WO2024118940A1 - Anti-psma antibodies and uses thereof - Google Patents

Abstract

Provided herein are methods and compositions relating to PSMA libraries having nucleic acids encoding for a scaffold comprising a prostate-specific membrane antigen (PSMA) binding domain. PSMA libraries described herein encode for immunoglobulins such as antibodies.

Description

ANTLPSMA ANTIBODIES AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Application No. 63/429,441, filed December 1, 2022, which is incorporated by reference herein in its entirety for any purpose.

BACKGROUND

[0002] Prostate-Specific Membrane Antigen (PSMA) is an type II membrane glycoprotein expressed in prostate tissue. PSMA catalyzes the hydrolysis of N-acetylaspartylglutamate to glutamate and N-acetylaspartate. PSMA is also highly specific for both benign and malignant prostatic tumors.

[0003] Due to its overexpression in the prostate, and particularly in prostate cancer, PSMA is used as a biomarker for therapy and imaging of prostatic cancer. 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 and/or antibody fragments comprising an amino acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 1-1450. In some examples, 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: 1-1450. In some examples, the antibody or antibody fragment comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1- 1450. In some examples, 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 camelid 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 examples, the antibody or antibody fragment binds to PSMA with a KD of less than 75 nM.

In some examples, the antibody or antibody fragment binds to PSMA with a KD of less than 50 nM.

In some examples, the antibody or antibody fragment binds to PSMA with a KD of less than 25 nM.

In some examples, the antibody or antibody fragment binds to PSMA with a KD of less than 10 nM.

[0006] Further provided herein are antibodies or antibody fragments that bind PSMA, comprising an immunoglobulin VDJ region comprising an amino acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 1-107. In some examples, the immunoglobulin VDJ region comprises an amino acid sequence at least about 95% identical to that set forth in any one of SEQ ID NOs: 1-107. In some examples, the immunoglobulin VDJ region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-107. In some examples, 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 examples, the antibody or antibody fragment thereof is chimeric, camelized, or humanized. In some examples, the antibody or antibody fragment binds to PSMA with a KD of less than 75 nM. In some examples, the antibody or antibody fragment binds to PSMA with a KD of less than 50 nM. In some examples, the antibody or antibody fragment binds to PSMA with a KD of less than 25 nM. In some examples, the antibody or antibody fragment binds to PSMA with a KD of less than 10 nM.

[0007] Further provided herein are antibodies or antibody fragments that bind PSMA, comprising a variable heavy chain (VHH) region comprising an amino acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 857-1153. In some examples, the VHH region comprises an amino acid sequence at least about 95% identical to that set forth in any one of SEQ ID NOs: 857-1153. In some examples, the VHH region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 857-1153. In some examples, 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 examples, the antibody or antibody fragment thereof is chimeric, camelized, or humanized. In some examples, the antibody or antibody fragment binds to PSMA with a KD of less than 75 nM. In some examples, the antibody or antibody fragment binds to

PSMA with a KD of less than 50 nM. In some examples, the antibody or antibody fragment binds to

PSMA with a KD of less than 25 nM. In some examples, the antibody or antibody fragment binds to

PSMA with a KD of less than 10 nM.

[0008] Further provided herein are methods for treating a disease comprising administering the antibodies or antibody fragments as described herein. In some examples, the disease is cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figures 1A-1C show examples of functional assays. Figure 1A depicts an assay that identifies antibodies capable of blocking a receptor-ligand interaction that leads to an intracellular signaling cascade. Figure IB depicts a pair of cell surface binding assays, in which secreted antibodies bind to target cells in the channel and are detected using a fluorescently labeled secondary reagent. Figure 1C depicts a ligand blocking assay performed using fluorescently labeled recombinant proteins.

[0010] Figure 2 depicts an example workflow.

[0011] Figures 3A-3C show results from a titer test assay for detecting heavy-chain-only antibodies (HCAb, IgG2/3 isotype) and total IgG in serum that bind PSMA (Figure 3A), an exemplary alternative target protein, Target B (Figure 3B) or HCAb independent of antigen binding, “total IgG2/3” (Figure 3C).

