 100 pL solution of perfluorooctyltri chlorosilane mixed with 10 pL light mineral oil. The device w as placed in a chamber, pumped for 10 min. and then the valve was closed to the pump and left to stand for 10 min. The chamber was vented to air. The device was resist stripped by performing two soaks for 5 min in 500 mL NMP at 70 °C with ultrasonication at maximum power (9 on Crest system). The device w as then soaked for 5 min in 500 mL isopropanol at room temperature with ultrasonication at maximum power. The device was dipped in 300 mL of 200 proof ethanol and blown dry with N2. The functionalized surface was activated to serve as a support for polynucleotide synthesis. [00148] Example 2: Synthesis of a 50-mer sequence on an oligonucleotide synthesis device [00149] A two-dimensional oligonucleotide synthesis device was assembled into a flowcell, which was connected to a flowcell (Applied Biosystems (ABI394 DNA Synthesizer"). The two- dimensional oligonucleotide synthesis device was uniformly functionalized with N-(3- TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest) was used to sy nthesize an exemplary polynucleotide of 50 bp ("50-mer polynucleotide") using polynucleotide synthesis methods described herein.
[00150] The sequence of the 50-mer was as described below: 5'AGACAATCAACCATTTGGGGTGGACAGCCTTGACCTCTAGACTTCGGCAT##TTTTTTT TTT3' (SEQ ID NO: 1), where # denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP -2244 from ChemGenes), which is a cleavable linker enabling the release of oligos from the surface during deprotection.
[00151] The synthesis was done using standard DNA synthesis chemistry (coupling, capping, oxidation, and deblocking) according to the protocol in Table 2 and an ABI synthesizer.
Table 2: Synthesis protocols
Figure imgf000044_0001
Figure imgf000045_0001
[00152] The phosphorami dite/activator combination was delivered similar to the delivery' of bulk reagents through the flowcell. No drying steps were performed as the environment stays "wet" with reagent the entire time. [00153] The flow restrictor was removed from the ABI 394 synthesizer to enable faster flow. Without flow restrictor, flow rates for amidites (0. IM in ACN), Activator. (0.25M Benzoylthiotetrazole ("BTT"; 30-3070-xx from GlenResearch) in ACN), and Ox (0.02M 12 in 20% pyridine, 10% water, and 70% THF) were roughly ~100uL/sec, for acetonitrile ("ACN") and capping reagents (1 :1 mix of CapA and CapB, wherein CapA is acetic anhydride in THF/Pyridine and CapB is 16% 1-methylimidizole in THF), roughly ~200uL/sec, and for Deblock (3% dichloroacetic acid in toluene), roughly ~300uL/sec (compared to ~50uL/sec for all reagents with flow restrictor). The time to completely push out Oxidizer was observed, the timing for chemical flow times was adjusted accordingly and an extra ACN wash was introduced between different chemicals. After polynucleotide synthesis, the chip was deprotected in gaseous ammonia overnight at 75 psi. Five drops of water were applied to the surface to recover polynucleotides. The recovered polynucleotides were then analyzed on a BioAnalyzer small RNA chip.
[00154] Example 3: Synthesis of a 100-mer sequence on an oligonucleotide synthesis device [00155] The same process as described in Example 2 for the synthesis of the 50-mer sequence was used for the synthesis of a 100-mer polynucleotide ("100-mer polynucleotide"; 5' CGGGATCCTTATCGTCATCGTCGTACAGATCCCGACCCATTTGCTGTCCACCAGTCATG CTAGCCATACCATGATGATGATGATGATGAGAACCCCGCAT##TTTTTTTTTT3' (SEQ ID NO: 2) , where # denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 from ChemGenes) on two different silicon chips, the first one uniformly functionalized with N-(3- TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE and the second one functionalized with 5/95 mix of 11 -acetoxyundecyltriethoxysilane and n-decyltri ethoxysilane, and the polynucleotides extracted from the surface were analyzed on a BioAnalyzer instrument.
[00156] All ten samples from the two chips were further PCR amplified using a forward (5'ATGCGGGGTTCTCATCATC3') (SEQ ID NO:3) and a reverse (5'CGGGATCCTTATCGTCATCG3') (SEQ ID NO:4) primer in a 50uL PCR mix (25uL NEB Q5 mastermix, 2.5uL lOuM Forward primer, 2.5uL lOuM Reverse primer, luL polynucleotide extracted from the surface, and water up to 50uL) using the following thermalcycling program: 98 °C, 30 sec 98 °C, 10 sec; 63 °C, 10 sec; 72 °C, 10 sec; repeat 12 cycles 72 °C, 2min
[00157] The PCR products were also run on a BioAnalyzer, demonstrating sharp peaks at the 100-mer position. Next, the PCR amplified samples were cloned, and Sanger sequenced. Table 3 summarizes the results from the Sanger sequencing for samples taken from spots 1-5 from chip 1 and for samples taken from spots 6-10 from chip 2. Table 3: Sequencing results
Figure imgf000047_0001
[00158] Thus, the high quality and uniformity of the synthesized polynucleotides were repeated on two chips with different surface chemistries. Overall, 89% of the 100-mers that were sequenced were perfect sequences with no errors, corresponding to 233 out of 262.
