WO2023209687A1

WO2023209687A1 - Cas9 antibodies and uses thereof

Info

Publication number: WO2023209687A1
Application number: PCT/IB2023/054484
Authority: WO
Inventors: John KULMAN; Lalit Kumar; Aditi SAKHI
Original assignee: Crispr Therapeutics Ag
Priority date: 2022-04-29
Filing date: 2023-04-29
Publication date: 2023-11-02

Abstract

Provided include antibodies or fragments thereof having specificity to a Cas9 protein. The present disclosure also relates to compositions, kits and methods of using the antibodies or fragments thereof, for example for detecting Cas9 in a biological sample.

Description

CAS9 ANTIBODIES AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63/336,968 filed on April 29, 2022, the content of which is incorporated herein by reference in its entirety for all purposes.

REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 80EM-341718-WO-SeqList, created March 14, 2023, which is 251 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND

Field

[0003] The present disclosure generally relates to the field of molecular biology and biotechnology.

Description of the Related Art

[0004] The targeting of DNA using the RNA-guided, DNA-targeting principle of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) systems has been widely used. The CRISP/Cas genome editing system uses a RNA-guided endonuclease technology which allows for inducing genomic modifications at any desired location in a genome.

[0005] Cas9 gene editing in clinical applications faces challenges including safe delivery, gene targeting efficiency and off-target effects. There is a need for developing sensitive antibody assays to detect residual Cas9 in the edited cells and tissues, as well as to control the precision of transfection and the level of Cas9 expression during and after the editing process.

SUMMARY

[0006] Disclosed herein includes an antibody or fragment thereof, where the antibody or fragment thereof has specificity to a Cas9 protein and comprises:

(a) a light chain variable region (VL) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 1-13;

(b) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-26 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 14-26;

(c) a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-39 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 27-39;

(d) a heavy chain variable region (VH) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-52 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 40-52;

(e) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-65 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 53-65; and

(f) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-78 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 66-78.

[0007] In some embodiments, the antibody or fragment thereof comprises:

(a) a VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13;

(b) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-26;

(c) a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-39;

(d) a VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-52;

(e) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-65; and

(f) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-78.

[0008] In some embodiments, the antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 1 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 1; a VL CDR2 of SEQ ID NO: 14 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 14; a VL CDR3 of SEQ ID NO: 27 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 27; a VH CDR1 of SEQ ID NO: 40 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 40; a VH CDR2 of SEQ ID NO: 53 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 53; and a VH CDR3 of SEQ ID NO: 66 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 66.

[0009] In some embodiments, the antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 4 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 4; a VL CDR2 of SEQ ID NO: 17 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 17; a VL CDR3 of SEQ ID NO: 30 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 30; a VH CDR1 of SEQ ID NO: 43 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 43; a VH CDR2 of SEQ ID NO: 56 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 56; and a VH CDR3 of SEQ ID NO: 69 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 69.

[0010] In some embodiments, the antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 7 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 7; a VL CDR2 of SEQ ID NO: 20 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 20; a VL CDR3 of SEQ ID NO: 33 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 33; a VH CDR1 of SEQ ID NO: 46 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 46; a VH CDR2 of SEQ ID NO: 59 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 59; and a VH CDR3 of SEQ ID NO: 72 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 72.

[0011] In some embodiments, the antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 8 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 8; a VL CDR2 of SEQ ID NO: 21 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 21; a VL CDR3 of SEQ ID NO: 34 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 34; a VH CDR1 of SEQ ID NO: 47 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 47; a VH CDR2 of SEQ ID NO: 60 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 60; and a VH CDR3 of SEQ ID NO: 73 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 73.

[0012] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-91, (ii) an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-91, or (iii) an amino acid sequence having one, two or three mismatches relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-91.

[0013] In some embodiments, the antibody or fragment thereof comprises a heavy chain variable region comprising (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-104, (ii) an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-104, (iii) an amino acid sequence having one, two or three mismatches relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-104.

[0014] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:79 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:79, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:92 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:92.

[0015] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:82 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 82, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:95 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 95.

[0016] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:85 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:85, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:98 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 98. [0017] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:86 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 86, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:99 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:99.

[0018] In some embodiments, the antibody or fragment thereof comprises a Fc domain. In some embodiments, the antibody or fragment thereof is a single-chain variable fragment (scFv), a single-domain antibody, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a Fv fragment, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, or a functionally active epitope-binding fragment thereof.

[0019] Disclosed herein includes a composition, where the composition comprises one or more of the antibodies disclosed herein or fragments thereof; and a buffer. In some embodiments, the composition comprises a Cas9 capable of specific binding to the one or more of the antibodies disclosed herein or fragments thereof. In some embodiments, the composition comprises a Cas9 or a variant or derivative thereof. In some embodiments, the Cas9 is selected from Streptococcus pyogenes Cas9, Streptococcus aureus Cas9, Staphylococcus lugdunensis Cas9, Neisseria meningitides Cas9, Streptococcus thermophilus CRISPR1 Cas9, Streptococcus thermophilus CRISPR3 Cas9, Treponema denticola Cas9, Campylobacter jejuni Cas9, Lachnospiraceae bacterium ND2006 Cpfl and Acidaminococcus sp. BV3L6 Cpfl.

[0020] In some embodiments, one of the one or more of the antibodies or fragments thereof is associated with a detectable label. The detectable label can be selected from: a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, an electrochemiluminescent label, and a combination thereof. The electrochemiluminescent label can be, for example, a sulfo-tag.

[0021] The composition can, for example, comprise a biological sample. In some embodiments, the composition comprises cells, cell supernatant, cell lysate, or a combination thereof; and optionally the cell supernatant is post-thaw cell supernatant. In some embodiments, the cells comprise immune cells, stem cells, peripheral blood mononuclear cells (PBMCs), or a combination thereof. In some embodiments, the immune cells comprise T cells, NK cells, or a combination thereof. The T cells can comprise CAR-T cells, the NK cells can comprise CAR-NK cells, or both. In some embodiments, the stem cells comprise hemopoietic stem cells (HSCs), hemopoietic progenitor cells (HPCs), induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells, mesenchymal stem cells (MSCs), tissue-specific stem cells, or a combination thereof.

[0022] In some embodiments, the composition comprises two of the antibodies disclosed herein or fragments thereof. In some embodiments, one of the antibody or fragment thereof is biotinylated, and optionally the other antibody or fragment thereof is sulfo-tagged.

[0023] Also provided herein includes a polynucleotide encoding the antibody disclosed herein or fragment thereof. Provided herein includes a kit for detecting Cas9, comprising one or more of the antibodies disclosed herein or fragments thereof. In some embodiments, one of the one or more of the antibodies or fragments thereof is associated with a detectable label. Non-limiting examples of detectable label include: a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, an electrochemiluminescent label, and a combination thereof. In some embodiments, the electrochemiluminescent label is a sulfo-tag.

[0024] In some embodiments, the kit comprises two of the antibody disclosed herein or fragment thereof, where the two of the antibody or fragment thereof are different from one another. In some embodiments, one of the antibody or fragment thereof is biotinylated, and optionally the other antibody or fragment thereof is sulfo-tagged. In some embodiments, one of the antibody or fragment thereof is sulfo-tagged, and optionally the other antibody or fragment thereof is biotinylated.

[0025] In some embodiments, a first antibody or fragment thereof of the two of the antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 1 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 1, a VL CDR2 of SEQ ID NO: 14 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 14, a VL CDR3 of SEQ ID NO: 27 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 27, a VH CDR1 of SEQ ID NO: 40 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 40, a VH CDR2 of SEQ ID NO: 53 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 53, and a VH CDR3 of SEQ ID NO: 66 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 66; and wherein a second antibody or fragment thereof of the two of the antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 4 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 17 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 17, a VL CDR3 of SEQ ID NO: 30 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 30, a VH CDR1 of SEQ ID NO: 43 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 43, a VH CDR2 of SEQ ID NO: 56 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 56, and a VH CDR3 of SEQ ID NO: 69 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 69.

[0026] In some embodiments, a first antibody or fragment thereof of the two of the antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 7 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 7, a VL CDR2 of SEQ ID NO: 20 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 20, a VL CDR3 of SEQ ID NO: 33 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 33, a VH CDR1 of SEQ ID NO: 46 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 46, a VH CDR2 of SEQ ID NO: 59 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 59, and a VH CDR3 of SEQ ID NO: 72 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 72; and wherein a second antibody or fragment thereof of the two of the antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 8 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 8, a VL CDR2 of SEQ ID NO: 21 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 21, a VL CDR3 of SEQ ID NO: 34 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 34, a VH CDR1 of SEQ ID NO: 47 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 47, a VH CDR2 of SEQ ID NO: 60 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 60, and a VH CDR3 of SEQ ID NO: 73 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 73.

[0027] Disclosed herein includes a method of using an antibody disclosed herein or fragment thereof, where the method comprises: contacting a biological sample with the antibody or fragment thereof, thereby detecting the presence or absence of Cas9 in the biological sample. Also disclosed herein includes a method of detecting Cas9 in a biological sample, where the method comprises: contacting a biological sample with one or more of the antibodies disclosed herein or fragments thereof under conditions allowing the one or more antibodies or fragments thereof to bind to Cas9, where the biological sample comprises Cas9 or is suspected of comprising Cas9, and detecting the binding of the one or more antibodies or fragments thereof to Cas9, wherein the detection of the binding is indicative of the presence of Cas9 in the biological sample.

[0028] In some embodiments, the biological sample comprises Cas9-gRNA ribonucleoprotein (RNP). In some embodiments, the biological sample comprises free Cas9 protein or a fragment thereof, for example the free Cas9 protein can be ApoCas9. In some embodiments, the Cas9 is a Cas9 having endogenous nuclease activity, a small Cas9, a dead Cas9 (dCas9), a fusion protein comprising Cas9, or a variant thereof; and optionally the fusion protein is a Cas9 base editor. In some embodiments, the Cas9 is fused with or linked with one or more polypeptide domains, and optionally the Cas9 is fused with or linked with one polypeptide domain or two polypeptide domains. The polypeptide domain can comprise a polypeptide domain having an activity, for example an enzymatic activity. The activity can be, for example, a deaminase activity, reverse transcriptase activity, transcription activation activity, a transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, methylase activity, demethylase activity, protease activity, transposase activity, recombinase activity, nuclease, or a combination thereof.

[0029] In some embodiments, the polypeptide domain comprises a tag, a label, a nucleic acid binding domain, a helix-turn-helix domain, a zinc finger domain, or a combination thereof. In some embodiments, the biological sample comprises cell lysate or cell supernatant, and optionally the cell supernatant is post-thaw cell supernatant. In some embodiments, the detecting the binding of the antibody or fragment thereof to the Cas9 is performed using an immunodetection assay. The immunodetection assay can be, for example, western blotting, enzyme-linked immunosorbent assay (ELISA), and/or bead-based luminescent amplification assay. The ELISA assay can be a electro chemiluminescent (ELC) ELISA assay. The bead-based luminescent amplification assay can be an ALPHALisa Assay. [0030] In some embodiments, contacting comprises: contacting the biological sample with a first antibody disclosed herein or fragment thereof under a condition allowing binding of the first antibody or fragment thereof to Cas9 to form a first antibody-Cas9 complex; contacting the first antibody-Cas9 complex with a second antibody disclosed herein or fragment thereof under a condition allowing binding of the second antibody or fragment thereof to the first antibody-Cas9 complex; and detecting the binding of the second antibody or fragment thereof to the first antibody-Cas9 complex.

[0031] In some embodiments, one or both of the first and second antibodies or fragments thereof is associated with a detectable label. The detectable label can be a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, an electrochemiluminescent label, or a combination thereof. In some embodiments, the electrochemiluminescent label is a sulfo-tag. In some embodiments, the first antibody or fragment thereof is biotinylated, and the second antibody or fragment thereof is sulfo-tagged.

[0032] In some embodiments, the first antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 1 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 1, a VL CDR2 of SEQ ID NO: 14 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 14, a VL CDR3 of SEQ ID NO: 27 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 27, a VH CDR1 of SEQ ID NO: 40 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 40, a VH CDR2 of SEQ ID NO: 53 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 53, and a VH CDR3 of SEQ ID NO: 66 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 66; and wherein the second antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 4 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 17 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 17, a VL CDR3 of SEQ ID NO: 30 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 30, a VH CDR1 of SEQ ID NO: 43 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 43, a VH CDR2 of SEQ ID NO: 56 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 56, and a VH CDR3 of SEQ ID NO: 69 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 69.

[0033] In some embodiments, the biological sample comprises a cell supernatant.

[0034] In some embodiments, the first antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 7 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 7, a VL CDR2 of SEQ ID NO: 20 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 20, a VL CDR3 of SEQ ID NO: 33 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 33, a VH CDR1 of SEQ ID NO: 46 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 46, a VH CDR2 of SEQ ID NO: 59 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 59, and a VH CDR3 of SEQ ID NO: 72 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 72; and wherein the second antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 8 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 8, a VL CDR2 of SEQ ID NO: 21 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 21, a VL CDR3 of SEQ ID NO: 34 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 34, a VH CDR1 of SEQ ID NO: 47 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 47, a VH CDR2 of SEQ ID NO: 60 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 60, and a VH CDR3 of SEQ ID NO: 73 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 73.

[0035] In some embodiments, the biological sample comprises a cell lysate.

[0036] In some embodiments, the first antibody or fragment thereof is immobilized on a solid phase.

[0037] In some embodiments, the method comprises washing away unbound Cas9 after the contacting the biological sample with the first antibody or fragment thereof and prior to the contacting the first antibody-Cas9 complex with the second antibody or fragment thereof. In some embodiments, the biological sample is a cell supernatant comprising Cas9 at a concentration in the range of 0.34-0.8 ng/mL. In some embodiments, the biological sample is a cell lysate comprising Cas9 at a concentration in the range of 1-34 ng/mL.

[0038] Also disclosed herein includes a method of detecting Cas9 in a biological sample, where the method comprises: contacting a biological sample with one or more of antibodies or fragments thereof having specificity to a Cas9 protein under conditions allowing the one or more antibodies or fragments thereof to bind to the Cas9, wherein the biological sample comprises Cas9 at a concentration of no more than 0.01 pg/mL, and detecting the binding of the one or more antibodies or fragments thereof to the Cas9, wherein the detection of the binding is indicative of the presence of Cas9 in the biological sample.

[0039] In some embodiments, the biological sample is a cell supernatant comprising Cas9 at a concentration of no more than 0.8 ng/mL. The biological sample can be cell lysate. In some embodiments, the biological sample is derived from a pharmaceutical composition comprising cells. In some embodiments, the cells comprise immune cells, liver cells, heart cells, skin cells, colon cells, epithelial cells, stem cells, peripheral blood mononuclear cells (PBMCs), or a combination thereof. In some embodiments, the immune cells comprise T cells, NK cells or a combination thereof; and optionally the T cells comprise CAR-T cells and/or the NK cells comprise CAR-NK cells. In some embodiments, the stem cells comprise hemopoietic stem cells (HSCs), hemopoietic progenitor cells (HPCs), induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells, mesenchymal stem cells (MSCs), tissue-specific stem cells, or a combination thereof. The detection methods disclosed herein can, in some embodiments, comprise determining the amount of Cas9 in the biological sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1 depicts a non-limiting exemplary binding kinetics experiment.

[0041] FIG. 2 shows results from a non-limiting exemplary western blot analysis on various Cas9 antibodies for detecting ApoCas9 in 100,000 cells equivalent T cell lysates spiked with or without 500 ng ApoCas9.

[0042] FIG. 3 shows results from a non-limiting exemplary western blot analysis on various Cas9 antibodies for detecting ApoCas9 in 100,000 cells equivalent T cell lysates spiked with or without 50 ng ApoCas9.

[0043] FIG. 4 shows results from a non-limiting exemplary western blot analysis on various Cas9 antibodies for detecting ApoCas9 in 100,000 cells equivalent T cell lysates spiked with or without 1 ng or 10 ng ApoCas9.

[0044] FIG. 5 depicts a plot from an exemplary ELISA assay using various Cas9 antibodies (4G10, 174, 453, 752, 3447, 5125, 7675 and 8817) at different concentrations to detect Cas9-gRNA ribonucleoprotein (RNP).