[0012] Figure 4A shows results from the capture and detection of secreted alpaca IgG2 and IgG3 subclasses (the first assay). Figure 4B shows results from the second assay in which the antialpaca secondary was replaced with AF647-labeled recombinant PSMA protein. Figure 4C shows that IgG2/3 secreting B cell population proportions and target-specific B cell binding population proportions remained above 10% and 0.1%, respectively, as long as 30 weeks after the beginning of the experiment. Figure 4D shows results of IgG2/3 secretion and target specificity over multiple target antigens.

[0013] Figures 5A-5D depict results from top hits of the binding assays. Figure 5A depict sensorgrams of top hits. Figure 5B shows clones binding PSMA in nanomolar affinity. Figure 5C evaluated the directional competition of the top hits using PSMA binding kinetics and epitope binning evaluations. Figure 5D exemplifies hits recognizing non-overlapping epitopes. Figure 5E shows recombinantly expressed VHH-Fc binding validation assay results for recombinant PSMA protein. [0014] Figures 6A depicts an exemplary workflow to identify additional related sequences from B cell repertoires.

[0015] Figure 6B shows a diversity tree of the top hits from both single B cell candidates and related repertoire sequences.

[0016] Figure 6C shows another diversity tree, with sequences, of the top hits from both single B cell candidates and related repertoire sequences.

[0017] Figure 6D shows the differentiation between the top hits from both single B cell candidates and related repertoire sequences.

[0018] Figure 7 shows humanization data of select hits.

[0019] Figure 8 shows an exemplary workflow of a single B cell screening process.

[0020] Figure 9 shows examples of bead-based and cell-based assays that can determine functionality of an antigen or antibody.

DETAILED DESCRIPTION

[0021] 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.

[0022] Definitions

[0023] 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.

[0024] 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 otherwise. 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.

[0025] 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.

[0026] 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.

[0027] Immunization-Based Antibody Discovery

[0028] Provided herein are methods, compositions, and systems for immunization-based antibody discovery. Methods, compositions, and systems described herein for the discovery of antibodies comprise a ratio-variant approach that takes advantage of the natural diversity of antibody sequences. In some instances, 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 using immunization-based methods. In some instances, such antibodies are purified and evaluated for activity (e.g., epitope binding). In some instances, fragments of sequences are purified 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 antibody pool 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.

[0029] Described herein are methods and systems of immunization-based antibody discovery. In some instances, the antibodies comprise a plurality of variant antibody sequences against various targets. In some instances, the pool of antibodies comprises at least 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more than 5000 antibody sequences. In some instances, the antibodies are sequenced and 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.

[0030] Described herein are antibodies 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 isolated antibodies comprise multiple variants encoding for CDR1, CDR2, or CDR3. In some instances, the isolated antibodies 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 isolated antibodies 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 isolated antibodies 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. Antibodies or antibody fragments are in some instances isolated, assembled, and/or enriched for desired sequences.

[0031] CDRs can be at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, or more amino acids in length. Long CDRs can access sterically hindered epitopes.

[0032] Variant sequences (e.g., VHH sequences) can be linked into dimers, trimers, tetramers, pentamers, hexamers, heptamers, octomers, or other multivalent groupings to create multispecific antibody sequences. Variant sequences can be fused with peptide ligands.

[0033] Antibody Discovery

[0034] Provided herein are antibodies 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.

[0035] 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 ability 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 -idiotypic (anti-Id) antibody, a nanobody (an antibody with a single variable domain located on a heavy chain, also known as a VHH 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 antigenrecognition 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 antigenbinding 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. In some instances, the antibodies 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.

[0036] In some embodiments, the immunoglobulins comprise heavy-chain-only antibodies (HCAb), which are uniquely able to recognize their cognate antigen without a light chain. In camelids, the HCAb subclasses IgG2 and IgG3 consist of a variable domain (VHH) linked via a hinge to the Fc domain comprised of two constant regions (CH2 and CH3).

[0037] In some embodiments, antibodies 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.

[0038] “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.

[0039] 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, non- human 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. [0040] “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 felinize antibody.

[0041] Methods as described herein may be used for generation of antibodies encoding a nonimmunoglobulin. In some instances, the antibodies 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. [0042] Antibodies described herein can be developed through immunization of a host animal. A host animal can be a dog, a cat, a rabbit, a sheep, a goat, a cow, a horse, a goat, a camel, a llama, an alpaca, a guanaco, a vicuna, a mouse, a rat, a donkey, a monkey, an ape, an amphibian, a reptile, a bird, or a fish. Antibodies described herein can be developed through immunization of a human. Immunized animals and/or humans can have peripheral blood mononuclear cells (PBMCs) isolated and screened to recover antibody sequences. Antibody sequences can be enriched, expressed in vectors, synthesized in vitro, and/or optimized for further study.