[00159] Table 4 summarizes error characteristics for the sequences obtained from the polynucleotide samples from spots 1-10.
Table 4: Error characteristics
Figure imgf000047_0002
Figure imgf000048_0001
[00160] Example 4: Antibody Libraries
[00161] Synthetic antibody libraries were developed, and SIRPa candidates were panned with three mixed libraries (FIG. 2). Phage ELISA conditions were antigen/BSA > 3.0. (FIGs. 3A-3D). The SIRPa in VHH Hi + VHH hShuffle library had a positive rate of 100% / 97% (FIG. 3A). The SIRPa in NAL + SAB + DeepCDR library had a positive rate of 100% / 97% (FIG. 3B). The SIRPa in HI NEW library had a positive rate of 97% / 100% (FIG. 3C).
[00162] Sequencing was performed on antibody candidates for SIRPa. 61 unique hits with 36 unique HCDR3s were identified across the three libraries (FIG. 4 and FIG. 5).
[00163] Top SIRPa candidates were reformatted into Fc/IgG antibodies. Kinetics studies were performed using Carterra SPR. In the lawn capture experiment, the antibody was coupled to a HC30M chip, with 0-500 nM antigen flow over the chip; HBSTE + 0.5% BSA run buffer; and had a 1: 1 binding fit in Carterra Kinetics software (FIGs. 6A-6F). Most binders were found to be between 10-100 nM (FIG. 6A).
[00164] Example 5. Exemplary Sequences
[00165] Sequences for SIRPa antibodies or immunoglobulins are seen in Table 5.
Table 5. SIRPa sequences
Figure imgf000048_0002
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
[00166] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.
[00167] ASPECTS OF THE INVENTION
[00168] The invention may be according to the following aspects.
[00169] Aspect 1. An antibody or antibody fragment comprising an amino acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 5-428.
[00170] Aspect 2. The antibody or antibody fragment of aspect 1, wherein the antibody or antibody fragment comprises an amino acid sequence at least about 95% identical to that set forth in any one of SEQ ID NOs: 5-428.
[00171] Aspect 3. The antibody or antibody fragment of aspect 1, wherein the antibody or antibody fragment comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 5- 428.
[00172] Aspect 4. The antibody or antibody fragment of any one of aspects 1-Error! Reference source not found., wherein the antibody is a monoclonal antibody, a polyclonal antibody, a bispecific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody. a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof.
[00173] Aspect 5. The antibody or antibody fragment of any one of aspects 1 -Error! Reference source not found., wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 75 nM.
[00174] Aspect 6. The antibody or antibody fragment of any one of aspects 1 -Error! Reference source not found., wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 50 nM.
[00175] Aspect 7. The antibody or antibody fragment of any one of aspects 1 -Error! Reference source not found., wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 25 nM.
[00176] Aspect 8. The antibody or antibody fragment of any one of aspects 1 -Error! Reference source not found., wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 10 nM.
[00177] Aspect 9. An antibody or antibody fragment that binds SIRPa, comprising an immunoglobulin heavy chain compnsing an ammo acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 182-240.
[00178] Aspect 10. The antibody or antibody fragment of aspect Error! Reference source not found., wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 95% identical to that set forth in any one of SEQ ID NOs: 182-240.
[00179] Aspect 1 1. The antibody or antibody fragment of aspect Error! Reference source not found., wherein the immunoglobulin heavy chain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 182-240.
[00180] Aspect 12. The antibody or antibody fragment of any one of claims Error! Reference source not found.-l l, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a bispecific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof.
[00181] Aspect 13. The antibody or antibody fragment of any one of aspects Error! Reference source not found.- 12, wherein the antibody or antibody fragment thereof is chimeric or humanized. [00182] Aspect 14. The antibody or antibody fragment of any one of aspects Error! Reference source not found.-13, wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 75 nM.
[00183] Aspect 15. The antibody or antibody fragment of any one of aspects Error! Reference source not found.-14, wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 50 nM.
[00184] Aspect 16. The antibody or antibody fragment of any one of aspects Error! Reference source not found.-15, wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 25 nM.