[0045] FIGS. 6A-B depict plots from exemplary ELISA assays using various Cas9 antibodies (7474, 8810, 8873, 9594, 11912, 12486 and 4G10) at different concentrations to detect ApoCas9.

[0046] FIGS. 7A-B depict plots from exemplary ELISA assays using various Cas9 antibodies (174, 453, 752, 3447, 5125, 7675, 8817 and 4G10) at 10 pg/mL to detect RNP (FIG 7A) and ApoCas9 (FIG. 7B) coated onto a microplate at different concentrations.

[0047] FIGS. 8A-B depict plots from exemplary ELISA assays using various Cas9 antibodies (4G10, 174, 453, 752, 3447, 5125, 7675 and 8817) to detect RNP (FIG. 8A) or ApoCas9 (FIG. 8B) in various lysis buffers (AlphaLISA, CS5, PBS and RIP A).

[0048] FIGS. 9A-B depict plots from exemplary ELISA assays using various Cas9 antibodies (4G10, 752, 3447, 5125 and 8817 at 1 pg/mL) to detect different concentrations of RNP (FIG. 9A) or ApoCas9 (FIG. 9B) in CS5 buffer.

[0049] FIGS. 10A-B depict plots from exemplary ELISA assays for the detection of various amounts of RNP (FIG. 10A) or ApoCas9 (FIG. 10B). The antibodies (4G10, 5125 and 9594) are provided at 0.25, 0.5, and 1.0 pg/mL, respectively.

[0050] FIGS. 11A-B depict plots showing electrochemiluminescence (ECL) signal of RNP (FIG. 11 A) and ApoCas9 (FIG. 1 IB) at different concentrations (pg/mL) using various pairs of capture and detection antibodies.

[0051] FIGS. 12A-E depict plots from exemplary sandwich Meso Scale Discovery Electrochemiluminescence (MSD) assays using a detection antibody (sulfo-tagged 5125 antibody) and a biotinylated capture antibody of 174 (FIG. 12A), 8873 (FIG. 12B), 11912 (FIG. 12C), 4G10 (FIG. 12D), or 9594 (FIG. 12E), to detect various amounts of Cas9 (pg/ml). FIG. 12F shows that the pair of 174 as capture and 5125 as detection provided a suitable range of detection.

[0052] FIGS. 13A-B depict plots from exemplary sandwich MSD assays with 0.5 or 1.0 pg/mL of sulfo-tagged 5125 antibody as detection antibody and varying amounts of biotinylated 174 antibody as coating/capture antibody to detect various amounts of Cas9 (pg/mL) with blocking with 3% Blocker A.

[0053] FIGS. 14A-D depict plots from exemplary sandwich MSD assays with 1 pg/mL of biotinylated 174 antibody (FIGs. 14A-B) or 8873 antibody (FIGs. 14C-D) as capture antibody and sulfo-tagged 5125 antibody (1 pg/mL) as detection antibody to detect various amounts of Cas9 (pg/mL) with one hour blocking (FIG. 14A and FIG. 14C) or overnight blocking (FIG. 14B and FIG. 14D).

[0054] FIGS. 15A-F depict plots from exemplary sandwich MSD assays with various unconjugated capture and sulfo-tagged detection antibodies (1 pg/mL) to detect various amounts of Cas9 (ng/mL) in a whole cell lysate generated in MPER lysis buffer.

[0055] FIGS. 16A-F depict plots from exemplary sandwich MSD assays with various unconjugated capture and sulfo-tagged detection antibodies (1 pg/mL) to detect various amounts of Cas9 (ng/mL) in a whole cell lysate generated in RIPA lysis buffer.

[0056] FIGS. 17A-F depict plots from exemplary sandwich MSD assays with various unconjugated capture and sulfo-tagged detection antibodies (1 pg/mL) to detect various amounts of Cas9 (ng/mL) in a whole cell lysate generated in AlphaLISA lysis buffer.

[0057] FIGS. 18A-D depict plots from exemplary sandwich MSD assays with biotinylated 8810 (FIGS. 18A-B) or 9594 (FIGS. 18C-D) antibodies as capture antibody and sulfo-tagged 4G10 (FIG. 18A and FIG. 18C) or 8873 (FIG. 18B and FIG. 18D) antibodies as detection antibody to detect various amounts of Cas9 (ng/mL) with blocking in a whole cell lysate generated in MPER lysis.

[0058] FIGS. 19A-D depict plots from exemplary sandwich MSD assays with 8810 (FIGS. 19A-B) or 9594 (FIGS. 19C-D) antibodies as capture antibody and sulfo-tagged 4G10 (FIG. 19A and FIG. 19C) or 8873 (FIG. 19B and FIG. 19D) antibodies as detection antibody to detect various amounts of Cas9 (ng/mL) without blocking in a whole cell lysate generated in MPER lysis.

[0059] FIGS. 20A-20D depict the results of FIGS. 19A-D with a narrower range of Cas9.

[0060] FIG. 21A depicts a plot showing residual SpCas9 quantification in cell supernatant (in freezing medium, Cryostor® CS5).

[0061] FIG. 21B depicts a plot showing residual SpCas9 quantification in whole cell lysates (in MPER buffer).

[0062] FIG. 22 provides a schematic illustrating how electrochemiluminescence works in Meso Scale Discovery (MSD) assay.

[0063] FIG. 23 provides a table showing the plate layout and the read out from an MSD assay.

[0064] FIGS. 24A-B are plots and tables showing the Cas9 protein amounts over time in liver (FIG. 24B) compared to QC standards (FIG. 24 A).

[0065] FIG. 25 is a schematic representation of the steps involved in an assay for KD determination using Octet® method.

[0066] FIG. 26A provides plots showing kinetic data for exemplary ApoCas9 antibodies. FIG. 26B provides plots showing kinetic data for exemplary RNP specific antibodies.

[0067] FIG. 27A depict plots of standard curves for exemplary Cas9 supernatant assays using 5125 detection antibody. FIG. 27B depicts plots of observed concentrations for the Cas9 supernatant assays using the 5125 detection antibody.

[0068] FIG. 28A depicts plots of standard curves for exemplary Cas9 supernatant assays using 174/5125 antibody pair. FIG. 28B depicts a plot of observed concentrations for the Cas9 supernatant assays using the 174/5125 antibody pair.

[0069] FIG. 29A depicts plots of standard curves for exemplary Cas9 cell lysate assays using 8873 detection antibody. FIG. 29B depicts plots of observed concentrations for the Cas9 cell lysate assays using the 8873 detection antibody.

[0070] FIG. 30A depicts plots of standard curves for exemplary Cas9 cell lysate assays using 8810/8873 antibody pair. FIG. 30B depicts plots of observed concentrations for the Cas9 cell lysate assays using the 8810/8873 antibody pair.

DETAILED DESCRIPTION

[0071] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein and made part of the disclosure herein.

[0072] All patents, published patent applications, other publications, and sequences from GenBank, and other databases referred to herein are incorporated by reference in their entirety with respect to the related technology.

[0073] Disclosed herein include antibodies and fragments thereof having specificity to a Cas9 protein or a fragment or derivative thereof, and related compositions, kits and methods of use. The present disclosure also provides a method of determining the presence/absence, amount and/or concentration of residual Cas9 alone or in complex with a guide RNA in a biological sample using the antibodies and fragments thereof disclosed herein. In some embodiments, the methods herein disclosed achieves a detection capability of detecting residual Cas9 at a low concentration (for example, below 0.8 ng/mL for a biological sample comprising a cell supernatant and below 34 ng/mL for a biological sample comprising a cell lysate). [0074] Disclosed herein includes an antibody or fragment thereof, wherein the antibody or fragment thereof has specificity to a Cas9 protein and comprises (a) a light chain variable region (VL) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 1-13; (b) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-26 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 14-26; (c) a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-39 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 27-39; (d) a heavy chain variable region (VH) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-52 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 40-52; (e) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-65 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 53-65; and (f) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-78 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 66-78.

[0075] Provided herein also includes a composition and kit comprising one or more of the antibodies or fragments thereof described herein and a polynucleotide encoding the antibody or fragment thereof.

[0076] Provided herein also includes a method of using the antibody or fragment thereof described herein. The method can comprise contacting a biological sample with the antibody or fragment thereof, thereby detecting the presence or absence of Cas9 in the biological sample. Provided herein also include a method of detecting Cas9 in a biological sample. The method comprises contacting a biological sample with one or more of the antibodies or fragments thereof under conditions allowing the one or more antibodies or fragments thereof to bind to Cas9, wherein the biological sample comprises Cas9 or is suspected of comprising Cas9. The method can further comprises detecting the binding of the one or more antibodies or fragments thereof to Cas9, wherein the detection of the binding is indicative of the presence of Cas9 in the biological sample. Provided herein also include a method of detecting Cas9 in a biological sample. The method can comprise contacting a biological sample with one or more of antibodies or fragments thereof having specificity to a Cas9 protein under conditions allowing the one or more antibodies or fragments thereof to bind to the Cas9, wherein the biological sample comprises Cas9 at a concentration of no more than 0.01 pg/mL. The method can further comprise detecting the binding of the one or more antibodies or fragments thereof to the Cas9, wherein the detection of the binding is indicative of the presence of Cas9 in the biological sample.

Definition

[0077] As used herein, the term “about” means plus or minus 5% of the provided value.

[0078] As used herein, the term “RNA-guided endonuclease” refers to a polypeptide capable of binding a RNA (e.g., a gRNA) to form a complex targeted to a specific DNA sequence (e.g., in a target DNA). A non-limiting example of RNA-guided endonuclease is a Cas polypeptide (e.g., a Cas endonuclease, such as a Cas9 endonuclease). In some embodiments, the RNA-guided endonuclease as described herein is targeted to a specific DNA sequence in a target DNA by an RNA molecule to which it is bound. The RNA molecule can include a sequence that is complementary to and capable of hybridizing with a target sequence within the target DNA, thus allowing for targeting of the bound polypeptide to a specific location within the target DNA.

[0079] As used herein, the term “guide RNA” or “gRNA” refers to a site-specific targeting RNA that can bind an RNA-guided endonuclease to form a complex, and direct the activities of the bound RNA-guided endonuclease (such as a Cas endonuclease) to a specific target sequence within a target nucleic acid. The guide RNA can include one or more RNA molecules.

[0080] As used herein, the term “Cas endonuclease” or “Cas nuclease” refers to an RNA-guided DNA endonuclease associated with the CRISPR adaptive immunity system.

[0081] Unless otherwise indicated “nuclease” and “endonuclease” are used interchangeably herein to refer to an enzyme which possesses endonucleolytic catalytic activity for polynucleotide cleavage.

[0082] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. A polynucleotide can be single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids/triple helices, or a polymer including purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0083] As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non- naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

[0084] As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the nucleotide bases or amino acid residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity or similarity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted with a functionally equivalent residue of the amino acid residues with similar physiochemical properties and therefore do not change the functional properties of the molecule.

[0085] A functionally equivalent residue of an amino acid used herein typically can refer to other amino acid residues having physiochemical and stereochemical characteristics substantially similar to the original amino acid. The physiochemical properties include water solubility (hydrophobicity or hydrophilicity), dielectric and electrochemical properties, physiological pH, partial charge of side chains (positive, negative or neutral) and other properties identifiable to a person skilled in the art. The stereochemical characteristics include spatial and conformational arrangement of the amino acids and their chirality. For example, glutamic acid is considered to be a functionally equivalent residue to aspartic acid in the sense of the current disclosure. Tyrosine and tryptophan are considered as functionally equivalent residues to phenylalanine. Arginine and lysine are considered as functionally equivalent residues to histidine.

[0086] As used herein, the term “binding” refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid or between a first protein and a second protein). While in a state of non-covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it means that the molecule X binds to molecule Y in a non-covalent manner). Binding interactions can be characterized by a dissociation constant (Kd), for example a Kd of, or a Kd less than, 10'⁶ M, 10'⁷ M, 10'⁸ M, 10'⁹M, 10'¹⁰ M, 10 ¹ M, 10'¹²M, 10'¹³ M, 10'¹⁴ M,10'¹⁵M, or a number or a range between any two of these values. Kd can be dependent on environmental conditions, e.g., pH and temperature. “Affinity” refers to the strength of binding, and increased binding affinity is correlated with a lower Kd.

[0087] As used herein, the term “hybridizing” or “hybridize” refers to the pairing of substantially complementary or complementary nucleic acid sequences within two different molecules. Pairing can be achieved by any process in which a nucleic acid sequence joins with a substantially or fully complementary sequence through base pairing to form a hybridization complex. “Hybridizing” or “hybridize” can comprise denaturing the molecules to disrupt the intramolecular structure(s) (e.g., secondary structure(s)) in the molecule. In some embodiments, denaturing the molecules comprises heating a solution comprising the molecules to a temperature sufficient to disrupt the intramolecular structures of the molecules. In some instances, denaturing the molecules comprises adjusting the pH of a solution comprising the molecules to a pH sufficient to disrupt the intramolecular structures of the molecules. For purposes of hybridization, two nucleic acid sequences or segments of sequences are “substantially complementary” if at least 80% of their individual bases are complementary to one another. In some embodiments, a splint oligonucleotide sequence is not more than about 50% identical to one of the two polynucleotides (e.g., RNA fragments) to which it is designed to be complementary. The complementary portion of each sequence can be referred to herein as a ‘segment’, and the segments are substantially complementary if they have 80% or greater identity.

[0088] The term “isolated” as used herein with respect to cells, proteins, nucleic acids (such as DNA or RNA), refers to molecules separated from other cells, proteins, nucleic acids, respectively, that are present in the natural source of the macromolecule. The term “isolated” as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. In some embodiments, an “isolated nucleic acid” refers to nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to cells or polypeptides which are isolated from other cellular proteins or tissues. Isolated polypeptides can encompass both purified and recombinant polypeptides.

[0089] As used herein, the term “recombinant” in the context of polypeptides or polynucleotides refers to a form of the polypeptide or polynucleotide that does not exist naturally, a non-limiting example of which can be created by combining polynucleotides or polypeptides or by combining different polypeptides that would not normally occur together.

[0090] As used herein, an “antibody” or “antigen-binding polypeptide” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen (e.g., Cas9). An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. Thus the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen. Examples of such include, but are not limited to, a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein.

[0091] The terms “antibody fragment” or “antigen-binding fragment”, as used herein, is a portion of an antibody such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term “antibody fragment” includes aptamers, L-RNA aptamers (also known as spiegelmers), and diabodies. The term “antibody fragment” also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0092] As used herein, a “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins. In some embodiments, the regions are connected with a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. ScFv molecules are known in the art and are described, e.g., in U.S. Patent No. 5,892,019.

[0093] As used herein, the term “antibody” encompasses various broad classes of polypeptides that can be distinguished biochemically. Those of skill in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (y, p, a, 5, or ε) with some subclasses among them (e.g., yl- y4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgGi, IgG2, IgG3, IgG4, IgG5, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight approximately 53,000-70,000 Daltons. The four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.

[0094] Antibodies, antigen-binding polypeptides, fragments, variants, or derivatives thereof of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VK or VH domain, fragments produced by a Fab expression library, and anti -idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to LIGHT antibodies disclosed herein). Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

[0095] Light chains are classified as either kappa or lambda (K, X). Each heavy chain class may be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

[0096] Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino-terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.

[0097] As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VL chains (i.e. VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3). In some instances, e.g., certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, a complete immunoglobulin molecule may consist of heavy chains only, with no light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993). [0098] In naturally occurring antibodies, the six “complementarity determining regions” or “CDRs” present in each antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domains, referred to as “framework” regions, show less inter- molecular variability. The framework regions largely adopt a P-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the P-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined (see “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0099] In the case where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term “complementarity determining region” (“CDR”) to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al., U.S. Dept, of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entireties. The CDR definitions according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth in the table below as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those of skill in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0100] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept, of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0101] In addition to table above, the Kabat number system describes the CDR regions as follows: VH CDR1 begins at approximately amino acid 31 (z.e., approximately 9 residues after the first cysteine residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. VH CDR2 begins at the fifteenth residue after the end of VH CDR1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. VH CDR3 begins at approximately the thirty third amino acid residue after the end of VH CDR2; includes 3-25 amino acids; and ends at the sequence W-G-X-G, where X is any amino acid. VL CDR1 begins at approximately residue 24 (z.e., following a cysteine residue); includes approximately 10- 17 residues; and ends at the next tryptophan residue. VL CDR2 begins at approximately the sixteenth residue after the end of VL CDR1 and includes approximately 7 residues. VL CDR3 begins at approximately the thirty third residue after the end of VL CDR2 (z.e., following a cysteine residue); includes approximately 7-11 residues and ends at the sequence F or W-G-X-G, where X is any amino acid.