[0043] Antibodies described herein can comprise nucleic acids encoding 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). In some instances, the variable region can be further divided into two regions, hypervariable regions (HV) and framework regions (FR).

[0044] Provided herein are antibodies comprising nucleic acids encoding for 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 antibodies 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 antibodies 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] Antibodies 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] Antibodies comprising nucleic acids encoding 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.

[0047] 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, heavy 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, IGHV3-30/33rn, 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.

[0048] Following the isolation of antibodies, antibodies may be used for screening and analysis. For example, antibodies can be assayed for displayability and panning. In some instances, displayability is assayed using a selectable tag. Exemplary 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, antibodies are assayed by sequencing using various methods including, but not limited to, single-molecule 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, antibodies are displayed on the surface of a cell or phage. In some instances, antibodies are enriched for sequences with a desired activity using phage display.

[0049] In some instances, the antibodies are assayed for functional activity, structural stability (e.g., thermal stable or pH stable), expression, binding, specificity, ligand blocking, apoptosis, antibody internalization, or a combination thereof. In some instances, the antibodies 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. Assays can be bead-based. Alternatively, assays can be cell-based. Exemplary types of assays are found in FIG. 9.

[0050] 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.

[0051] 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.

[0052] In some embodiments, the variant antibodies or IgGs generated by methods as described herein result in a decreased ECso in a T-cell cytotoxicity assay as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an ECso in a T-cell cytotoxicity assay that is at least 5X decreased as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an ECso in a T-cell cytotoxicity assay that is at least 8X decreased as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an ECso in a T-cell cytotoxicity assay that is at least 10X decreased as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an ECso in a T-cell cytotoxicity assay that is at least 20X decreased as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an ECso in a T-cell cytotoxicity assay that is at least 25X decreased as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an ECso in a T-cell cytotoxicity assay that is at least 3 OX decreased as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an ECso in a T-cell cytotoxicity assay that is at least 40X decreased as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an ECso in a T-cell cytotoxicity assay that is at least 50X decreased as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgGs.

[0053] 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).

[0054] Diseases and Disorders

[0055] Provided herein are antibodies 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, a respiratory disease or disorder, pain, a digestive disease or disorder, a reproductive disease or disorder, an endocrine 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 cancer is prostate cancer. In some instances, the disease or disorder is an autoimmune disease. In some instances, the disease or disorder is diabetes. In some instances, the disease or disorder is Type I diabetes. 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.

[0056] In some instances, the disease or disorder is associated with prostate-specific membrane antigen (PSMA) dysfunction. In some instances, the disease or disorder is associated with upregulation of PSMA. In some instances, the disease or disorder is associated with downregulation of PSMA. In some instances, the disease or disorder is associated with aberrant signaling via PSMA. In some instances, the disease or disorder is associated with a glutamate imbalance. In some instances, the disease or disorder is cancer. In some instances, the disease or disorder is a neural based or neurological disease. In some instances, the disease or disorder is a neurodegenerative disease. In some instances, the disease or disorder is an inflammatory disorder. In some instances, the disease is diabetes.

[0057] Protein Targets

[0058] Provided herein are antibodies comprising nucleic acids encoding for antibodies or immunoglobulins that target prostate-specific membrane antigen (PSMA). [0059] Provided herein are PSMA antibodies or immunoglobulins, wherein the PSMA antibody or immunoglobulin comprises a sequence at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-1450. In some instances, the antibody or immunoglobulin sequence comprises at least or about 95% sequence identity to any one of SEQ ID NOs: 1-1450. In some instances, the antibody or immunoglobulin sequence comprises at least or about 97% sequence identity to any one of SEQ ID NOs: 1-1450. In some instances, the antibody or immunoglobulin sequence comprises at least or about 99% sequence identity to any one of SEQ ID NOs: 1-1450. In some instances, the antibody or immunoglobulin sequence comprises at least or about 100% sequence identity to any one SEQ ID NOs: 1-1450. [0060] In some embodiments, the PSMA 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: 215-321, 429-535, and 643-749. 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: 215-321, 429-535, 643-749, or 1154-1450. 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: 215-321, 429-535, 643-749, or 1154-1450. 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: 215-321, 429-535, 643-749, or 1154-1450. 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: 215-321, 429-535, 643-749, or 1154-1450. 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: 215-321, 429-535, 643-749, or 1154-1450.