[00185] Aspect 17. The antibody or antibody fragment of any one of aspects Error! Reference source not found.- 16, wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 10 nM.
[00186] Aspect 18. An antibody or antibody fragment that binds SIRPa, comprising an immunoglobulin light chain comprising an amino acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 382-428.
[00187] Aspect 19. The antibody or antibody fragment of aspect 18, wherein the immunoglobulin light chain comprises an amino acid sequence at least about 95% identical to that set forth in any one of SEQ ID NOs: 382-428.
[00188] Aspect 20. The antibody or antibody fragment of aspect 18, wherein the immunoglobulin light chain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 382-428.
[00189] Aspect 21. The antibody or antibody fragment of any one of aspects 18-20, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity7 determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof.
[00190] Aspect 22. The antibody or antibody fragment of any one of aspects 18-21, wherein the antibody or antibody fragment thereof is chimeric or humanized.
[00191] Aspect 23. The antibody or antibody fragment of any one of aspects 18-22, wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 75 nM. [00192] Aspect 24. The antibody or antibody fragment of any one of aspects 18-23, wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 50 nM.
[00193] Aspect 25. The antibody or antibody fragment of any one of aspects 18-24, wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 25 nM.
[00194] Aspect 26. The antibody or antibody fragment of any one of aspects 18-25, wherein the antibody or antibody fragment binds to SIRPa with a KD of less than 10 nM.
[00195] Aspect 27. A method of treating a disease comprising administering the antibody or antibody fragment of any one of aspects 1-26.
[00196] Aspect 28. The method of aspect 27, wherein the disease is cancer.
[00197] Aspect 29. An antibody or antigen-binding fragment thereof that binds SIRPa, wherein the antibody or antigen-binding fragment thereof comprises the following combinations of complementary determining regions (CDR) sequences (or a functional variant thereof having one, two or three amino acid substitutions with respect to each amino acid sequence):
Table 6. SIRPa sequences combination
Figure imgf000062_0001
Figure imgf000063_0001
[00198] Aspect 30. An antibody or antigen-binding fragment thereof that binds SIRPa, wherein the antibody or antigen-binding fragment thereof comprises the following combinations of complementary determining regions (CDR) sequences (or a functional variant thereof having one, two or three amino acid substitutions with respect to each amino acid sequence):
Table 7. SIRPa sequences combinations
Figure imgf000063_0002
[00199] Aspect 31. The antibody or antigen-binding fragment thereof of aspect 30, wherein the antibody or antigen-binding fragment thereof is a VHH antibody. [00200] Aspect 32. An antibody or antigen-binding fragment thereof that binds SIRPa, wherein the antibody or antigen-binding fragment thereof comprises the following combinations of variable domain, heavy chain (VH) and variable domain, light chain (VL) sequences (or a functional variant thereof having one, two or three amino acid substitutions with respect to each amino acid sequence):
Table 8. SIRPa sequences combinations
Figure imgf000064_0001
Figure imgf000065_0001
[00201] Aspect 33. An antibody or antigen-binding fragment thereof that binds SIRPa. wherein the antibody or antigen-binding fragment thereof is a VHH antibody comprising the variable domain, heavy chain region (VH) sequences (or a functional variant thereof having one, two or three amino acid substitutions with respect to each amino acid sequence) of any one of SEQ ID NOs: 229-240.

Claims

1. An antibody, or antigen-binding fragment thereof, that binds SIRPa, wherein the antibody or fragment comprises one of the following complementary determining region (CDR) sets: a) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 5,
64 and 123, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 241, 288 and 335, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; b) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 6,
65 and 124, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 242, 289 and 336, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; c) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 7,
66 and 125, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 243, 290 and 337, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; d) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 8,
67 and 126, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 244, 291 and 338, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; e) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 9,
68 and 127, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 245, 292 and 339, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; f) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 10.
69 and 128, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 246, 293. and 340, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; g) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 11.
70 and 129, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 247, 294 and 341, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; h) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 12,
71 and 130, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 248, 295 and 342, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; i) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 13,
72 and 131, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 249, 296 and 343, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; j) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 14.