[0102] Antibodies disclosed herein can be from any animal origin including vertebrates (e.g., birds, reptiles, amphibians, and mammals). In some embodiments, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In some embodiments, the variable region is condricthoid in origin (e.g., from sharks).

[0103] As used herein, the term “heavy chain constant region” includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of a CHI domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the disclosure may comprise a polypeptide chain comprising a CHI domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CHI domain and a CH3 domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the disclosure comprises a polypeptide chain comprising a CH3 domain. Further, an antibody for use in the disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As set forth above, it will be understood by one of skill in the art that the heavy chain constant region may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.

[0104] The heavy chain constant region of an antibody disclosed herein may be derived from different immunoglobulin molecules. For example, a heavy chain constant region of a polypeptide may comprise a CHI domain derived from an IgGi molecule and a hinge region derived from an IgGs molecule. In another example, a heavy chain constant region can comprise a hinge region derived, in part, from an IgGi molecule and, in part, from an IgGs molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGi molecule and, in part, from an IgG4 molecule.

[0105] As used herein, the term “light chain constant region” includes amino acid sequences derived from antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or constant lambda domain.

[0106] A “light chain-heavy chain pair” refers to the collection of a light chain and heavy chain that can form a dimer through a disulfide bond between the CL domain of the light chain and the CHI domain of the heavy chain.

[0107] As previously indicated, the subunit structures and three dimensional configuration of the constant regions of the various immunoglobulin classes are well known. As used herein, the term “VH domain” includes the amino terminal variable domain of an immunoglobulin heavy chain and the term “CHI domain” includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain. The CHI domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.

[0108] As used herein the term “CH2 domain” includes the portion of a heavy chain molecule that extends, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., U.S. Dept, of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983). The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 residues. [0109] As used herein, the term “hinge region” includes the portion of a heavy chain molecule that joins the CHI domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol 161 :4083 (1998)).

[0110] As used herein the term “disulfide bond” includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CHI and CH2 regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).

[oni] As used herein, the term “chimeric antibody” will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the instant disclosure) is obtained from a second species. In some embodiments, the target binding region or site is from a non-human source (e.g. mouse or primate) and the constant region is human.

[0112] By “specifically binds” or “has specificity to,” it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody “A” may be deemed to have a higher specificity (e.g., greater binding affinity) for a given epitope than antibody “B,” or antibody “A” may be said to bind to epitope “C” with a higher specificity (e.g., greater binding affinity) than it has for related epitope “D ”

CRISPR-Cas Gene Editing System and RNA-Guided Nuclease

[0113] Provided herein include antibodies or fragments thereof having specificity to a Cas9 protein, and related compositions, kits and methods of use. The antibodies or fragments thereof and related compositions, kits and methods can be used in an immunoassay to detect Cas9 and/or Cas9-gRNA complex known as ribonucleoprotein (RNP) employed in a CRISPR-Cas gene editing system.

[0114] The CRISPR-Cas9 gene editing system is a naturally-occurring defense mechanism in prokaryotes that has been repurposed as a RNA-guided DNA-targeting platform used for gene editing. It relies on the DNA nuclease Cas9, and two noncoding RNAs-crisprRNA (crRNA) and trans-activating RNA (tracrRNA) to target the cleavage of DNA. crRNA drives sequence recognition and specificity of the CRISPR-Cas9 complex through Watson-Crick base pairing typically with a 20 nucleotide (nt) sequence in the target DNA. The CRISPR-Cas9 complex only binds DNA sequences that contain a sequence match to the first 20 nt of the crRNA, single-guide RNA (sgRNA), if the target sequence is followed by a specific short DNA motif (with the sequence NGG) referred to as a protospacer adjacent motif (PAM). TracrRNA hybridizes with the 3’ end of crRNA to form an RNA-duplex structure that is bound by the Cas9 endonuclease to form the catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA. Once the CRISPR-Cas9 complex is bound to DNA at a target site, two independent nuclease domains within the Cas9 enzyme each cleave one of the DNA strands upstream of the PAM site, leaving a double-strand break (DSB) where both strands of the DNA terminate in a base pair (a blunt end). After binding of CRISPR-Cas9 complex to DNA at a specific target site and formation of the site-specific DSB, the next key step is repair of the DSB. Cells use two main DNA repair pathways to repair the DSB: non-homologous end-joining (NHEJ) and homology- directed repair (HDR). In some embodiments, CRISPR-Cas9 gene editing system comprises an RNA-guided nuclease and one or more guide RNAs targeting one or more target genes.

[0115] As described herein, the RNA-guided endonuclease can be naturally-occurring or non-naturally occurring. The Non-limiting Examples of RNA-guided endonuclease include a Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslOO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cpfl endonuclease, and functional derivatives thereof.

[0116] In some instances, the antibodies or fragments thereof and related compositions, kits and methods can be used to detect a Cas9 endonuclease or a fragment or a derivative or a complex thereof capable of specifically binding to the one or more of the antibodies or fragments thereof of the present disclosure. The Cas9 endonuclease can be, for example, Streptococcus pyogenes Cas9, Streptococcus aureus Cas9, Staphylococcus lugdunensis Cas9, Neisseria meningitides Cas9, Streptococcus thermophilus CRISPR1 Cas9, Streptococcus thermophilus CRISPR3 Cas9, Treponema denticola Cas9, Campylobacter jejuni Cas9, Lachnospiraceae bacterium ND2006 Cpfl and Acidaminococcus sp. BV3L6 Cpfl. In some embodiments, the Cas9 endonuclease is Streptococcus pyogenes (SpyCas9), Staphylococcus lugdunensis (SluCas9), or Staphylococcus aureus (SaCas9). In some embodiments, the Cas9 is a Cas9 having endogenous nuclease activity (/.< ., Cas9 endonuclease). In some embodiments, the Cas9 described herein is a variant of Cas9 endonuclease, including but not limited to, a Cas9 fragment, a small Cas9, a dead Cas9 (dCas9), a fusion protein comprising Cas9 or a fragment or a variant thereof, and a Cas9 nickase. In some embodiments, the fusion protein comprising Cas9 is a Cas9 base editor (e.g., cytosine base editors and adenine base editors). In some embodiments, a Cas nuclease can comprise a RuvC or RuvC-like nuclease domain (e.g., Cpfl) and/or a HNH or HNH-like nuclease domain e.g., Cas9).

[0117] As described herein, the Cas9 (e.g., a dCas9) or a fragment thereof can be fused with or linked with one or more polypeptide domains, for example, one, two, three, four or more polypeptide domains. For example, the Cas9 can be fused with or linked with one polypeptide domain or two polypeptide domains. In some embodiments, the Cas9 is present in a fusion protein comprising the Cas9 and one or more polypeptide domains. The polypeptide domain fused or linked with the Cas9 can be another Cas protein, a non-Cas protein, or a portion or fragment thereof. The polypeptide domain described herein can, for example, have an activity (e.g., an enzymatic activity, and a nucleic acid (DNA and/or RNA) binding activity). In some embodiments, the polypeptide domain can possess one or more of the activities selected from deaminase activity, reverse transcriptase activity, transcription activation activity, a transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, methylase activity, demethylase activity, protease activity, transposase activity, recombinase activity, nuclease, and any combination thereof. In some embodiments, the polypeptide domain comprises or is a deaminase, reverse transcriptase, transcription activator, transcription repressor, transcription release factor, histone modification factor, nuclease, nucleic acid association factor, methylase, demethylase, protease, transposase, recombinase, nuclease, transferase, glycosylase, demethylase (e.g., lysine-specific demethylase), nuclear factor (e.g., P65, NF-kappa-B), methyltransferase (e.g., DNA methyltransferase (Dnmt)), any combination thereof, or a portion or a fragment of the aforementioned enzymes/factors. Examples of deaminase include, but are not limited to, APOBEC1, APOBEC3A-H, APOBEC3G, activation-induced cytidine deaminase (AICDA), cytidine deaminase (CDA), dCMP deaminase (DCTD), AMP deaminase (AMPD1), adenosine deaminase acting on tRNA (AD AT), adenosine deaminase acting on dsRNA (ADAR), double-stranded RNA-specific editase 1 (AD ARBI), adenosine deaminase acting on mononucleotides (ADA), guanine deaminase (GDA), or any combination thereof. In some embodiments, the polypeptide domain comprises a tag, a label, a nucleic acid binding domain, a helix-turn-helix domain, a zinc finger domain, or a combination thereof. Non-limiting examples of transcription activator include VP 16, VP64, P65, replication and transcription activator (Rta), p300, or any combination thereof. Non-limiting examples of transcription repressor include Kruppel-associated box (KRAB). In some embodiments, the polypeptide domain(s) fused or linked with Cas9 comprises Heat Shock Factorl (HSF1), P65, cytosine demethylase ten-eleven translocation methylcytosine dioxygenase 1 (Tetl), human histone deacetylase3 (HDAC3), chromatin loop, DNA adenine methyltransfer, KRAB, methyl- CpG-binding protein (MeCp2), a fragment or variant thereof, or any combination thereof.

[0118] A Cas9 nuclease can be a small Cas9. The small Cas9 can be engineered from portions of Cas9 endonucleases derived from any of the RNA-guided Cas9 endonucleases described herein and known in the art. In some cases, a small Cas9 is shorter than 1,100 amino acids in length.

[0119] A Cas9 can be a mutant Cas9 endonuclease or a derivative thereof. For example, the Cas9 can be a mutant of a naturally occurring Cas9 endonuclease. The mutant Cas9 can also be a mutant Cas9 endonuclease with altered activity compared to a naturally occurring Cas9 endonuclease, such as altered endonuclease activity (e.g., altered or abrogated DNA endonuclease activity without substantially diminished binding affinity to DNA). Such modification can allow for the sequence-specific DNA targeting of the mutant Cas9 for the purpose of transcriptional modulation (e.g., activation or repression); epigenetic modification or chromatin modification by methylation, demethylation, acetylation or deacetylation, or any other modifications of DNA binding and/or DNA-modifying proteins known in the art. In some embodiments, the mutant Cas9 has no DNA endonuclease activity.

[0120] The Cas9 described herein can be a nickase that cleaves the complementary strand of the target DNA but has reduced ability to cleave the non-complementary strand of the target DNA, or that cleaves the non-complementary strand of the target DNA but has reduced ability to cleave the complementary strand of the target DNA. In some embodiments, the Cas9 has a reduced ability to cleave both the complementary and the non-complementary strands of the target DNA.

Cas9-Specific Antibody

[0121] The present disclosure provides Cas9 specific antibodies or fragments thereof with high affinity and specificity to a Cas9 protein. The Cas9 protein can be a Cas9 protein alone or a fragment or a derivative thereof or a Cas9-gRNA ribonucleoprotein (RNP). The Cas9 specific antibodies herein described can provide sensitive and selective detection of Cas9 in a biological sample being assayed. In some embodiments, the Cas9 antibodies herein described can detect the presence, amount and/or concentration of residual Cas9 alone or in complex with a guide RNA (e.g., RNP) in a sample. In some embodiments, the Cas9 antibodies or fragments thereof can be used in an assay to detect residual Cas9 at a concentration about, at most, at most about, at least, or at least about, 35 ng/mL, 30 ng/mL, 25 ng/mL, 20 ng/mL, 15 ng/mL, 10 ng/mL, 5 ng/mL, 4 ng/mL, 3 ng/mL, 2 ng/mL, 1 ng/mL, 0.8 ng/mL, 0.6 ng/mL, 0.4 ng/mL, 0.2, ng/mL 0.1 ng/mL or a number or a range between any two of these values.

[0122] In some embodiments, the antibodies or fragments thereof disclosed herein contain CDR regions defined in SEQ ID NOs: 1-78 or variants thereof having one, two or three mismatches (e.g., a single substitution, deletion or insertion) in any one of SEQ ID NOs: 1-78.

[0123] In some embodiments, the antibody or fragment thereof comprises (a) a light chain variable region (VL) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13 or a variant thereof having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-13; (b) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-26 or a variant thereof having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 14-26; (c) a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-39 or a variant thereof having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 27-39; (d) a heavy chain variable region (VH) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-52 or a variant thereof having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 40-52; (e) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-65 or a variant thereof having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 53- 65; and/or (f) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-78 or a variant thereof having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 66-78.

[0124] In some embodiments, the antibody or fragment thereof comprises (a) a light chain variable region (VL) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13 or a variant thereof having one, two or three mismatches in any one of SEQ ID NOs: 1-13; (b) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-26 or a variant thereof having one, two or three mismatches in any one of SEQ ID NOs: 14-26; (c) a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-39 or a variant thereof having one, two or three mismatches in any one of SEQ ID NOs: 27-39; (d) a heavy chain variable region (VH) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-52 or a variant thereof having one, two or three mismatches in any one of SEQ ID NOs: 40-52; (e) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-65 or a variant thereof having one, two or three mismatches in any one of SEQ ID NOs: 53-65; and/or (f) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-78 or a variant thereof having one, two or three mismatches in any one of SEQ ID NOs: 66-78.

[0125] In some embodiments, the antibody or fragment thereof comprise (a) a light chain variable region (VL) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 1-13; (b) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-26 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 14-26; and (c) a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-39 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 27-39.

[0126] In some embodiments, the antibody or fragment thereof comprise (d) a heavy chain variable region (VH) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-52 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 40-52; (e) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-65 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 53-65; and (f) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-78 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 66-78.

[0127] In some embodiments, the antibody or fragment thereof comprises (a) a light chain variable region (VL) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 1-13; (b) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-26 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 14-26; (c) a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-39 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 27-39; (d) a heavy chain variable region (VH) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-52 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 40-52; (e) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-65 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 53-65; and (f) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-78 or a variant thereof having a single substitution, deletion or insertion from any one of SEQ ID NOs: 66-78.

[0128] In some embodiments, the substitutions used herein are conservative substitutions or substitutions with a functionally equivalent residue. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain and/or an amino acid having physiochemical and stereochemical characteristics substantially similar to the original amino acid. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0129] In some embodiments, the antibody or fragment thereof comprise (a) a VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1- 13; (b) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-26; (c) a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-39; (d) a VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-52; (e) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-65; and (f) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-78.

[0130] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 1 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 1; a VL CDR2 of SEQ ID NO: 14 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 14; a VL CDR3 of SEQ ID NO: 27 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 27; a VH CDR1 of SEQ ID NO: 40 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 40; a VH CDR2 of SEQ ID NO: 53 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 53; and a VH CDR3 of SEQ ID NO: 66 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 66.

[0131] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 1; a VL CDR2 of SEQ ID NO: 14; a VL CDR3 of SEQ ID NO: 27; a VH CDR1 of SEQ ID NO: 40; a VH CDR2 of SEQ ID NO: 53; and a VH CDR3 of SEQ ID NO: 66.

[0132] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 2 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 2; a VL CDR2 of SEQ ID NO: 15 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 15; a VL CDR3 of SEQ IDNO: 28 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 28; a VH CDR1 of SEQ ID NO: 41 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 41; a VH CDR2 of SEQ ID NO: 54 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 54; and a VH CDR3 of SEQ ID NO: 67 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 67.

[0133] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 2; a VL CDR2 of SEQ ID NO: 15; a VL CDR3 of SEQ ID NO: 28; a VH CDR1 of SEQ ID NO: 41; a VH CDR2 of SEQ ID NO: 54; and a VH CDR3 of SEQ ID NO: 67.

[0134] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 3 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 3; a VL CDR2 of SEQ ID NO: 16 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 16; a VL CDR3 of SEQ ID NO: 29 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 29; a VH CDR1 of SEQ ID NO: 42 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 42; a VH CDR2 of SEQ ID NO: 55 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 55; and a VH CDR3 of SEQ ID NO: 68 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 68.