[0061] In some embodiments, the PSMA 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: 215-321. In some instances, the antibody or immunoglobulin sequence comprises HCDR1 comprising at least or about 95% homology of any one of SEQ ID NOs: 215-321. In some instances, the antibody or immunoglobulin sequence comprises HCDR1 comprising at least or about 97% homology to any one of SEQ ID NOs: 215-321. In some instances, the antibody or immunoglobulin sequence comprises HCDR1 comprising at least or about 99% homology to any one of SEQ ID NOs: 215- 321. In some instances, the antibody or immunoglobulin sequence comprises HCDR1 comprising at least or about 100% homology to any one of SEQ ID NOs: 215-321. 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: 215-321. [0062] In some embodiments, the PSMA 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 to any one of SEQ ID NOs: 429-535. In some instances, the antibody or immunoglobulin sequence comprises HCDR2 comprising at least or about 95% homology to any one of SEQ ID NOs: 429-535. In some instances, the antibody or immunoglobulin sequence comprises HCDR2 comprising at least or about 97% homology to any one of SEQ ID NOs: 429-535. In some instances, the antibody or immunoglobulin sequence comprises HCDR2 comprising at least or about 99% homology to any one of SEQ ID NOs: 429- 535. In some instances, the antibody or immunoglobulin sequence comprises HCDR2 comprising at least or about 100% homology to any one of SEQ ID NOs: 429-535. 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: 429-535.

[0063] In some embodiments, the PSMA 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: 643-749 or 1154-1450. In some instances, the antibody or immunoglobulin sequence comprises HCDR3 comprising at least or about 95% homology to any one of SEQ ID NOs: 643-749 or 1154-1450. In some instances, the antibody or immunoglobulin sequence comprises HCDR3 comprising at least or about 97% homology to any one of SEQ ID NOs: 643-749 or 1154-1450. In some instances, the antibody or immunoglobulin sequence comprises HCDR3 comprising at least or about 99% homology to any one of SEQ ID NOs: 643-749 or 1154-1450. In some instances, the antibody or immunoglobulin sequence comprises HCDR3 comprising at least or about 100% homology to any one of SEQ ID NOs: 643-749 or 1154-1450. 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: 643-749 or 1154-1450. [0064] In some embodiments, the PSMA antibody or immunoglobulin sequence comprises N- terminal segments (FRs) 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: 108-214, 322- 428, 536-642, and 750-856. In some instances, the antibody or immunoglobulin sequence comprises N-terminal framework region (FR) comprising at least or about 95% homology to any one of SEQ ID NOs: 108-214, 322-428, 536-642, and 750-856. In some instances, the antibody or immunoglobulin sequence comprises N-terminal segments (FRs) comprising at least or about 97% homology to any one of SEQ ID NOs: 108-214, 322-428, 536-642, and 750-856. In some instances, the antibody or immunoglobulin sequence comprises N-terminal segments (FRs) comprising at least or about 99% homology to any one of SEQ ID NOs: 108-214, 322-428, 536-642, and 750- 856. In some instances, the antibody or immunoglobulin sequence comprises N-terminal segments (FRs) comprising at least or about 100% homology to any one of SEQ ID NOs: 108-214, 322-428, 536-642, and 750-856. In some instances, the antibody or immunoglobulin sequence comprises N- terminal segments (FRs) 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: 1 OS- 214, 322-428, 536-642, and 750-856.

[0065] In some embodiments, the PSMA antibody or immunoglobulin sequence comprises an FR1 of the heavy chain (HFR1) 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: 108-214. In some instances, the antibody or immunoglobulin sequence comprises HFR1 comprising at least or about 95% homology of any one of SEQ ID NOs: 108-214. In some instances, the antibody or immunoglobulin sequence comprises HFR1 comprising at least or about 97% homology to any one of SEQ ID NOs: 108-214. In some instances, the antibody or immunoglobulin sequence comprises HFR1 comprising at least or about 99% homology to any one of SEQ ID NOs: 108-214. In some instances, the antibody or immunoglobulin sequence comprises HFR1 comprising at least or about 100% homology to any one of SEQ ID NOs: 108-214. In some instances, the antibody or immunoglobulin sequence comprises HFR1 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: 108- 214.