73 and 132, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 250, 297 and 344, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; k) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 15,
74 and 133, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 251, 298 and 345, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; l) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 16,
75 and 134, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 252, 299 and 346, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; m) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 17,
76 and 135, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 253, 300 and 347, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; n) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 18,
77 and 136, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 254, 301 and 348, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; o) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 19,
78 and 137, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 255, 302 and 349, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; p) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 20,
79 and 138, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 256, 303 and 350, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; q) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 21,
80 and 139, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 257, 304 and 351, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; r) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 22,
81 and 140, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 258, 305 and 352, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; s) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 23,
82 and 141, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 259, 306 and 353, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; t) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 24,
83 and 142, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 260, 307 and 354, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; u) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 25,
84 and 143, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 261, 308 and 355, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; v) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 26,
85 and 144, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 262, 309 and 356, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; w) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 27,
86 and 145, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 263, 310 and 357, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; x) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 28 . 87 and 146, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 264, 311 and 358, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; y) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 29,
88 and 147, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 265, 312 and 359, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; z) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 30,
89 and 148, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 266, 313 and 360, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; aa) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 31.
90 and 149, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 267, 314 and 361, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; bb) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 32,
91 and 150, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 268, 315 and 362, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; cc) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 33,
92 and 151, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 269, 316 and 363, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; dd) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 34.
93 and 152, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 270, 317 and 364, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; ee) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 35,
94 and 153, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 271, 318 and 365, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; ff) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 36,
95 and 154, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 272, 319 and 366, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; gg) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 37,
96 and 155, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 273, 320 and 367, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; hh) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 38,
97 and 156, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 274, 321 and 368, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; ii) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 39,
98 and 157, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 275, 322 and 369, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; jj) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 40,
99 and 158, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 276, 323 and 370, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; kk) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 41,
100 and 159, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 277, 324 and 371, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences;
11) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 42,
101 and 160, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 278, 325 and 372, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; mm) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 43,
102 and 161, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 279, 326 and 373, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; nn) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 44,
103 and 162, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 280, 327 and 374, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; oo) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 45,
104 and 163, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 281, 328 and 375, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; pp) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 46,
105 and 164, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 282, 329 and 376, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; qq) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 47,
106 and 165, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 283, 330 and 377, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; rr) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 48,
107 and 166, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 284, 331 and 378, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; ss) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 49,
108 and 167, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 285, 332 and 379, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; tt) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 50,
109 and 168, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 286, 333 and 380, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; uu) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequence of SEQ ID NO: 51. 110 and 169, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences and light chain CDRs 1-3 (CDRL1-3) comprising the amino acid sequence of SEQ ID NO: 287, 334 and 381, respectively, or functional variants thereof having one or two amino acid substitutions with respect to these amino acid sequences; vv) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequences of SEQ ID NO: 5, 190 and 375, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences; ww) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequences of SEQ ID NO:
52, 111 and 170, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences; xx) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequences of SEQ ID NO:
53, 112 and 171, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences; yy) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequences of SEQ ID NO:
54, 113 and 172, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences; zz) heavy chain CDRs 1 -3 (CDRH1 -3) comprising the amino acid sequences of SEQ ID NO:
55, 114 and 173, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences; aaa) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequences of SEQ ID NO:
56, 115 and 174, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences; bbb) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequences of SEQ ID NO:
57, 116 and 175, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences; ccc) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequences of SEQ ID NO:
58, 117 and 176, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences; ddd) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequences of SEQ ID NO:
59, 118 and 177, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences; eee) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequences of SEQ ID NO:
60, 119 and 178, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences; fff) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequences of SEQ ID NO:
61, 120 and 179, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences; ggg) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequences of SEQ ID NO:
62, 121 and 180, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences; or hhh) heavy chain CDRs 1-3 (CDRH1-3) comprising the amino acid sequences of SEQ ID NO:
63, 122 and 181, respectively, or functional variants thereof having one or two amino acid substitutions with respect to such amino acid sequences.
2. An antibody, or antigen-binding fragment thereof, that binds SIRPa, wherein the antibody or fragment comprises a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to any one of SEQ ID NO: 229-240.