[0135] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 3; a VL CDR2 of SEQ ID NO: 16; a VL CDR3 of SEQ ID NO: 29; a VH CDR1 of SEQ ID NO: 42; a VH CDR2 of SEQ ID NO: 55; and a VH CDR3 of SEQ ID NO: 68.

[0136] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 4 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 4; a VL CDR2 of SEQ ID NO: 17 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 17; a VL CDR3 of SEQ ID NO: 30 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 30; a VH CDR1 of SEQ ID NO: 43 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 43; a VH CDR2 of SEQ ID NO: 56 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 56; and a VH CDR3 of SEQ ID NO: 69 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 69.

[0137] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 4; a VL CDR2 of SEQ ID NO: 17; a VL CDR3 of SEQ ID NO: 30; a VH CDR1 of SEQ ID NO: 43; a VH CDR2 of SEQ ID NO: 56; and a VH CDR3 of SEQ ID NO: 69.

[0138] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 5 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 5; a VL CDR2 of SEQ ID NO: 18 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 18; a VL CDR3 of SEQ ID NO: 31 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 31; a VH CDR1 of SEQ ID NO: 44 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 44; a VH CDR2 of SEQ ID NO: 57 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 57; and a VH CDR3 of SEQ ID NO: 70 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 70.

[0139] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 5; a VL CDR2 of SEQ ID NO: 18; a VL CDR3 of SEQ ID NO: 31; a VH CDR1 of SEQ ID NO: 44; a VH CDR2 of SEQ ID NO: 57; and a VH CDR3 of SEQ ID NO: 70.

[0140] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 6 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 6; a VL CDR2 of SEQ ID NO: 19 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 19; a VL CDR3 of SEQ ID NO: 32 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 32; a VH CDR1 of SEQ ID NO: 45 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 45; a VH CDR2 of SEQ ID NO: 58 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 58; and a VH CDR3 of SEQ ID NO: 71 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 71.

[0141] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 6; a VL CDR2 of SEQ ID NO: 19; a VL CDR3 of SEQ ID NO: 32; a VH CDR1 of SEQ ID NO: 45; a VH CDR2 of SEQ ID NO: 58; and a VH CDR3 of SEQ ID NO: 71.

[0142] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 7 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 7; a VL CDR2 of SEQ ID NO: 20 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 20; a VL CDR3 of SEQ ID NO: 33 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 33; a VH CDR1 of SEQ ID NO: 46 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 46; a VH CDR2 of SEQ ID NO: 59 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 59; and a VH CDR3 of SEQ ID NO: 72 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 72.

[0143] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 7; a VL CDR2 of SEQ ID NO: 20; a VL CDR3 of SEQ ID NO: 33; a VH CDR1 of SEQ ID NO: 46; a VH CDR2 of SEQ ID NO: 59; and a VH CDR3 of SEQ ID NO: 72.

[0144] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 8 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 8; a VL CDR2 of SEQ ID NO: 21 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 21; a VL CDR3 of SEQ ID NO: 34 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 34; a VH CDR1 of SEQ ID NO: 47 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 47; a VH CDR2 of SEQ ID NO: 60 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 60; and a VH CDR3 of SEQ ID NO: 73 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 73.

[0145] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 8; a VL CDR2 of SEQ ID NO: 21; a VL CDR3 of SEQ ID NO: 34; a VH CDR1 of SEQ ID NO: 47; a VH CDR2 of SEQ ID NO: 60; and a VH CDR3 of SEQ ID NO: 73.

[0146] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 9 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 9; a VL CDR2 of SEQ ID NO: 22 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 22; a VL CDR3 of SEQ ID NO: 35 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 35; a VH CDR1 of SEQ ID NO: 48 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 48; a VH CDR2 of SEQ ID NO: 61 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 61; and a VH CDR3 of SEQ ID NO: 74 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 74.

[0147] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 9; a VL CDR2 of SEQ ID NO: 22; a VL CDR3 of SEQ ID NO: 35; a VH CDR1 of SEQ ID NO: 48; a VH CDR2 of SEQ ID NO: 61; and a VH CDR3 of SEQ ID NO: 74.

[0148] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 10 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 10; a VL CDR2 of SEQ ID NO: 23 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 23; a VL CDR3 of SEQ ID NO: 36 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 36; a VH CDR1 of SEQ ID NO: 49 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 49; a VH CDR2 of SEQ ID NO: 62 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 62; and a VH CDR3 of SEQ ID NO: 75 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 75.

[0149] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 10; a VL CDR2 of SEQ ID NO: 23; a VL CDR3 of SEQ ID NO: 36; a VH CDR1 of SEQ ID NO: 49; a VH CDR2 of SEQ ID NO: 62; and a VH CDR3 of SEQ ID NO: 75. [0150] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 11 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 11; a VL CDR2 of SEQ ID NO: 24 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 24; a VL CDR3 of SEQ ID NO: 37 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 37; a VH CDR1 of SEQ ID NO: 50 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 50; a VH CDR2 of SEQ ID NO: 63 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 63; and a VH CDR3 of SEQ ID NO: 76 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 76.

[0151] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 11; a VL CDR2 of SEQ ID NO: 24; a VL CDR3 of SEQ ID NO: 37; a VH CDR1 of SEQ ID NO: 50; a VH CDR2 of SEQ ID NO: 63; and a VH CDR3 of SEQ ID NO: 76.

[0152] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 12 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 12; a VL CDR2 of SEQ ID NO: 25 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 25; a VL CDR3 of SEQ ID NO: 38 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 38; a VH CDR1 of SEQ ID NO: 51 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 51 ; a VH CDR2 of SEQ ID NO: 64 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 64; and a VH CDR3 of SEQ ID NO: 77 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 77.

[0153] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 12; a VL CDR2 of SEQ ID NO: 25; a VL CDR3 of SEQ ID NO: 38; a VH CDR1 of SEQ ID NO: 51; a VH CDR2 of SEQ ID NO: 64; and a VH CDR3 of SEQ ID NO: 77.

[0154] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 13 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 13; a VL CDR2 of SEQ ID NO: 26 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 26; a VL CDR3 of SEQ ID NO: 39 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 39; a VH CDR1 of SEQ ID NO: 52 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 52; a VH CDR2 of SEQ ID NO: 65 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 65; and a VH CDR3 of SEQ ID NO: 78 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 78.

[0155] In some embodiments, the antibody or fragment thereof comprises a VL CDR1 of SEQ ID NO: 13; a VL CDR2 of SEQ ID NO: 26; a VL CDR3 of SEQ ID NO: 39; a VH CDR1 of SEQ ID NO: 52; a VH CDR2 of SEQ ID NO: 65; and a VH CDR3 of SEQ ID NO: 78.

[0156] The antibody or fragment thereof can comprise a light chain variable region and/or a heavy chain variable region herein described. In some embodiments, the light chain variable region comprises an amino acid sequence having about, at least or at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-91. In some embodiments, the light chain variable region comprises an amino acid sequence having one, two or three mismatches relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-91. In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-91. The heavy chain variable region can comprise an amino acid sequence having about, at least or at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-104. In some embodiments, the heavy chain variable region comprises an amino acid sequence having one, two or three mismatches relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-104. In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-104.

[0157] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 79 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 79, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:92 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 92.

[0158] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 80 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 80, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 93 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 93.

[0159] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 81 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 81, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 94.

[0160] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 82 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 82, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 95.

[0161] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 83 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 83, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 96 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 96.

[0162] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 84 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 84, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 97 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 97.

[0163] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 85 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 85, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 98 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 98.

[0164] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 86 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 86, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 99 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 99.

[0165] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 87 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 87, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 100 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 100. [0166] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 88 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 88, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 101 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 101.

[0167] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 89 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 89, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 102 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 102.

[0168] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 90 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 90, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 103 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 103.

[0169] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 91 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 91, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 104 or a peptide having about, at least, or at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 104.

[0170] In some embodiments, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 79 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 92. In some embodiments, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 82 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 85 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 86 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 99.

[0171] In some embodiments, the antibody or fragment thereof can further comprises a Fc domain. The antibody or fragment thereof disclosed herein can be, or can comprise, for example, a single-chain variable fragment (scFv), a single-domain antibody, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a Fv fragment, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual specific antibody, an anti -idiotypic antibody, a bispecific antibody (e.g., the Cas9 specific antibody or fragment thereof can comprise another binding site directed at a different antigen), or a functionally active epitope-binding fragment thereof.

[0172] In some embodiments, the antibody or fragment thereof is a single-chain variable fragment (scFv) or a single-domain antibody. Single-chain variable fragment can be formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge (e.g., a disulfide linkage), resulting in a single-chain fusion peptide. The heavy and light chain fragments of the Fv region can be selected from any of the heavy and light chain fragments and variants thereof described herein (e.g., SEQ ID NOs: 72-91, SEQ ID NOs: 92-104 or variants thereof). Examples of techniques which can be used to produce scFvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., Proc. Natl. Sci. USA 90: 1995-1999 (1993); and Skerra c/ a/., Science 240: 1038-1040 (1988). In some embodiments, the antibody or fragment thereof is a single-domain antibody comprising a heavy chain variable region or a variant thereof described herein. For example, the single-domain antibody can comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-104 or variants thereof having about, at least or at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 92-104 or fragments thereof. The single-domain antibody can comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-78 or variants having one, two or three mismatches in one of SEQ ID NOs: 40-78.

[0173] In some embodiments, the antibodies or fragments thereof do not elicit an undesirable (e.g., deleterious) immune response in a subject to be treated, e.g., in a human. In some embodiments, antibodies, fragments, variants, or derivatives thereof of the disclosure are modified to reduce their immunogenicity using techniques recognized in the art. For example, antibodies can be humanized, primatized, deimmunized, or chimeric antibodies.

[0174] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies, fragments, variants or derivatives thereof of the disclosure. The polynucleotides of the present disclosure can encode the heavy and light chain variable regions of the antibodies, fragments, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. In some embodiments, the polynucleotides of the present disclosure can encode portions of the heavy and light chain variable regions of the antibodies (e.g., the CDR regions), fragments, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules.

[0175] Methods of making antibodies are well known in the art and described herein. For example, polynucleotides encoding desired antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The isolated and subcloned hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into antibody-producing cells including prokaryotic or eukaryotic host cells such as E. coll cells, simian COS cells, Chinese Hamster Ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulins. The isolated DNA can be used to clone constant and variable region sequences for the manufacture antibodies as described in Newman et al., U.S. Pat. No. 5,658,570 which is incorporated by reference herein. Essentially, this entails extraction of RNA from the selected cells, conversion to cDNA, and amplification by PCR using Ig specific primers. Suitable primers for this purpose are also described in U.S. Pat. No. 5,658,570. As described herein, transformed cells expressing the desired antibody can be grown up in relatively large quantities to provide clinical and commercial supplies of the immunoglobulin.

[0176] In some embodiments, mutations can be introduced in the nucleotide sequence encoding an antibody of the present disclosure using standard techniques known to those of skill in the art, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions.

[0177] In some embodiments, the antibodies, fragments, variants, or derivatives thereof can further comprise a chemical moiety not naturally associated with an antibody. For example, the antibody or fragment thereof can comprise a flexible linker or can be modified to add a functional moiety such as a detectable label. The antibodies, fragments, variants, or derivatives thereof can be modified, i.e., by the covalent or non-covalent attachment of a chemical moiety to the antibody such that the attachment does not interfere or prevent the antibody from binding to the epitope. The chemical moiety can be conjugated to an antibody using any technique known in the art.

[0178] A detectable label can be any moiety attached to a specific binding partner (e.g., an antibody and/or Cas9) to render the reaction between members of a specific binding pair (e.g., the antibody and Cas9) detectable. A label can produce a signal that is detectable by visual or instrumental means. In some embodiments, a moiety itself may not be detectable but can become detectable upon reaction with another moiety. In some embodiments, the detectable label can be selected from the group consisting of: a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, and electrochemiluminescent label, or a combination thereof. In some embodiments, the electrochemiluminescent label is a sulfo-tag. Sulfo-tag NHS-ester is an N-hydroxy succinimide ester which can couple to primary amine groups of proteins and sulfo-tag conjugated proteins can used as detection reagents in immunoassays. The signal generated from the detectable label moiety can be detected and the presence of the signal indicates presence of Cas9 in a sample.

Compositions and Kits for Detecting Cas9

[0179] Provided herein also include compositions, reagents and kits for the detection of Cas9 (alone or in a RNP) in a biological sample. The compositions can comprises one or more antibodies or fragments thereof of the present disclosure and a buffer (e.g., an assay buffer, a wash buffer, a kinetic buffer and/or a read buffer).

[0180] For example, a composition can comprise one or more antibodies or fragments thereof, having one or more of the CDR regions having SEQ ID NOs: l-78 or variants thereof having a single substitution, deletion or insertion in any one of SEQ ID Nos: 1-78. A composition can comprise one or more antibodies or fragments thereof, having one or more of the light chain variable regions having SEQ ID Nos: 79-91 or variants thereof having about, at least, or at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of SEQ ID Nos: 79- 91. A composition can comprise one or more antibodies or fragments thereof, having one or more of the light chain variable regions having SEQ ID Nos: 79-91 or variants thereof having one, two or three mismatches relative to any one of SEQ ID Nos: 79-91. A composition can comprise one or more antibodies or fragments thereof, having one or more of the heavy chain variable regions having SEQ ID NOs: 92-104 or variants thereof having about, at least, or at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of SEQ ID Nos: 92-104. A composition can comprise one or more antibodies or fragments thereof, having one or more of the heavy chain variable regions having SEQ ID Nos: 92-104 or variants thereof having one, two or three mismatches relative to any one of SEQ ID Nos: 92-104.

[0181] In some embodiments, the compositions or kits can comprise two of the antibodies or fragments thereof, the two antibodies or fragments thereof being different from one another.

[0182] For example, a composition or a kit can comprise a first antibody or fragment thereof, having: a VL CDR1 of SEQ ID NO: 1 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 1, a VL CDR2 of SEQ ID NO: 14 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 14, a VL CDR3 of SEQ ID NO: 27 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 27, a VH CDR1 of SEQ ID NO: 40 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 40, a VH CDR2 of SEQ ID NO: 53 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 53, and a VH CDR3 of SEQ ID NO: 66 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 66. The composition or kit can comprise a second antibody or fragment thereof having: a VL CDR1 of SEQ ID NO: 4 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 17 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 17, a VL CDR3 of SEQ ID NO: 30 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 30, a VH CDR1 of SEQ ID NO: 43 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 43, a VH CDR2 of SEQ ID NO: 56 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 56, and a VH CDR3 of SEQ ID NO: 69 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 69.

[0183] In some embodiments, the first antibody or fragment thereof can comprise a light chain having an amino acid sequence of SEQ ID NO: 79 or a peptide having about, at least or at least about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO:79, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:92 or a peptide having about, at least or at least about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO:92. In some embodiments, the second antibody or fragment thereof can comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO:82 or a peptide having about, at least or at least about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO:82, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:95 or a peptide having about, at least or at least about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 95.

[0184] In another example, the composition or kit can comprise a first antibody or fragment thereof having: a VL CDR1 of SEQ ID NO: 7 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 7, a VL CDR2 of SEQ ID NO: 20 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 20, a VL CDR3 of SEQ ID NO: 33 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 33, a VH CDR1 of SEQ ID NO: 46 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 46, a VH CDR2 of SEQ ID NO: 59 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 59, and a VH CDR3 of SEQ ID NO: 72 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 72. The composition or kit can comprise a second antibody or fragment thereof having: a VL CDR1 of SEQ ID NO: 8 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 8, a VL CDR2 of SEQ ID NO: 21 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 21, a VL CDR3 of SEQ ID NO: 34 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 34, a VH CDR1 of SEQ ID NO: 47 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 47, a VH CDR2 of SEQ ID NO: 60 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 60, and a VH CDR3 of SEQ ID NO: 73 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 73.

[0185] In some embodiments, the first antibody or fragment thereof can comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO: 85 or a peptide having about, at least or at least about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 85, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 98 or a peptide having about, at least or at least about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the second antibody or fragment thereof can comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO: 86 or a peptide having about, at least or at least about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 86, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 99 or a peptide having about, at least or at least about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 99.