[0066] In some embodiments, the PSMA antibody or immunoglobulin sequence comprises a FR2 of the heavy chain (HFR2) 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: 322-428. In some instances, the antibody or immunoglobulin sequence comprises HFR2 comprising at least or about 95% homology to any one of SEQ ID NOs: 322-428. In some instances, the antibody or immunoglobulin sequence comprises HFR2 comprising at least or about 97% homology to any one of SEQ ID NOs: 322-428. In some instances, the antibody or immunoglobulin sequence comprises HFR2 comprising at least or about 99% homology to any one of SEQ ID NOs: 322-428. In some instances, the antibody or immunoglobulin sequence comprises HFR2 comprising at least or about 100% homology to any one of SEQ ID NOs: 322-428. In some instances, the antibody or immunoglobulin sequence comprises HFR2 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: 322-428.

[0067] In some embodiments, the PSMA antibody or immunoglobulin sequence comprises a FR3 of the heavy chain (HFR3) 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: 536-642. In some instances, the antibody or immunoglobulin sequence comprises HFR3 comprising at least or about 95% homology to any one of SEQ ID NOs: 536-642. In some instances, the antibody or immunoglobulin sequence comprises HFR3 comprising at least or about 97% homology to any one of SEQ ID NOs: 536-642. In some instances, the antibody or immunoglobulin sequence comprises HFR3 comprising at least or about 99% homology to any one of SEQ ID NOs: 536-642. In some instances, the antibody or immunoglobulin sequence comprises HFR3 comprising at least or about 100% homology to any one of SEQ ID NOs: 536-642. In some instances, the antibody or immunoglobulin sequence comprises HFR3 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: 536-642.

[0068] In some embodiments, the PSMA antibody or immunoglobulin sequence comprises a FR4 of the heavy chain (HFR4) 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: 750-856. In some instances, the antibody or immunoglobulin sequence comprises HFR3 comprising at least or about 95% homology to any one of SEQ ID NOs: 750-856. In some instances, the antibody or immunoglobulin sequence comprises HFR3 comprising at least or about 97% homology to any one of SEQ ID NOs: 750-856. In some instances, the antibody or immunoglobulin sequence comprises HFR3 comprising at least or about 99% homology to any one of SEQ ID NOs: 750-856. In some instances, the antibody or immunoglobulin sequence comprises HFR3 comprising at least or about 100% homology to any one of SEQ ID NOs: 750-856. In some instances, the antibody or immunoglobulin sequence comprises HFR3 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: 750-856. [0069] In some instances, the PSMA antibody or immunoglobulin sequence comprises a variable diversity and joining region (VDJ region) 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: 1-107. In some instances, the PSMA antibody or immunoglobulin sequence comprises a VDJ region comprising at least or about 95% sequence identity to any one of SEQ ID NOs: 1-107. In some instances, the PSMA antibody or immunoglobulin sequence comprises a VDJ region comprising at least or about 97% sequence identity to any one of SEQ ID NOs: 1-107. In some instances, the PSMA antibody or immunoglobulin sequence comprises a VDJ region comprising at least or about 99% sequence identity to any one of SEQ ID NOs: 1-107. In some instances, the PSMA antibody or immunoglobulin sequence comprises a VDJ region comprising at least or about 100% sequence identity to any one of SEQ ID NOs: 1-107. In some instances, the PSMA antibody or immunoglobulin sequence comprises a VDJ region 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: 1-107.

[0070] In some instances, the PSMA antibody or immunoglobulin sequence comprises a variable heavy chain region (VHH region) 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: 857-1153. In some instances, the PSMA antibody or immunoglobulin sequence comprises a VHH region comprising at least or about 95% sequence identity to any one of SEQ ID NOs: 857-1153. In some instances, the PSMA antibody or immunoglobulin sequence comprises a VHH region comprising at least or about 97% sequence identity to any one of SEQ ID NOs: 857-1153. In some instances, the PSMA antibody or immunoglobulin sequence comprises a VHH region comprising at least or about 99% sequence identity to any one of SEQ ID NOs: 857-1153. In some instances, the PSMA antibody or immunoglobulin sequence comprises a VHH region comprising at least or about 100% sequence identity to any one of SEQ ID NOs: 857-1153. In some instances, the PSMA antibody or immunoglobulin sequence comprises a VHH region 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: 857-1153.