3. An antibody, or antigen-binding fragment thereof, that binds SIRPa. wherein the antibody or fragment comprises one of the following heavy chain (VH) and light chain (VL) sets: a) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to any one of SEQ ID NO: 229-240; b) a VH comprising an amino acid sequence at least 90%. at least 95% identical or 100% identical to SEQ ID NO: 182 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 382; c) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 183 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 383; d) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 184 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 384; e) a VH comprising an amino acid sequence at least 90%. at least 95% identical or 100% identical to SEQ ID NO: 185 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 385; f) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 186 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 386; g) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 187 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 387; h) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 188 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 388; i) a VH comprising an amino acid sequence at least 90%. at least 95% identical or 100% identical to SEQ ID NO: 189 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 389; j) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 190 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 390; k) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 191 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 391; l) a VH comprising an amino acid sequence at least 90%. at least 95% identical or 100% identical to SEQ ID NO: 192 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 392; m) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 193 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 393; n) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 194 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 394; o) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 195 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 395; p) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 196 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 396; q) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 197 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 397; r) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 198 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 398; s) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 199 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 399; t) a VH comprising an amino acid sequence at least 90%. at least 95% identical or 100% identical to SEQ ID NO: 200 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 400; u) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 201 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 401; v) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 202 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 402; w) a VH comprising an amino acid sequence at least 90%. at least 95% identical or 100% identical to SEQ ID NO: 203 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 403; x) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 204 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 404; y) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 205 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 405; z) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 206 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 406; aa) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 207 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 407; bb) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 208 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 408; cc) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 209 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 409; dd) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 210 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 410; ee) a VH comprising an amino acid sequence at least 90%. at least 95% identical or 100% identical to SEQ ID NO: 211 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 411; fl a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 212 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 412; gg) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 213 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 413; hh) a VH comprising an amino acid sequence at least 90%. at least 95% identical or 100% identical to SEQ ID NO: 214 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 414; ii) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 215 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 415; jj) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 216 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 416; kk) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 217 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 417;
11) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 218 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 418; mm) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 219 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 419; nn) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 220 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 420; oo) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 221 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 421; pp) a VH comprising an amino acid sequence at least 90%. at least 95% identical or 100% identical to SEQ ID NO: 222 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 422; qq) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 223 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 423; rr) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 224 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 424; ss) a VH comprising an amino acid sequence at least 90%. at least 95% identical or 100% identical to SEQ ID NO: 225 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 425; tt) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 226 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 426; uu) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 227 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 427; or vv) a VH comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 228 and a VL comprising an amino acid sequence at least 90%, at least 95% identical or 100% identical to SEQ ID NO: 428.
4. The antibody or fragment of any one of claim 1-3 2, wherein the antibody or fragment is a humanized or chimeric antibody or an antigen-binding fragment thereof.
5. The antibody or fragment of any one of claims 1-4, that is a monoclonal antibody or an antigen-binding fragment thereof.
6. The antibody or fragment of any one of claims 1-5, wherein the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody; or wherein the antigen-binding fragment thereof is a single-chain Fv (scFv), a disulfide-linked Fv (sdFv), a Fab fragment, a F(ab')2 fragment.
7. The antibody or fragment of any one of claims 1-6, wherein the antibody is a VHH antibody comprising at least one variable domain, heavy chain region (VH) wherein VH comprises complementarity' determining regions CDRH1, CDRH2, and CDRH3.
8. The antibody or antibody fragment of any one of claims 1-7. wherein the antibody or antibody fragment binds SIRPa with a KD of less than 75 nM, less than 50 nM, less than 25 nM, or less than 10 nM.
9. An isolated nucleic acid that encodes the antibody, or antigen-binding fragment thereof, of any one of claims 1-8.
10. An expression vector comprising the nucleic acid of claim 9.
11. An isolated host cell comprising the nucleic acid of claim 9 or the expression vector of claim 10.
12. An isolated host cell that expresses the antibody or antigen-binding fragment thereof of any one of claims 1-11.
13. A library’ comprising nucleic acids, wherein at least one of the nucleic acids encodes for at least one of the antibodies or antigen-binding fragments thereof of any one of claims 1-8.
14. The library of claim 13, yvherein at least one of the antibodies or antigen-binding fragments thereof is a single-chain Fv (scFv) fragment or a VHH antibody.
15. A method of producing an antibody or antigen-binding fragment thereof that binds SIRPa, comprising incubating the host cell of claim 11 or 12 under conditions suitable for producing the antibody or antigen-binding fragment thereof.
16. The method of claim 15, further comprising isolating the antibody or antigen-binding fragment thereof.
17. A method of treating a disease or disorder associated with SIRPa dysfunction in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-8.
18. The method of claim 17, yvherein the disease is cancer.
19. The antibody or antigen-binding fragment thereof of any one of claims 1-8 for use in a method of treating a disease or disorder associated with SIRPa dysfunction in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody, or antigen-binding fragment thereof, of any one of claims 1 -8.
PCT/US2024/025535 2023-04-20 2024-04-19 Antibodies and variant nucleic acid libraries for sirp-alpha WO2024220895A1 (en)

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US20220195063A1 (en) * 2017-05-16 2022-06-23 Byondis B.V. Anti-sirp alpha antibodies
US20220226469A1 (en) * 2018-11-14 2022-07-21 Arch Oncology, Inc. Therapeutic sirp-alpha antibodies
US20230106247A1 (en) * 2020-03-20 2023-04-06 L&L Biopharma Co., Ltd. Sirpalpha-targeting antibody or antigen binding fragment thereof, and preparation and application thereof
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