[0186] For purposes of capturing Cas9, an antibody or fragment thereof can be coated on a solid support such as for example, a microparticle (e.g., magnetic particle), bead, test tube, microtiter plate, cuvette, membrane, scaffolding molecule, film, filter paper, disc or chip. Another antibody or fragment thereof can be on separate solid supports or provided in a solution.

[0187] One or more of the antibody or fragment thereof in the composition or kit can be associated with a detectable label. The detectable label can be selected from the group comprising of a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, and electrochemiluminescent label (e.g., a sulfo-tag), or a combination thereof. In some embodiments, one of the antibodies or fragments thereof can be labeled with an N-hydroxysuccinimide ester (e.g., sulfo-tagged). For example, a detection antibody or fragment thereof can be sulfo-tagged and used as a detection antibody to generate electrochemiluminescence in an immunoassay (e.g., in a Meso Scale Discovery assay). Optionally, a first antibody or fragment thereof is biotinylated, and a second antibody or fragment thereof is sulfo-tagged..

[0188] The compositions and kits herein described can be used in any biological sample comprising or suspected of comprising a Cas9 or a variant or a derivative thereof. The term “biological sample” as used herein includes, but is not limited to, any quantity of a substance from a living thing or formerly living thing. Such living things include, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits and other animals. Such substances include, but are not limited to, blood, (e.g., whole blood or components thereof), plasma, serum, urine, saliva, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes and spleen. In some embodiments, the biological sample comprises cells, cell supernatant, cell lysate, or a combination thereof. In some embodiments, the cell supernatant is post-thaw cell supernatant. The cell supernatant can comprise, for example, in CS5 (Cryostor®) cell cryopreservation/freezing medium. The cell lysate can comprise, for example, lysis buffer and Halt inhibitor cocktail (e.g., Halt Protease Inhibitor Cocktail or Halt Protease and Phosphatase Inhibitor Cocktail).

[0189] The biological sample can be fixed or not fixed, or comprises fixed biological materials (e.g., cells) and/or biological materials that are not fixed. In some embodiments, the biological sample is fixed to preserve the biological materials such as cells from decay and/or degradation and/or to maintain the antigenicity of the target molecules. Fixation can be accomplished using any convenient protocols known in the art such as immunohistochemistry (IHC) fixation protocol. IHC fixation immobilizes antigens while retaining cellular and subcellular structure. In some embodiments, fixation can be a chemical fixation using a fixation reagent (e.g. a reagent that contains at least one fixative). Common fixatives include crosslinking fixatives, precipitating fixatives, oxidizing fixatives, Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE) fixatives, or a combination thereof. Examples of suitable cross-liking fixatives include, but are not limited to, aldehydes (e.g., formaldehyde, also commonly referred to as “formalin”; paraformaldehyde, the polymeric form of formaldehyde; and glutaraldehyde), imidoesters, NHS (N-Hydroxysuccinimide) esters, and the like. Examples of suitable precipitating fixatives include, but are not limited to, alcohols (e.g., methanol, ethanol, etc.), acetone, acetic acid, and others known in the art.

[0190] The biological sample can be obtained or derived from human or non-human organisms (including but are not limited to primates, birds, fish, reptiles, amphibians, or plants). The biological sample can be, or be derived from, a clinical sample, a pharmaceutical product, an environmental sample, a nutraceutical product, a animal or veterinary product, or any combination thereof. The biological sample can be, or can comprise, tissue(s), cell(s), biological fluid(s), or any combination thereof. The tissue can, for example, comprise cells transplanted into the tissue, and optionally the cells have been gene edited. In some embodiments, the tissue can comprise cells that have grown into the tissue, and optionally the cells have been gene edited. In some embodiments, the biological sample is derived from a pharmaceutical composition comprising cells. The biological sample can comprise any cells capable of being edited by CRISPR/Cas9 gene editing system. In some embodiments, the biological sample comprises immune cells, stem cells, blood cells (e.g., peripheral blood mononuclear cells (PBMCs)), or a combination thereof. In some embodiments, the biological sample comprises T cells, B cells, neutrophils, monocytes, lymphocytes, and dendritic cells. In some embodiments, the biological sample comprises immune cells such as a leukopak sample. A leukopak sample can be an enriched leukapheresis product collected from peripheral blood, which typically contains a variety of blood cells including monocytes, lymphocytes, platelets, plasma, and red cells. Examples of immune cells include, but are not limited to, T cells, B cells, dendritic cells, natural killer cells (NK cells), or any combination thereof. The cells disclosed herein can comprise chimeric antigen receptors (CARs). For example, the T cells can comprise CAR-T cells, the NK cells can comprise CAR-NK cells, or a combination thereof. The stem cells can comprise hemopoietic stem cells (HSCs), hemopoietic progenitor cells (HPCs), induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells, mesenchymal stem cells (MSCs), tissue-specific stem cells, or a combination thereof. In some embodiments, the biological sample is obtained or derived from a subject (e.g., a mammal, a primate, a human) prior to, subject to, or after receiving a treatment involving the use of Cas9 endonuclease. In some embodiments, the biological sample is obtained or derived from a subject (e.g., a mammal, a primate, a human) prior to, subject to, or after receiving a gene therapy treatment, e.g., a gene therapy based on CRISPR/Cas9 gene editing. For example, the CRISPR/Cas9 gene editing can be an in vivo gene editing administrated to the subject. The gene(s) being edited can include, but are not limited to, cardiovascular disease related genes (e.g., ANGPTL3, PCSK9 and LPA), genes related to diabetes (e.g., insulin, GLP-1), and oncology genes. The methods disclosed herein can be used to detect Cas9 protein at, at about, at least, or at most, 30 minutes, one hour, two hours, three hours, six hours, nine hours, twelve hours, eighteen hours, one day, thirty-six hours, two days, three days, four days, five days, six days, a week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, nine months, one year, two years, three years, four years, five years, or a number or a range between any two of these values, after the gene therapy. The Cas9 antibodies, methods, compositions, and kits disclosed herein can be used to detect Cas9 in various tissues, organs and systems of the subject, for example, liver, lung, brain, central neuronal system (CNS), heart, intestines, blood, plasma, stomach, prostate, bladder, kidney, colon, pancreas, skin, thyroidjoins, spleen, bones, anus, appendix, bone marrow, cervix, clitoris, eyes, hair follicle, spinal cord, placenta, rectum, tonsils, testes, salivary glands, ovary, and any combination thereof. In some embodiments, the antibodies, methods, compositions, and kits disclosed herein is used to detect Cas9 in one or more of bodily fluids, including but not limited to, intravascular fluid (blood plasma), interstitial fluid, lymphatic fluid, and transcellular fluid. The bodily fluids include, but are not limited to, cerebrospinal fluid, synovial fluid, pleural fluid, amniotic fluid, pericardial fluid, peritoneal fluid, saliva, blood, and semen.

[0191] The composition described herein can, for example, comprise a biological sample, cells, cell supernatant, cell lysate or a combination thereof. The biological sample can comprise or is suspected of comprising a Cas9 or a variant or a derivative thereof. The Cas9 or a variant or a derivative thereof can a free Cas9 (e.g., ApoCas9), a Cas9 fragment, a small Cas9, a dead Cas9 (dCas9), a fusion protein comprising Cas9 or a fragment or a variant thereof (e.g., a Cas9 base editor such as cytosine base editors and adenine base editors), a Cas9 nickase, or a mutant thereof. In some embodiments, the Cas9 or a variant or a derivative thereof can bind with a guide RNA to form a Cas9-gRNA ribonucleoprotein (RNP). In some embodiments, the Cas9 can be selected from the group consisting of Streptococcus pyogenes Cas9, Streptococcus aureus Cas9, Staphylococcus lugdunensis Cas9, Neisseria meningitides Cas9, Streptococcus thermophilus CRISPR1 Cas9, S Streptococcus thermophilus CRISPR3 Cas9, Treponema denticola Cas9, Campylobacter jejuni Cas9, Lachnospiraceae bacterium ND2006 Cpfl and Acidaminococcus sp. BV3L6 Cpfl.

[0192] The antibodies and fragment thereof described herein can be provided in a kit and used in any immunoassay format known to those skilled in the art. The kit can comprise purified, or isolated, and optionally lyophilized, Cas9 specific antibody, and/or one or more containers such as tube, microtiter plates or stripes which can be already coated with one of the antibodies (e.g., a coating/capture antibody) for conducting the assay, and/or a buffer such as an assay buffer, a wash buffer, or a read buffer. In some embodiments, the kit can comprise a kinetic buffer. A kinetic buffer can be used to inhibit non-specific binding to surfaces and other proteins. In some embodiments, a kinetic buffer can contain a blocking agent, bovine serum albumin, and surfactant (e.g., Tween-20) as will be understood by a skilled person in the art. The components can be provided as a concentration solution, a working solution, a substrate solution, or a stop solution.

[0193] The antibodies can be provided in separate containers or pre-dispensed into an appropriate assay format, for example, into microtiter plates. In some preferred embodiments, there are two or more containers provided, each container comprising an antibody or fragment thereof of the present disclosure. For example, in some embodiments, the kit can comprise two containers: the first container contains a first antibody or fragment thereof for capturing Cas9 (e.g., a capture antibody) and the second container contains a second antibody or fragment thereof for detecting the captured Cas9 (e.g., a detection antibody). One or both of the capture and detection antibodies can be associated with a detectable label known in the art and described herein. For example, the first antibody or fragment thereof is biotinylated, and the second antibody or fragment thereof is labeled with a sulfo-tagged (e.g., sulfo-tagged streptavidin). In some embodiments, the kit can comprise a solid support coated with an agent that allows binding of a capture antibody and the kit further comprises each of the individual capture and detection antibody pairs in separate containers. The kit can optionally include other reagents required to conduct an assay such as buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents, and the like.

[0194] In some embodiments, a kit can further include instructions for using the components of the kit to practice the method s described herein such as to assay a sample for the presence, amount and/or concentration of Cas9 by immunoassay. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions can be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging), etc. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD- ROM, diskette, flash drive, etc. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the Internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

Methods of Detecting Cas9

[0195] Provided herein includes methods of using the antibodies or fragments thereof, and related compositions and kits. For example, the methods, compositions and kits can be used to detect the presence/absence, amount and/or concentration of Cas9 in a biological sample. In some embodiments, the method comprises contacting a biological sample with the antibody or fragment thereof of the present disclosure, thereby detecting the presence or absence of Cas9 in the biological sample.

[0196] The biological sample can be pretreated prior to contacting with the antibody or fragment thereof, for example, using a pretreatment reagent. A pretreatment reagent can be any reagent appropriate for use with the immunoassay and kits of the present disclosure, including for example one or more solvents, salts, and/or detergents. In some embodiments, a pretreatment step can comprise collecting a cell supernatant of a biological sample by removing a precipitate from a mixture formed by addition of a pretreatment agent to the biological sample. In some embodiments, a pretreatment step can comprise lysing the cells in a biological sample to produce a cell lysate. Any cell lysis techniques suitable to effectively release the cell content can be used herein. Cell lysis can comprise physical approaches such as mechanical disruption, liquid homogenization, high frequency sound waves (sonication), freeze/thaw cycles and manual grinding, or detergent- or solution-based cell lysis methods as will be understood by a person skilled in the art. In some embodiments, lysing the cells comprises freezing and thawing the cells one or more times. %

[0197] In some embodiments, the method can further comprise fixing the biological sample prior to contacting the biological sample with the antibody or fragment thereof. For example, the biological sample can be contacted with a fixation reagent comprising one or more fixatives herein described under a condition for a certain period time. The time period depends on the temperature, the nature of the sample and the choice of fixatives as will be understood by a person skilled in the art.

[0198] The antibody or fragment thereof can be any of the antibodies or fragments thereof described in the present disclosure, including for example antibodies or fragments thereof having one or more of SEQ ID Nos: 1-39 (VL CDRs), SEQ ID Nos: 40-78 (VH CDRs), SEQ ID Nos: 79-91 (VL regions), SEQ ID Nos: 92-104 (VH regions), or variants or fragments thereof described herein.

[0199] The antibody or fragment thereof can be immobilized (e.g., bound) to a solid support or a solid phase directly or indirectly (e.g., using a linking agent). A “solid phase” or a “solid support” refers to any material that is insoluble, or can be made insoluble by a subsequent reaction. The solid phase can be chosen for its intrinsic ability to attract and immobilize a capture agent (e.g., a capture antibody). Alternatively, the solid phase can have affixed thereto a linking agent that has the ability to attract and immobilize the capture agent. The linking agent can, for example, include a charged substance that is oppositely charged with respect to the capture agent itself or to a charged substance conjugated to the capture agent. In general, the linking agent can be any binding partner (preferably specific) that is immobilized on (attached to) the solid phase and that has the ability to immobilize the capture agent through a binding reaction. The linking agent enables the indirect binding of the capture agent to a solid phase material before the performance of the assay or during the performance of the assay. The solid phase can, for example, be plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon, including, for example, a test tube, microtiter well, sheet, bead, microparticle, chip, and other configurations known to those of ordinary skill in the art.

[0200] The biological sample can be any biological sample described herein, such as cell lysate or cell supernatant, and optionally the cell supernatant is post-thaw cell supernatant. The biological sample can comprise Cas9 described herein or is suspected of comprising Cas9. For example, the biological sample can comprise a free Cas9 protein (ApoCas9) or a fragment thereof, a Cas9 having endogenous nuclease activity, a small Cas9, a dead Cas9 (dCas9), a fusion protein comprising Cas9 (e.g., Cas9 base editor), or a variant thereof. The biological sample can comprise a Cas9-gRNA ribonucleoprotein.

[0201] The methods, compositions and kits described herein are capable of detecting Cas9 with high sensitivity, high specificity, and/or high accuracy. The methods, compositions and kits can be used, in some embodiments, to detect Cas9 in samples with low concentrations or low amount of Cas9. For example, in some embodiments, the biological sample comprises Cas9 at a concentration of about, at least, at least about, at most, or at most about 0.0001 pg/mL, 0.0005 pg/mL, 0.001 pg/mL, 0.0015 pg/mL, 0.002 pg/mL, 0.0025 pg/mL, 0.003 pg/mL, 0.0035 pg/mL, 0.004 pg/mL, 0.0045 pg/mL, 0.005 pg/mL, 0.0055 pg/mL, 0.006 pg/mL, 0.0065 pg/mL, 0.007 pg/mL, 0.0075 pg/mL, 0.008 pg/mL, 0.0085 pg/mL, 0.009 pg/mL, 0.0095 pg/mL, 0.01 pg/mL, 0.015 pg/mL, 0.02 pg/mL, 0.025 pg/mL, 0.03 pg/mL, 0.035 pg/mL, 0.04 pg/mL, 0.045 pg/mL, 0.05 pg/mL, 0.06 pg/mL, 0.07 pg/mL, 0.08 pg/mL, 0.09 pg/mL, 0.1 pg/mL, 0.15 pg/mL, 0.2 pg/mL, 0.25 pg/mL, 0.3 pg/mL, 0.4 pg/mL, 0.5 pg/mL, 0.6 pg/mL, 0.7 pg/mL, 0.8 pg/mL, 0.9 pg/mL, 1 pg/mL, 1.2 pg/mL, 1.4 pg/mL, 1.6 pg/mL, 1.8 pg/mL, 2 pg/mL, 2.5 pg/mL, 3 pg/mL, 3.5 pg/mL, 4 pg/mL, 4.5 pg/mL, 5 pg/mL or a number or range between any two of these values. In some embodiments, the biological sample comprises Cas9 at a concentration no more than 0.01 pg/mL.

[0202] In some embodiments, the biological sample is a cell supernatant (e.g., a postthaw cell supernatant) comprising Cas9 at a concentration about, at least, at least about, at most, or at most about 0.1 ng/mL, 0.15 ng/mL, 0.2 ng/mL, 0.25 ng/mL, 0.3 ng/mL, 0.35 ng/mL, 0.4 ng/mL, 0.45 ng/mL, 0.5 ng/mL, 0.55 ng/mL, 0.6 ng/mL, 0.65 ng/mL, 0.7 ng/mL, 0.75 ng/mL, 0.8 ng/mL, 0.85 ng/mL, 0.9 ng/mL, or a number or a range between any two of these values. In some embodiments, the Cas9 is at a concentration in the range of 0.34-0.8 ng/mL, for example 0.34-0.7 ng/ml, 0.35-0.6 ng/ml, 0.4-0.5 ng/ml. In some embodiments, the biological sample is a cell supernatant comprising Cas9 at a concentration of no more than 0.8 ng/mL.