[0071] Variant Libraries

[0072] Codon variation

[0073] Libraries of variant nucleic acids isolated and sequenced from immunization-based antibody discovery methods as 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

[0074] 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.

EXAMPLES

[0075] 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.

[0076] Example 1: PSMA Monoclonal Antibody Discovery

[0077] In this experiment, antibodies were secreted from single B cells to be interrogated for biologically relevant functions such as target binding, ligand blocking, and inhibition of signal transduction.

[0078] As shown in FIG. 8, structured light was used to segregate antibody-secreting cells into thousands of individual nanopens on an OptoSelect chip in a process called ‘optoelectrical positioning’. Each nanopen held a single B cell, and opened at one end into a common channel. As the secreted antibodies continuously diffused from the nanopen into the channel, their functional antigen-binding properties were measured using time-lapse fluorescent imaging assays containing capture beads, fluorescently-labeled target molecules, target-expressing cells, or other assay components. A positive result was observed as a spreading “bloom” of fluorescent signal in the channel area above the source pen. After each assay was completed, it was flushed out and a new assay imported; multiple assays were performed sequentially to measure different functions of antibodies secreted by a single B cell. After the assay results were analyzed to identify B cells secreting antibodies of interest, the B cells were individually exported and the mRNA encoding the paired heavy and light chains was sequenced. The entire screening workflow was completed in one day, rapidly providing a comprehensive functional profile that was used to select promising antibody sequences for further characterization.

[0079] A functional assay was developed to identify antibodies capable of blocking a receptorligand interaction that leads to an intracellular signaling cascade (FIG. 1A). The assay design relied upon a cell line in which a ligand binding to a receptor triggered a signaling cascade that led to the secretion of a reporter molecule. The reporter cells were penned and incubated together with antibody-secreting B cells prior to ligand exposure. The assay was subsequently imported in the channel to capture and detect the secreted reporter molecule. In this assay setup, active signaling through the receptor was indicated by the fluorescent detection of secreted reporter in the channel above the source pen. In contrast, blocked signaling was distinguished by the absence of a fluorescent signal in the channel above the source pen, indicating a lack of reporter secretion. After validation using control antibodies, this assay was used in a live screen to identify multiple signaling antagonists of interest.

[0080] A second functional assay shows a pair of cell surface binding assays, in which secreted antibodies bound to target cells in the channel and were detected using a fluorescently labeled secondary reagent (FIG. IB). By carefully choosing the positive and counter-screen cell lines, this screening strategy was used to identify target-specific cell binders.

[0081] A third assay shows a ligand blocking assay performed using fluorescently labeled recombinant proteins (FIG. 1C). In this setup, the ligand and target are each labeled with a different fluorophore and imported into the channel with mouse IgG capture beads. A secreted antibody that blocks the interaction was identified by positive signal from the fluorophore corresponding to the target only, while a non-blocker appeared as a positive signal from both fluorophores, indicating simultaneous binding of the antibody and the ligand to the target protein. [0082] The three assays described above can be performed sequentially, providing an unparalleled level of detail regarding antibody function for thousands of single B cells per chip.

[0083] To address whether the platform could be adapted for screening primary camelid B cells to facilitate in vivo VHH discovery a study was performed in which an immunized alpaca served as a source of antibody-secreting B cells for development and optimization of the screening workflow. Prostate-specific membrane antigen (PSMA) was used as the model target for the purposes of this study.

[0084] Beginning after 2-3 months of immunization and concurrently with repeated boosts, peripheral blood mononuclear cells (PBMCs) were collected at intervals spanning up to 9 months of sampling (FIG. 2). At each time point, antibody-secreting B cells were screened and targetspecific HCAb-expressing single B cells were sequenced. A subset of VHH sequences was chosen for recombinant expression, and the resulting VHH-Fc constructs were validated for target binding and epitope binning by SPR. Finally, the VHHs were humanized using a rapid and high-throughput process, and the humanized variants were characterized using a suite of developability assays. [0085] A serum titer test was performed to identify alpacas enriched for IgG2/3 isotype targetspecific B cells following immunization at various time points (FIGs. 3A-3C). On the day of each alpaca production bleed, the PBMCs were processed to enrich alpaca memory B cells, which were then stimulated in culture using an optimized method to secrete IgG. On the day of the screen, the cultured B cells were imported onto the chip, and single cells were segregated into nanopens in preparation for analysis. Custom anti-alpaca IgG capture beads were developed and, after premixing with anti-alpaca IgG2/3-AF488 secondary antibody, imported into the channel. FIG. 3A shows positive IgG2/3 seroconversion during an immunization time course. FIG. 3B shows negligible IgG2/3 seroconversion during an immunization time course. FIG. 3C shows that the total IgG2/3 remains constant through immunization and is target agnostic.

[0086] Longitudinal imaging in the FITC channel was performed to visualize the signal from the capture and detect secreted alpaca IgG2 and IgG3 subclasses (FIG. 4A). Following the first assay, the chip was flushed and a second assay was performed in which the anti-alpaca secondary was replaced with AF647-labeled recombinant PSMA protein. In this format, the secreted antibodies were captured on the beads, and binding to the fluorescent PSMA antigen resulted in a positive signal above the pen (FIG. 4B). Desired hits were chosen for export and sequencing based on a positive readout in both assays. Combining the results of both assays guided the identification of PSMA-binding, and IgG2/3+ candidates were exported for single cell sequencing, downstream expression, and validation.

[0087] A distinct advantage of alpacas is their large size, which enables repeated longitudinal sampling and screening. To investigate the IgG2/3 secretion rates and target binding rates over time, the screening workflow was repeated using PBMC collected at different intervals after the start of immunization for multiple projects using different alpacas, targets, and immunization regimens. IgG2/3 secretion rates followed a consistent pattern, with HCAb secretion rates reaching a peak of -30% around 18 weeks after the start of immunization and declining somewhat thereafter, but remaining above 10% as long as 30 weeks out (FIG. 4C). While target binding rates were considerably lower than overall IgG2/3 secretion rates, the longitudinal pattern tracked closely with the HCAb secretion rates, also peaking around week 18. Screening results showed that distinct antigens revealed similar trends and that HCAb (IgG2/3) and target-specific responses were sustained over the course of immunization (FIG. 4D). Results also showed a tendency for a peak response after 4-5 months of immunization.

[0088] To determine the sequences and evaluate the sequence diversity of PSMA-binding hits identified during the screens, target-binding hits were exported, and their heavy chains sequenced using custom-designed IgG2/3 -specific forward and reverse primers. Analyses of the resulting VHH sequences indicated a high degree of diversity among the subset of 25 sequences analyzed, with the greatest diversity seen in HCDR1 and HCDR3.

[0089] In addition to sequencing the exported hits, the antibody repertoire was also analyzed by performing next-generation sequencing on additional B cells enriched from PBMCs harvested at various production bleed time points, but that were not used in single B cell screening assays (FIGs. 6A-6D). VHH sequences were expressed in the format of VHH-Fc with an average yield of 0.6 mg using a HEK expression system. A high degree of diversity was observed among the 27 most abundant sequences, with most of the diversity concentrated in the HCDR1 and HCDR3 (FIG. 6D). Repertoire analysis was performed using R scripts and other available software packages.

[0090] To further evaluate the antibodies discovered during the screens, a subset of 13 clones was selected for recombinant expression and detailed characterization. Each clone was expressed as a - VHH-human IgGl Fc (hFc) fusion protein and purified using a protein affinity column. The purified VHH-hFc clones were analyzed for target binding kinetics by biolayer interferometry on a Carterra LSA instrument. Briefly, VHH-hFc mAb was captured on an anti-human Fc surface followed by injecting an ascending concentration series of dimeric PSMA. All clones except for one bound PSMA with single digit nanomolar affinity in this format (FIG. 5B). PSMA binding kinetics and epitope binning were also evaluated (FIG. 5A). 92% of clones had single digit nanomolar or better affinity in a monovalent binding format.

[0091] A subset of clones with high monovalent affinity were assessed for epitope diversity. Clones were selected across multiple timepoints in proportion to immune response. Several clones were identified as bidirectional competitors, and one clone (clone PSMA83) was a unidirectional competitor, blocking three other clones without being blocked by those clones (FIG. 5C). Overall, the 13 clones recognized largely non-overlapping epitopes and exhibited high epitopic breadth, with several epitopes represented by just one clone in this set (FIG. 5D). FIG. 5E also shows a 65% positive binding validation rate when looking at PSMA binding vs off-target binding using ELISA.