[0203] In some embodiments, the biological sample is a cell lysate comprising Cas9 at a concentration about, at least, at least about, at most or at most about 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL, 11 ng/mL, 12 ng/mL, 13 ng/mL, 14 ng/mL, 15 ng/mL, 16 ng/mL, 17 ng/mL, 18 ng/mL, 19 ng/mL, 20 ng/mL, 21 ng/mL, 22 ng/mL, 23 ng/mL, 24 ng/mL, 25 ng/mL, 26 ng/mL, 27 ng/mL, 28 ng/mL, 29 ng/mL, 30 ng/mL, 31 ng/mL, 32 ng/mL, 33 ng/mL, 34 ng/mL, 35 ng/mL, or a number or a range between any two of these values. In some embodiments, the Cas9 is at a concentration in the range of 1-34 ng/mL, for example 2-30 ng/mL, 5-25 ng/mL, or 10-20 ng/mL. In some embodiments, the biological sample is a cell lysate comprising Cas9 at a concentration of no more than 34 ng/mL.

[0204] The method can comprise contacting a biological sample with one or more of antibodies or fragments thereof having specificity to a Cas9 protein under conditions allowing the one or more antibodies or fragments thereof to bind to the Cas9, wherein the biological sample comprises Cas9 at a concentration of no more than 0.01 pg/mL. The method can further comprise detecting the binding of the one or more antibodies or fragments thereof to the Cas9, wherein the detection of the binding is indicative of the presence of Cas9 in the biological sample.

[0205] In some embodiments, a method can comprise contacting a biological sample with one or more of the antibodies or fragments thereof of the present disclosure under conditions allowing the one or more antibodies or fragments thereof to bind to Cas9, wherein the biological sample comprises Cas9 or is suspected of comprising Cas9, and detecting the binding of the one or more antibodies or fragments thereof to Cas9, wherein the detection of the binding is indicative of the presence of Cas9 in the biological sample.

[0206] The antibodies or fragments thereof can be used in molar excess amounts of the maximum amount of Cas9 expected in the sample. For example, the antibody or fragment thereof can be provided at a concentration in a range of 10 to 10⁷ pg/ml. In some embodiments, the antibody or fragment thereof is provided at a concentration of about 0.001 pg/ml, 0.01 pg/ml, 0.1 pg/ml, 0.2 pg/ml, 0.4 pg/ml, 0.6 pg/ml, 0.8 pg/ml, 1 pg/ml, 2 pg/ml, 3 pg/ml, 4 pg/ml, 5 pg/ml, 6 pg/ml, 7 pg/ml, 8 pg/ml, 9 pg/ml, 10 pg/ml, 20 pg/ml, 30 pg/ml, 40 pg/ml, 50 pg/ml, or a number or a range between any two of these values.

[0207] The mixture of the antibody or fragment thereof and the biological sample can be incubated for a time period to allow for the formation of an antibody-Cas9 complex. The incubation can be carried out at a pH of from about 4.5 to about 10.0, at a temperature of from about 2° C to about 45° C, and for a time period from at least about one minute to about 24 hours, preferably from about 1 hour to 24 hours. The incubation can be conducted in one step (e.g., the sample and at least one antibody or fragment thereof are added sequentially or simultaneously to a reaction vessel) or in more than one step (e.g., mixing the sample and a first antibody or fragment thereof to form a first mixture, incubating the first mixture, adding a second antibody or fragment thereof to the first mixture to form a second mixture, incubating the second mixture, and so on).

[0208] The binding of the one or more antibodies or fragments thereof to Cas9 can be detected using any suitable assay known in the art. In some embodiments, detecting the binding of the antibody or fragment thereof to the Cas9 is performed using an immunodetection assay. Examples of such assays include, but are not limited to, immunoassay, such as sandwich immunoassay (e.g., monoclonal-polyclonal sandwich immunoassays, including radioisotope detection (radioimmunoassay (RIA)), enzyme detection (enzyme immunoassay (EIA) or enzyme- linked immunosorbent assay (ELISA) (e.g., Quantikine ELISA assays, R&D Systems, Minneapolis, Minn.), and Mesosacale Discovery (MSD) Electro chemiluminescent ELISA), competitive inhibition immunoassay (e.g., forward and reverse), fluorescence polarization immunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT), bioluminescence resonance energy transfer (BRET), and homogeneous chemiluminescent assay. In some embodiments, the immunodetection assay is selected from the group consisting of: western blotting, enzyme-linked immunosorbent assay (ELISA), and bead-based luminescent amplification assay. In some embodiments, the ELISA assay is a electro chemiluminescent (ELC) ELISA assay. In some embodiments, the bead-based luminescent amplification assay is an ALPHALisa Assay. In the Alpha-LISA assay, biotinylated antibodies are associated with a streptavidin-coated alpha donor bead, and used to bind to an anti-analyte-conjugated AlphaLISA acceptor bead. In the MSD assay described herein, a sulfo-tag conjugated detection antibody and a biotinylated capture antibody are used in a pair to bind and detect the presence of an analyte (e.g., an analyte immobilized to a streptavidin coated plate) to which the detection antibody and the capture antibody can specifically bind to. One of the advantages of the MSD assay described herein is that the lack of need for secondary detection antibody is needed.

[0209] In some embodiments, the method uses two or more antibodies or fragments thereof to detect the presence, amount or concentration of Cas9 in a biological sample, one antibody or fragment thereof being a capture antibody for capturing Cas9 and another being a detection antibody to generate a detectable signal (e.g., electrochemiluminescence) in an immunoassay, thereby detecting the presence, amount or concentration of Cas9. For example, a biological sample can be first contacted with at least one capture antibody and then with at least one detection antibody. In another example, the sample can be contacted simultaneously with a capture antibody and a detection antibody. In some embodiments, a sample suspected of containing Cas9 or a fragment thereof is first contacted with at least a first antibody or fragment thereof under conditions that allow the formation of a first antibody-Cas9 complex. This step can be repeated or simultaneously conducted with at least a second antibody or fragment thereof. In some embodiments, the order in which the biological sample, the first antibody or fragment thereof, and the second antibody or fragment thereof are added to form a reaction mixture is not critical.

[0210] In some embodiments, the first antibody or fragment thereof (e.g., the capture antibody) can comprise a VL CDR1 of SEQ ID NO: 1 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 1, a VL CDR2 of SEQ ID NO: 14 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 14, a VL CDR3 of SEQ ID NO: 27 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 27, a VH CDR1 of SEQ ID NO: 40 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 40, a VH CDR2 of SEQ ID NO: 53 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 53, and a VH CDR3 of SEQ ID NO: 66 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 66. The second antibody or fragment thereof (e.g. the detection antibody) can comprise a VL CDR1 of SEQ ID NO: 4 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 17 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 17, aVL CDR3 of SEQ ID NO: 30 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 30, a VH CDR1 of SEQ ID NO: 43 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 43, a VH CDR2 of SEQ ID NO: 56 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 56, and a VH CDR3 of SEQ ID NO: 69 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 69. In some embodiments, the first and second antibody or fragment thereof are used to detect a biological sample comprises a cell supernatant.

[0211] In some embodiments, the first antibody or fragment thereof can comprise a light chain having an amino acid sequence of SEQ ID NO: 79 or a peptide having about, at least or at least about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 79, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 92 or a peptide having about, at least or at least about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 92. In some embodiments, the second antibody or fragment thereof can comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO: 82 or a peptide having about, at least or at least about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 82, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95 or a peptide having about, at least or at least about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 95. In some embodiments, the first and second antibody or fragment thereof are used to detect Cas9 in a biological sample comprises a cell supernatant.

[0212] In some embodiments, the first antibody or fragment thereof (e.g., the capture antibody) can comprise a VL CDR1 of SEQ ID NO: 7 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 7, a VL CDR2 of SEQ ID NO: 20 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 20, a VL CDR3 of SEQ ID NO: 33 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 33, a VH CDR1 of SEQ ID NO: 46 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 46, a VH CDR2 of SEQ ID NO: 59 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 59, and a VH CDR3 of SEQ ID NO: 72 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 72. The second antibody or fragment thereof (e.g. the detection antibody) can comprise a VL CDR1 of SEQ ID NO: 8 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 8, a VL CDR2 of SEQ ID NO: 21 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 21, a VL CDR3 of SEQ ID NO: 34 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 34, a VH CDR1 of SEQ ID NO: 47 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 47, a VH CDR2 of SEQ ID NO: 60 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 60, and a VH CDR3 of SEQ ID NO: 73 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 73. In some embodiments, the first and second antibody or fragment thereof are used to detect a biological sample comprises a cell lysate.

[0213] In some embodiments, the first antibody or fragment thereof can comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO: 85 or a peptide having about, at least or at least about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 85, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 98 or a peptide having about, at least or at least about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the second antibody or fragment thereof can comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO: 86 or a peptide having about, at least or at least about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 86, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 99 or a peptide having about, at least or at least about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the first and second antibody or fragment thereof are used to detect Cas9 in a biological sample comprises a cell lysate.

[0214] Accordingly, in some embodiments, contacting a biological sample with one or more of the antibodies or fragments thereof comprises contacting the biological sample with a first antibody or fragment thereof of the present disclosure under a condition allowing binding of the first antibody or fragment thereof to Cas9 to form a first antibody-Cas9 complex and contacting the first antibody-Cas9 complex with a second antibody or fragment thereof of the present disclosure under a condition allowing binding of the second antibody or fragment thereof to the first antibody-Cas9 complex. For example, the first antibody or fragment thereof can be a capture/coating antibody and the second antibody or fragment thereof can be a detection antibody (e.g., associated with a detectable label such as a sulfo-tag). The method can detect the presence or absence of Cas9 in the biological sample by detecting the binding of the second antibody or fragment thereof to the first antibody-Cas9 complex. In some embodiments, the first antibody or fragment thereof is a capture/coating antibody and the second antibody or fragment thereof is a detection antibody.

[0215] In some embodiments, the first antibody or fragment thereof (e.g., the coating/capture antibody) is immobilized on a solid phase. The method can further comprise attaching the first antibody or fragment thereof to the solid phase directly or indirectly (e.g., using a linking agent). In some embodiments, none of the antibodies or fragments thereof are immobilized but are instead added to the sample simultaneously or sequentially.

[0216] In some embodiments, the method can comprise removing unbound Cas9 after contacting the biological sample with the first antibody or fragment thereof and prior to contacting the first antibody-Cas9 complex with the second antibody or fragment thereof using any technique known in the art. For example, the unbound Cas9 can be removed by washing (e.g., using a wash buffer).

[0217] In some embodiments, the method can comprise removing unbound second antibody or fragment thereof after contacting the first antibody-Cas9 complex with a second antibody or fragment thereof of the present disclosure under a condition allowing binding of the second antibody or fragment thereof to the first antibody-Cas9 complex (e.g., after incubating the first antibody-Cas9 complex and the second antibody or fragment thereof for a time period). The unbound second antibody or fragment thereof can be removed by washing (e.g. using a wash buffer).

[0218] In some embodiments, one or both of the first and second antibodies or fragments thereof is associated with a detectable label or labeled with a specific partner that allows capture or detection. For example, a detection antibody can be associated with a detectable label. The detectable label can be any moiety capable of producing a signal (e.g., optical, electrical, and/or fluorescent signal) that can be detected by visual or instrumental means. A detectable label can be selected from the group consisting of a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, an electrochemiluminescent label, or a combination thereof. For example, the detectable label can be a radioactive label (such as ³H, ¹²⁵1, ³⁵S, ¹⁴C, ³²P, and ³³P), an enzymatic label (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, and the like), a chemiluminescent label (such as acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, and the like), a fluorescent label (such as fluorescein (e.g., 5 -fluorescein, 6-carboxyfluorescein, 3'6- carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmium selenide), a thermometric label, or an immunopolymerase chain reaction label. In some embodiments, the detectable label is a sulfo-tag. The detectable label can be associated with the antibodies either directly or indirectly through a coupling agent known in the art.

[0219] In some embodiments, binding of an antibody or fragment thereof generates a detectable signal such as a chemiluminescent signal which can be detected using routine techniques known to those skilled in the art. Based on the intensity of the signal generated, the presence/absence of Cas9 in the sample can be determined and the amount can be quantified. In some embodiments, the amount of Cas9 can be quantified by comparing the amount of signal generated to a standard curve for Cas9 or by comparison to a reference standard as will be understood by a person skilled in the art.

[0220] The antibody or fragment thereof, and related compositions, kits and methods of use provide a sensitive and selective detection of Cas9 in a sample being assayed. In some embodiments, the antibody or fragment thereof, and related compositions, kits and methods of use can provide improved residual Cas9 detection with increased sensitivity compared to existing assays (e.g., a combination of mouse and rabbit anti-Cas9 antibodies with different binding characteristics). In some embodiments, the antibody or fragment thereof, and related compositions, kits and methods of use can have a detection capability of detecting residual Cas9 at a concentration of about, at most, or at most about 34 ng/mL, 33 ng/mL, 32 ng/mL, 31 ng/mL, 30 ng/mL, 29 ng/mL, 28 ng/mL, 27 ng/mL, 26 ng/mL, 25 ng/mL, 24 ng/mL, 23 ng/mL, 22 ng/mL, 21 ng/mL, 20 ng/mL, 19 ng/mL, 18 ng/mL, 17 ng/mL, 16 ng/mL, 15 ng/mL, 14 ng/mL, 13 ng/mL, 12 ng/mL, 11 ng/mL, 10 ng/mL, 9 ng/mL, 8 ng/mL, 7 ng/mL, 6 ng/mL, 5 ng/mL, 4 ng/mL, 3 ng/mL, 2 ng/mL, 1 ng/mL, 0.8 ng/mL, 0.7 ng/mL, 0.6 ng/mL, 0.5 ng/mL, 0.4 ng/mL, 0.3 ng/mL, 0.2 ng/mL, 0.1 ng/mL or a number or a range between any two of these values. Also disclosed herein includes instructions and manuals for using the antibodies, compositions, kits and systems disclosed herein for detecting Cas9.

EXAMPLES

[0221] Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure.

Example 1

Production of Antibodies Against Cas9

[0222] This Example provides the procedure and initial characterization for antibodies that recognize one or more forms of Cas9. A Beacon B-cell screening method (Abveris) was utilized to generate antibodies that recognize one or more forms of Cas9. Mice were immunized with weekly injections for three to six weeks with either SpCas9 (SEQ ID NO: 209) or with RNP of Cas9 and a gRNA (SEQ ID NO: 201), using a rapid immunization protocol. At the end of immunization protocol, mice were screened for the presence of specific antibodies in their serum by ELISA and isolated plasma B cells from the selected immunized mice were screened in OptoSelect Chip on Berkeley Lights Optofluidic Beacon platform. The positive plasma B cells were identified, and the antibody variable light and heavy chain sequences from the antibodies produced by these cells were sequenced. A total of 96 different recombinant antibodies were generated and expressed in cell lines and affinity purified by column chromatography.

Binding Characterization of Antibodies that Recognize Cas9

[0223] The 96 antibodies were screened on a BLI (Bio-layer Light interferometry) based Octet platform for their binding characteristics to Cas9 (SEQ ID NO: 209) alone (“ApoCas9”), a guide RNA (SEQ ID NO: 210), and RNP of Cas9 and gRNA. The BLI technology-based Octet detected label-free real time molecular interaction analysis and measured the difference in reflected light’ s wavelength (AZ) between the two surfaces on the biosensor. The conditions for loading antibody and analyte concentrations, binding time, biosensor probe selection and other assay steps were established using commercial Cas9 antibodies.

[0224] 40 mL of IX OCTET® Kinetics buffer was prepared by adding 4 mL of stock

(10X buffer) to 36 mL of IX DPBS and mixing well. This buffer was used to make all antigen and antibody solutions as shown in Table 1 for binding experiments and in Table 2 for Epitope binning experiments. The biosensor hydration plate was prepared by adding 225 pL of IX OCTET® binding buffer to each well. An example assay plate using a commercial Cas9 antibody (4G10) was prepared based on plate layout defined in Table 1. An exemplary result of a typical assay step in a binding experiment is shown in FIG. 1.

Table 1: Binding Experiment

* Volume required for each well to be final volume of 225 pL was calculated.

Table 2: Epitope Binning Experiment

* Volume required for each well to be final volume of 225 pL was calculated.

[0225] 10.0 nM of each clone antibody was tested using a Protein G biosensor or AMC with the analyte/antigen being a buffer control (i.e., no antigen), ApoCas9, RNP, or gRNA. The screening on octet resulted in dividing recombinant into three groups: those that bind RNP, those that bind ApoCas9, and those that could bind both. 46 antibodies were positive to ApoCas9, RNP, or both, 38 antibodies were negative to ApoCas9 or RNP, and 12 were undetermined due to low antibody concentration. Table 3 below lists exemplary antibodies.

Table 3: Exemplary antibodies

[0226] Antibodies that had fast on-rate binding with higher affinity and were stable during disassociation were selected for further studies. Six ApoCas9 specific antibodies that had stable on-rate and stable off-rate were selected for further studies. Similarly, RNP specific antibodies were selected that had stable on-rates and off-rates. For RNP and ApoCas9 cross- reactive antibodies, antibodies that were stable for one or the other (typically RNP) and dissociated rapidly were selected.

[0227] RNPs comprising SpCas9 (SEQ ID NO: 209) with other gRNAs (e.g., B2M gRNA, VEGFA22 gRNA and IL2RG gRNA) were also tested. The data (not shown) suggested that the Cas9 specific antibodies with stable on-rates and off-rates were also found to be specific to RNPs comprising the Cas9 regardless of the gRNAs in the RNPs. These data demonstrated that the anti-Cas9 antibodies disclosed herein exhibit comparative binding specificity to Cas9 alone and to RNPs of Cas9 and a gRNA.

Example 2

Use of Cas9 Antibodies in Western blotting

[0228] Various Cas9 antibodies, as described in Example 1, were used in Western blotting to determine if they could recognize and bind ApoCas9 (e.g., SpCas9) in 100,000 cells equivalent T cell lysates that have been spiked with 500 ng (FIG. 2), 50 ng (FIG. 3), 1 ng (FIG. 4), or 10 ng of SpCas9 (FIG. 4). FIGS. 2 and 3 show detection in the T cell lysates spiked with or without SpCas9 by Western blot (on Jess). FIG. 4 shows detection of spiked SpCas9 in the T cell lysates by Western blot (on Jess) with SpCas9 specific antibodies

Example 3

Use of Cas9 Antibodies in ELISA

[0229] Various SpCas9 antibodies, as described in Example 1, were used in indirect and sandwich ELISA formats. In the indirect ELISA, individual untagged or tagged antibodies were titrated for sensitivity in a residual SpCas9 detection assay. In the sandwich ELISA, paired antibodies were used where one antibody was tagged and empirical testing was performed to identify antibody pairs. Untagged or tagged antibodies were titrated for sensitivity in a residual SpCas9 detection assay. Candidate antibodies were biotinylated for these experiments.

[0230] Various Cas9 antibodies were used in an indirect ELISA format to detect RNP or ApoCas9 (1 pg/mL in PBS). Different concentrations of antibodies were incubated with RNP (FIG. 5) or ApoCas9 (FIGS. 6A and 6B) that was coated to a microplate for 1 hr. A goat antimouse IgG-HRP antibody was used as the secondary antibody.

[0231] Different concentrations of RNP (FIG. 7A) or ApoCas9 (FIG. 7B) were coated overnight onto a microplate and then incubated with various Cas9 antibodies (10 pg/mL). A goat anti-mouse IgG-HRP antibody was used as the secondary antibody.

[0232] Various Cas9 antibodies were used in an indirect ELISA format to detect 1 pg/mL or RNP (FIG. 8A) or ApoCas9 (FIG. 8B) in various lysis buffers. The antibodies were incubated with RNP or Cas9 that was coated overnight onto a microplate. A goat anti-mouse IgG- HRP antibody was used as the secondary antibody. [0233] Various Cas9 antibodies were used in an indirect ELISA format to detect different concentrations of RNP (FIG. 9A) or ApoCas9 (FIG. 9B) in CS5 buffer. 1 pg/mL of antibody was incubated with RNP or Cas9 that was coated overnight onto a microplate. A goat anti-mouse IgG-HRP antibody was used as the secondary antibody.

[0234] Various Cas9 antibodies were used in an indirect ELISA format to detect various amounts (0-2000 pg/mL) of RNP (FIG. 10A) or ApoCas9 (FIG. 10B) with 15 min of color development time. 0.25, 0.5, and 1.0 pg/mL of antibody was incubated with various amounts of RNP or Cas9 that was coated overnight in PBS onto a microplate. A goat anti-mouse IgG-HRP antibody was used as the secondary antibody.

Example 4

Development of a Sensitive Residual Cas9 Detection Assay

[0235] The current assay for detection of residual SpCas9 uses a combination of mouse and rabbit anti-Cas9 antibodies with different binding characteristics and has a detection capability of 0.843 ng/mL for post-thaw supernatants and 34.8 ng/mL for cell lysates. Therefore, a more sensitive assay is required. This Example provides the protocol for detection of residual SpCas9 (including apoCas9 and RNPs) in cell supernatants as well as in whole cell lysates using the antibodies developed in Example 1. Also performed in this example, the non-biotinylated antibodies did not perform well with the cell lysate samples (data not shown).

[0236] The antibodies described in Table 4 were selected for RNP and apoCas9- specific residual Cas9 assay development. A commercially available antibody, 4G10 was used as a control.

Table 4: Antibodies targeting SpCas9

MSD Assay

[0237] This example provides a method for the quantitation of residual SpCas9 in the cell supernatant or cell lysate of a cell based drug product. Residual Streptococcus pyogenes derived CRISPR associated protein 9 (SpCas9) in the drug product was quantitated against a reference SpCas9 protein using a Mesoscale Discovery (MSD) Electro chemiluminescent (ECL) ELISA method. The MSD platform used a plate-based ELISA method, with an electrochemiluminescence (ECL) signal to quantify analyte across various matrices. This platform used low sample volume, had rapid read times and the ability to multiplex, if needed.

[0238] Various pairs of antibodies (1 pg/mL each) were used in a MSD format with RNP (FIG. 11 A) and Cas9 (FIG. 11B) as analyte. Antibodies were tagged with biotin and sulfo for testing in a Sandwich MSD assay format (Table 5).

Table 5. Results from Sandwich MSD assay

[0239] Antibodies were tagged with biotin and sulfo for testing in a Sandwich MSD assay format. A blocking with 3% blocker A step was added to the MSD assay format. Table 6 shows the of biotinylated and Sulfo-tagged antibody pairs in Sandwich MSD assay (20,000 counts being a predetermined cutoff).

Table 6. Results from Sandwich MSD assay

Examples testing different pairs:

[0240] Pairing of 5125 antibody as detection antibody with various antibodies as capture/coating antibody. 1 pg/mL of biotinylated coating antibody (174, 8873, 11912, 4G10, and 9594 antibodies) was tested with 1 pg/mL of detection antibody (sulfo-tagged 5125 antibody) in a sandwich MSD assay format to detect various amounts of Cas9 (pg/mL) with blocking with 3% Blocker A (FIGS. 12A-12E). FIG. 12F shows that the pair of 174 as capture and 5125 as detection provided a suitable range of detection.

[0241] 0.5 and 1.0 pg/mL of sulfo-tagged 5125 antibody as detection antibody was tested with varying amounts of biotinylated 174 antibody as coating antibody in a sandwich MSD assay format to detect various amounts of Cas9 (pg/mL) with blocking with 3% Blocker A (FIGS. 13A-13B). 1 pg/mL of 5125 and 1 pg/mL of 174 was chosen so that neither antibody was limiting in the detection of Cas9.

[0242] Optimization of length of blocking before coating antibody. 1 hr vs overnight blocking was tested using 1 pg/mL of biotinylated 174 antibody or 8873 antibody as capture antibody and 1 pg/mL of sulfo-tagged 5125 antibody as detection antibody in a sandwich MSD assay format to detect various amounts of Cas9 (pg/mL) with blocking with 3% Blocker A (FIGS. 14A-14D)

[0243] Lysate buffer determination. Whole cell lysate was generated in MPER lysis buffer (FIGS. 15A-15F), RIPA lysis buffer (FIGS. 16A-16F), and AlphaLISA lysis buffer (FIGS. 17A-17F) and 1 pg/mL of various unconjugated capture and sulfo-tagged detection antibodies were used to detect various amounts of Cas9 (ng/mL) in the whole cell lysate in the sandwich MSD assay format. Tables 7-9 show the derived Cas9 concentrations (ng/mL) detected by the assays.

Table 7. Results from Sandwich MSD assay performed for whole cell lysates

Table 8. Results from Sandwich MSD assay performed for whole cell lysates

Table 9. Results from Sandwich MSD assay performed for whole cell lysates

[0244] Testing whole cell lysate with biotinylated capture (with blocking). 1 pg/mL of biotinylated 8810 and 9594 antibodies was used as capture antibody and sulfo-tagged 4G10 and 8873 antibodies were used to detect various amounts of Cas9 (ng/mL) in the whole cell lysate generated in MPER lysis in a sandwich MSD assay format (FIGS. 18A-18D). Table 10 shows assay results using QC samples prepared in 10% whole cell lysate (prepared in MPER buffer) and that none of the detected Cas9 for the QC samples were within range showing that biotinylated capture antibodies were not useful in detecting Cas9 in whole lysates.

Table 10. Results from Sandwich MSD assay performed for whole cell lysates

[0245] Testing whole cell lysate with unconjugated capture (no blocking). 1 pg/mL of 8810 and 9594 antibodies was used as capture antibody and sulfo-tagged 4G10 and 8873 antibodies were used to detect various amounts of Cas9 (ng/mL) in 10% whole cell lysate generated in MPER lysis in a sandwich MSD assay format (FIGS. 19A-19D). Table 11 shows assay results using QC samples prepared in 10% whole cell lysate (prepared in MPER buffer).

[0246] FIGS. 20A-20D and Table 12 show the results with a narrower range of Cas9. Table 11. Results using QC samples from Sandwich MSD assay

Table 12. Results using QC samples from Sandwich MSD assay

[0247] Supernatant Assay Pairing'. Antibody pair of antibody 174 as coating antibody (biotinylated capture anti-SpCas9 antibody) and antibody 5125 as detection antibody (sulfotagged anti-SpCas9 antibody) were used in a residual Cas9 quantitation assay in cell supernatant (freezing medium; Cryostor® CS5) (Table 13, FIG. 21A). The assay dynamic range was from 40 pg/ml to 100000 pg/ml. It was determined that for detection of residual Cas9 in cell supernatant, antibody 174 as coating/capture antibody and antibody 5125 as detection antibody was the most optimal pair. The sensitivity was determined to be 373 pg/ml (lower limit of quantitation)

Table 13. Results of supernatant assay pairing

[0248] Cell Lysate Pairing. Antibody pair of antibody 8810 as coating antibody (biotinylated capture anti-SpCas9 antibody) and antibody 8873 as detection antibody (sulfotagged anti-SpCas9 antibody) were used in a residual Cas9 quantitation assay in whole cell lysates (in MPER buffer) (Table 14, FIG. 21B). The assay dynamic range was from 1 ng/ml to 100 ng/ml. In case of cell lysate, antibody 8810 as coating/capture antibody and antibody 8873 as detection antibody was considered the most optimal pair. Sensitivity was determined to be 7 ng/ml (lower limit of quantitation).

Table 14. Results of cell lysate assay pairing

[0249] The sensitivity of this assay was described by its limit of detection, as well as the ULOQ and LLOQ, which were the highest and lowest standard curve points (Table 15). In comparison to the currently available assay that utilizes rabbit, mouse, and goat antibodies, the MSD assay for detecting residual SpCas9 is far more sensitive (as shown in Table 16, the sensitivity for the cell supernatant assay was increased 2.2-fold using the MSD assay described herein over the current assay, and the sensitivity for the cell lysate assay was increased 5 -fold using the MSD assay described herein over the current assay).

Table 15. Sensitivity Data of MSD assay with SpCas9 antibodies (ng/mL)

Table 16. Comparison of assays disclosed herein and currently assays

Example 5

Cas9 Assay in Cell Supernatant [0250] For the sample testing, supernatant was aspirated from the centrifuged cells, the cell pellet was lysed, and both analytes (supernatant and cell lysate) were assayed for any residual amount of SpCas9 that was present in the final drug product. This method was specific to the supernatant portion only for the testing of samples and was developed using the anti-SpCas9 antibodies disclosed herein and optimized for greater sensitivity. For this assay, the anti-SpCas9 antibodies were biotinylated.

[0251] Coating Antibody Preparation. 150 pl of 3% Blocker A (100 mL of DPBS (Gibco 14190-144) per 3 g of Blocker A (MSD; R93BA-4) was added to each well of a MSD GOLD 96-well Streptavidin SECTOR Plate. The plate was covered with an adhesive plate sealer and incubated at 2-8°C overnight to block the plate. After overnight incubation, the adhesive plate sealer was removed from the MSD coated plate, and the plate was washed 3 times with 200 pl per well of Wash buffer. The plate was tapped out on an absorbent towel after the final wash.

[0252] The coating antibody (for example, #174 Biotinylated anti-SpCas9 antibody) was diluted to 1 pg/mL in 1% Blocker A (2 ml of 3% Blocker A to 4 ml of DPBS). 50 pl of 1 pg/mL coating antibody solution was added per well of the blocked plate. The plate was covered with adhesive plate sealer and incubated for 1 hr at room temperature on a plate shaker set at 600 rpm.

[0253] While the plate incubated, the reference, Quality controls (QCs) and sample analytes were prepared. The supernatant samples were prepared by thawing vials of drug product in a water bath, mixing the vial content by gentle pipetting/vortexing, centrifuging the vials for 5 minutes at 16000 RPM (room temperature), and collecting the supernatants. A serial dilution of the reference/QCs was performed with a diluent (e.g., CS5) to generate a standard curve ranging from 46 pg/ml to 100,000 pg/ml (Tables 17 and 18).

Table 17: Standard curve generated for references

Table 18: Standard curve generated for QCs

[0254] Drug Product Samples were diluted as necessary to an appropriate concentration to fit within the standard curve. Samples were run in triplicates.

[0255] The adhesive plate sealer was removed and the plates were washed 3 times with 200 pl per well of Wash buffer. The plate was tapped out on an absorbent towel after final wash. 50 pl per well of Reference SpCas9, Blanks (CS5), QCs and Drug product samples was added to the plate in triplicate. The plate was covered with an adhesive plate sealer and incubated for 1 hr at room temperature on a plate shaker set at 600 rpm.

[0256] Addition of SULFO-Tagged Detection Antibody to plate. The adhesive plate sealer was removed and the plate was washed 3 times with 200 pl per well of Wash buffer. The plate was tapped out on an absorbent towel after final wash. The detection antibody (for example #5125, Sulfo-tagged anti-SpCas9 antibody) was diluted to 1 pg/mL in 1% Blocker A. 50 pl per well of detection antibody was added to the plate. The plate was covered with adhesive plate sealer and incubated for 1 hr at room temperature on a plate shaker set at 600 rpm.

[0257] Read Plate. 20 mL of 2xMSD Read Buffer (for one 96 well plate) was prepared by mixing 10 mL of 4x MSD Read Buffer (cat # R92TC-2) to 2x with 10 mL Milli-Q water. The adhesive plate sealer was removed and the plate was washed 3 times with 200 pl per well of Wash buffer. The plate was tapped out on an absorbent towel after final wash. 150 pl of 2x Read Buffer was added per well. ECL counts in the plate were acquired by reading the plate immediately on an MSD plate reader.

[0258] Data Analysis. Analysis was performed on Graph-Pad Prism. The analysis parameters were as follows: Standard curve fit was set (default) as a 4-parameter, non-linear regression curve. Standards/QCs were defined at concentrations listed in the table above.

[0259] Samples were named appropriately, assigned to the respective standard, and the dilution factor was defined (if neat, set at 1). Any additional dilution was added within the software (i.e.-if diluted at 1 : 100 to fit within the curve, the dilution entered into the software would be “100”).