[0092] Finally, 12 of the parental clones were humanized in bulk using five different methods (FIG. 7). The humanness of the parental clones ranged from 68.0 - 82.7% (average 73.9%). After humanization, average humanness increased by several percentage points to varying degrees, while the KD decreased by varying degrees, depending on the humanization method used. Importantly, some of the humanized variants achieved substantial increases in humanness with minimal to no perturbation of target binding KD.

[0093] Example 2. Exemplary Sequences

[0094] Sequences for PSMA immunoglobulins are seen in Tables 2-3.

Table 2. PSMA sequences

Table 3. Additional PSMA sequences

[0095] 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.

Claims

CLAIMS WHAT IS CLAIMED IS:

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: 1-1450.

2. The antibody or antibody fragment of claim 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: 1-1450.

3. The antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-1450.

4. The antibody or antibody fragment of any one of claims 1-3, 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 camelid 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.

5. The antibody or antibody fragment of any one of claims 1-4, wherein the antibody or antibody fragment binds to PSMA with a KD of less than 75 nM.

6. The antibody or antibody fragment of any one of claims 1-4, wherein the antibody or antibody fragment binds to PSMA with a KD of less than 50 nM.

7. The antibody or antibody fragment of any one of claims 1-4, wherein the antibody or antibody fragment binds to PSMA with a KD of less than 25 nM.

8. The antibody or antibody fragment of any one of claims 1-4, wherein the antibody or antibody fragment binds to PSMA with a KD of less than 10 nM.

9. An antibody or antibody fragment that binds PSMA, comprising an immunoglobulin VDJ region comprising an amino acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 1-107.

10. The antibody or antibody fragment of claim 9, wherein the immunoglobulin VDJ region comprises an amino acid sequence at least about 95% identical to that set forth in any one of SEQ ID NOs: 1-107.

11. The antibody or antibody fragment of claim 9, wherein the immunoglobulin VDJ region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-107.

12. The antibody or antibody fragment of any one of claims 9-11, 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 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.

13. The antibody or antibody fragment of any one of claims 9-12, wherein the antibody or antibody fragment thereof is chimeric, camelized, or humanized.

14. The antibody or antibody fragment of any one of claims 9-12, wherein the antibody or antibody fragment binds to PSMA with a KD of less than 75 nM.

15. The antibody or antibody fragment of any one of claims 9-12, wherein the antibody or antibody fragment binds to PSMA with a KD of less than 50 nM.

16. The antibody or antibody fragment of any one of claims 9-12, wherein the antibody or antibody fragment binds to PSMA with a KD of less than 25 nM.

17. The antibody or antibody fragment of any one of claims 9-12, wherein the antibody or antibody fragment binds to PSMA with a KD of less than 10 nM.

18. An antibody or antibody fragment that binds PSMA, comprising a variable heavy chain (VHH) region comprising an amino acid sequence at least about 90% identical to that set forth in any one of SEQ ID NOs: 857-1153.

19. The antibody or antibody fragment of claim 18, wherein the VHH region comprises an amino acid sequence at least about 95% identical to that set forth in any one of SEQ ID NOs: 857- 1153.

20. The antibody or antibody fragment of claim 18, wherein the VHH region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 857-1153.

21. The antibody or antibody fragment of any one of claims 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 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.

22. The antibody or antibody fragment of any one of claims 18-20, wherein the antibody or antibody fragment thereof is chimeric, camelized, or humanized.

23. The antibody or antibody fragment of any one of claims 18-20, wherein the antibody or antibody fragment binds to PSMA with a KD of less than 75 nM.

24. The antibody or antibody fragment of any one of claims 18-20, wherein the antibody or antibody fragment binds to PSMA with a KD of less than 50 nM.

25. The antibody or antibody fragment of any one of claims 18-20, wherein the antibody or antibody fragment binds to PSMA with a KD of less than 25 nM.

26. The antibody or antibody fragment of any one of claims 18-20, wherein the antibody or antibody fragment binds to PSMA with a KD of less than 10 nM.

27. A method of treating a disease comprising administering the antibody or antibody fragment of any one of claims 1-26.

28. The method of claim 27, wherein the disease is cancer.