Example 6

Cas9 Assay in Cell Lysate

[0260] For the sample testing, supernatant was aspirated from the centrifuged cells, the cell pellet was lysed, and both analytes (supernatant and cell lysate) were assayed for any residual amount of SpCas9 that was present in the final drug product. This method was specific to the cell lysate portion only for the testing of samples and was developed using the anti-SpCas9 antibodies disclosed herein and optimized for greater sensitivity. For this assay, the anti-SpCas9 antibodies were not biotinylated.

[0261] Coating Antibody Preparation. The coating antibody (for example, #8810 anti- SpCas9 antibody) was diluted to 1 pg/mL in DPBS. 50 pl of 1 pg/mL coating antibody solution was added per well of the Mesoscale Standard Multi-Array plate. The plate was covered with adhesive plate sealer and incubated for 30 seconds at room temperature on a plate shaker set at 600 rpm. Each well was visually inspected to ensure that the coating antibody solution was evenly distributed across the bottom of each well. The coated plate was incubated at 2-8°C overnight.

[0262] Blocking Plate . After overnight incubation of coating antibody, the plate and all other reagents were allowed to equilibrate to room temp. The adhesive plate sealer was removed from the MSD coated plate, and the plate was washed 3 times with 200 pl per well of Wash buffer. The plate was tapped out on an absorbent towel after final wash. 200 pl of Superblock was added to each well. The plate was covered with adhesive plate sealer and incubated for 1 hr at room temperature on a plate shaker set at 600 rpm.

[0263] Cell lysate Preparation. Each vial of drug product and cells (e.g., unedited T- cells, e.g., 50 x 106 cells/mL) was thawed in a 37°C water bath for 2-3 minutes. The vial’s content was mixed by gentle pipetting/vortexing. If multiple aliquots of unedited T-cells were thawed, the aliquots were pooled together into a homogenous solution. The vials were centrifuged for 5 minutes at 1600 RPM, room temperature (RT). The supernatants were aspirated entirely and retained for processing to be used in the cell supernatant protocol. The Drug Product and unedited T-cell pellets were restored to the original aliquot volume with ice-cold MPER PI Lysis buffer (10 pL of HALT Protease Inhibitor was added per 990 pL of MPER buffer and stored on ice until lysate preparation). The concentration of the resuspended solution should be equal to 50 xlO⁶ cells/mL.

[0264] The cell pellet was resuspended by pipetting and the resuspended pellet was incubated for 15 minutes on ice to lyse the cells. The lysate solution was diluted 1 : 10 in Amersham ECL Block/Blocker B (100 mL of DPBS was added per 1 g of Amersham ECL block/Blocker B), or equivalent. The 1: 10 diluted lysate solutions were stored on ice when not in use.

[0265] A serial dilution of the reference/QCs was performed with a diluent to generate a standard curve ranging from 1 ng/ml to 128 ng/ml (Tables 19 and 20).

Table 19

Table 20

[0266] Drug Product Samples were diluted as necessary to an appropriate concentration to fit within the standard curve. Samples were run in triplicates.

[0267] The adhesive plate sealer was removed and the plates were washed 3 times with 200 pl per well of Wash buffer. The plate was tapped out on an absorbent towel after final wash. 50 pl per well of Reference SpCas9, Blanks (CS5), QCs and Drug product samples was added to the plate in triplicate. The plate was covered with an adhesive plate sealer and incubated for 1 hr at room temperature on a plate shaker set at 600 rpm.

[0268] Addition of SULFO-Tagged Detection Antibody to plate. The adhesive plate sealer was removed and the plate was washed 3 times with 200 pl per well of Wash buffer. The plate was tapped out on an absorbent towel after final wash. The detection antibody (for example #8873, Sulfo-tagged anti-SpCas9 antibody) was diluted to 1 pg/mL in 1% Blocker A. 50 pl per well of detection antibody was added to the plate. The plate was covered with adhesive plate sealer and incubated for 1 hr at room temperature on a plate shaker set at 600 rpm.

[0269] Read Plate. 20 mL of 2x MSD Read Buffer (for one 96 well plate) was prepared by mixing 10 mL of 4x MSD Read Buffer (cat # R92TC-2) to 2x with 10 mL Milli-Q water. The adhesive plate sealer was removed and the plate was washed 3 times with 200 pl per well of Wash buffer. The plate was tapped out on an absorbent towel after final wash. 150pl of 2x Read Buffer was added per well. ECL counts in the plate were acquired by reading the plate immediately on an MSD plate reader.

[0270] Data Analysis. Analysis was performed on Graph-Pad Prism. The analysis parameters were as follows: Standard curve fit was set (default) as a 4-parameter, non-linear regression curve. Standards/QCs were defined at concentrations listed in the table above.

[0271] Samples were named appropriately, assigned to the respective standard, and the dilution factor was defined (if neat, set at 1). Any additional dilution was added within the software (i.e.-if diluted at 1 : 100 to fit within the curve, the dilution entered into the software would be “100”).

Example 7

Prediction of CDR and Framework Regions from Antibody Sequences

[0272] Computational modeling tools can be used to predict the structure of antibodies, such as the CDR and framework regions (the scaffolding that enables CDRs to be properly accessible for binding antigens) from an antibody sequence. Any molecular modeling software known in the art for antibody or protein structure prediction can be used in the present disclosure to predict the CDR and framework regions, including for example, RosettaAntibody, SCALOP (Sequence-based antibody Canonical loop structure annotation), DeepAb, AlphaFold, ABodyBuilder, ABlooper and others identifiable to a skilled person. Tables 21-25 below provide sequences of exemplary VL CDR and VH CDR regions that were predicted.

Table 21

Table 22

Table 23

Table 24

Table 25

Table 26

Example 8

Cas9 protein quantification in liver

[0273] In this example, an assay was developed for quantification of Cas9 protein in liver using meso scale discovery (MSD). MSD assay uses high binding carbon electrodes in the bottom of MULTI-ARRAY and MULTI-SPOT microplates allowing for easy attachment of biological reagents (10X greater binding capacity than polystyrene). MSD assays use electrochemiluminescent labels that are conjugated to detect antibodies. The labels are called SULFO-TAG and allow for ultra-sensitive detection. Electricity is applied to the plate electrodes by an MSD instrument leading to light emission by SULFO-TAG labels. Light intensity is then measured to quantify analytes in the sample. Additional details about MSD assay and electrochemiluminescent labels can found at www.mesoscale.com/en/technical_resources/our_technology/. FIG. 22 provides a schematic illustrating how electrochemiluminescence works in an MSD assay.

[0274] An exemplary MSD assay procedure is briefly described as follows. A 96-well standard multi-array plate was coated with anti-SpCas9 antibody(capture antibody, #8810) overnight at 4°C. The plate was washed and blocked with Superblock for 1 h at room temperature. The plate was then washed with a wash buffer and loaded with samples or standards. The plate was covered with an adhesive plate sealer and incubated for 1 h at room temperature. Next, the adhesive plate sealer was removed and the plate was washed with a wash buffer. Sulfo-tagged anti-SpCas9 antibody (detection antibody, #8873) was added to the plate. The plate was covered with adhesive plate sealer and incubated for 1 h at room temperature. The adhesive plate sealer was then removed and the plate was washed with a wash buffer. MSD read buffer was added to the plate. ECL counts in the plate were acquired by reading the plate on an MSD plate reader. FIG. 23 provides a table showing the plate layout and the read out from an MSD assay.

[0275] 20 C57BL/6 female mice were dosed with lipid nanoparticles encapsulating a gRNA targeting ANGPTL3 gene. End-point measurement was collected at baseline, 3, 6, 12, 24, 48, 72 h post-dose. The measurement includes liver editing efficiency and liver Cas9 protein quantification using the MSD assay procedure described above. FIG. 24A-B are plots and tables showing the Cas9 protein amounts over time in liver (FIG. 24B) compared to a standard curve and QC standards (FIG. 24A). Values for samples within a yellow box were not able to interpolated. The results demonstrate that the Cas9 protein amount in liver decreases over time and drops to about 2-4 ng Cas9/mg protein. No residual Cas9 protein was detected 72 hours postdose.

Example 9

Determination binding activities of Cas9 antibodies

[0276] This example describes experiments performed to determine KD values of exemplary Cas9 antibodies disclosed herein. The methods used herein are developed using Octet® BLI system. FIG. 25 is a schematic representation of all the steps involved in an assay for KD determination using Octet® method. KD, the equilibrium dissociation constant, is a ratio of k_Off and k_On (i.e., KD = k_Off/ k_on). KD and affinity are inversely related. The KD value relates to the concentration of antibody. The lower the KD value, the higher the affinity of the antibody.

[0277] A total of 13 antibodies were selected for KD determination. Based on preliminary characterization, these antibodies were classified into two categories: ApoCas9 (gRNA free Cas9) binders and RNP (ApoCas9 and TAI sgRNA) binders. Sample IDs 7474, 8810, 8873, 9594, 11912, and 12486 are ApoCas9 binders. Sample IDs 174, 453, 753, 3447, 5125, 7675, and 8817 are RNP binders.

[0278] Briefly, biotinylated SpCas9 are loaded on Streptavidin Sensor Tips at 50 nM. Tips are blocked in BSA blocking buffer to avoid non-specific binding. SpCas9 associates with antibodies at 8 different concentrations (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM and 0 nM) for 120 sec. Binding affinity is measured and KD value is calculated using Octet HT analysis software using a 1 :2 binding model.

[0279] FIG. 26A provides plots showing kinetic data for ApoCas9 antibodies. FIG. 26B provides plots showing kinetic data for RNP specific antibodies. Table 27 below provides the KD values of the 13 antibodies. The %² (Chi-square) value (last column) indicates the goodness of the fit. All the antibodies listed have great KD values and show great affinity to SpCas9. This data is consistent with the ELISA and MSD binding data seen previously.

Table 27. KD values of exemplary antibodies

Example 10

Exemplary residual Cas9 assays

[0280] This example describes non-limiting exemplary residual Cas9 assays performed for cell supernatant samples and cell lysate samples using detection antibodies only or pairs of capture and detection antibodies disclosed herein.

Supernatant samples using a detection antibody

[0281] Abeam antibody (cat# ab210752) at 1 pg/mL in DPBS was added at 50 pl/well to a microplate and coated for at least 12 hours at 2-8 °C. Superblock™ blocking agent was added to the microplate at 200 pl/well and incubated for about Ihr (± 15 mins). Cas9 from different suppliers (Suppliers A and B) were diluted to obtain a serial 1 :3.3 dilution of calibrators from 100 ng/mL from 0.077 ng/mL in appropriate diluent (e.g., CryoStor CSB/CS10 containing 50% CSB and 50% CS10 solution, such as 100 mL CSB + 100 mL CS10), then added at 50 pl/well to the microplate and incubated for about Ihr (± 15 mins). Sulfo-tagged 5125 antibody was diluted at 0.5 pg/mL in Superblock™, added to the microplate at 50 pl/well, and incubated for about Ihr (± 15 mins). The plate was then read on an MSD plate reader using MSD read buffer. Table 28 below provides results of the supernatant assays, including expected concentrations and coefficient of variation (CV) for each Cas9 sample.

Table 28. Results of supernatant assay using 5125 detection antibody

[0282] FIG. 27A depicts plots of standard curves for Supplier A Cas9 supernatant assays (left panel) and Supplier B Cas9 supernatant assays (right panel) using the 5125 detection antibody. FIG. 27B provides plots of observed concentrations for Supplier A Cas9 supernatant assays (left panel) and Supplier B Cas9 supernatant assays (right panel) using the 5125 detection antibody. The observed limit of quantification (LOQ) for both Cas9 samples is 0.843 ng/mL. Supernatant samples using a capture/detection antibody pair

[0283] Capture antibody (biotinylated 174 antibody) at 1 pg/mL in DPBS was added at 50 pl/well to a MSD Gold Streptavidin Sector Plate and coated for 1 hr at room temperature on a plate shaker set at 600 rpm. Superblock™ blocking agent was added to the microplate at 200 pl/well and incubated for about Ihr (± 15 mins). Cas9 from different suppliers (Suppliers A and B) were diluted to obtain a serial 1 :2.25 dilutions of calibrators from 100 ng/mL from 0.077 ng/mL in appropriate diluent (e.g., CryoStor CSB/CS10 containing 50% CSB and 50% CS10 solution, such as 100 mL CSB ± 100 mL CS10), then added at 50 pl/well to the microplate and incubated for about Ihr (± 15 mins). Sulfo-tagged 5125 antibody was diluted at 0.5 pg/mL in Superblock™, added to the microplate at 50 pl/well, and incubated for about Ihr (± 15 mins). The plate was then read on an MSD plate reader using MSD read buffer. Table 29 below provides results of the supernatant assays for each Cas9 sample. Table 29. Results of supernatant assay using the 174/5125 antibody pair

[0284] FIG. 28A depicts plots of standard curves for Supplier A Cas9 supernatant assays (left panel) and Supplier B Cas9 supernatant assays (right panel) using the 174/5125 antibody pair. FIG. 28B is a plot of observed concentrations for Supplier A Cas9 supernatant assays and Supplier B Cas9 supernatant assays using the 174/5125 antibody pair. The observed LOQ for Supplier A Cas9 curve is 0.771 ng/mL and 1.734 ng/mL for Supplier B Cas9. Cell lysate samples using a detection antibody

[0285] Abeam antibody (cat# ab210752) at 1 pg/mL in DPBS was added to a plate and coated for at least 12 hours at 2-8 °C. Superblock™ blocking agent was added to the microplate at 200 pl/well and incubated for about Ihr (± 15 mins). To prepare unedited T-cell lysates, cells are thawed in water bath at 32 °C for 3 mins and then spun down at 1600 rpm (~300xG). The supernatant was removed. Cells were then lysed using MPER and PI buffer (10 pL of protease inhibitor per ImL MPER extraction buffer) for 15 mins at room temperature and then diluted 1 : 10 in 1% Amersham (1g of Amersham (ECL blocking buffer) + 99 mL DPBS). Serial 1 : 1.75 dilutions from 100 ng/mL to 3.48 ng/mL were prepared by diluting Cas9 (Supplier A Cas9 and Supplier B Cas9)) in lysates and then added at 50 pl/well to the plate and incubated for about 1 hr (± 15 mins). 8873 antibody was diluted at 0.5 pg/mL in 1% Amersham, added to the plate at 50 pl/well, and incubated for about Ihr (± 15 mins). The plate was then read on an MSD plate reader using MSD read buffer. Tables 30-31 below provide results of the cell lysate assays for Cas9 from different suppliers.

Table 30. Results of cell lysate assays for Supplier A Cas9

Table 31. Results of cell lysate assays for Supplier B Cas9

[0286] FIG. 29A depicts plots of standard curves for Supplier B Cas9 cell lysate assays (top panel) and Supplier A Cas9 cell lysate assays (bottom panel) using the 8873 detection antibody. FIG. 29B provides plots of observed concentrations for Supplier B Cas9 cell lysate assays (left panel) and Supplier A Cas9 cell lysate assays (right panel) using the 8873 detection antibody. The observed LOQ for Supplier B Cas9 is 58.5 ng/mL and 60.9 ng/mL for Supplier A Cas9. The LOQ values reported for the lysate standard curve are multiplied by a factor of 10 to compensate for the 10-fold dilution of the samples being analyzed.

Cell lysate samples using a capture/detection antibody pair

[0287] Capture antibody (8810) at 1 pg/mL in DPBS was added to a MSD plate and coated for at least 12 hours at 2-8 °C. Superblock™ blocking agent was added to the microplate at 200 pl/well and incubated for about Ihr (± 15 mins). To prepare unedited T-cell lysates, cells are thawed in water bath at 32 °C for 3 mins and then spun down at 1600 rpm (~300xG). The supernatant was removed. Cells were then lysed using MPER and PI buffer (10 pL of protease inhibitor per ImL MPER extraction buffer) for 15 mins at room temperature and then diluted 1 : 10 in 1% Amersham (1g of Amersham (ECL blocking buffer) + 99 mL DPBS). Serial 1 :3 dilutions from 100 ng/mL to 0.137 ng/mL were prepared by diluting Cas9 (Supplier A Cas9 and Supplier B Cas9) in lysates and then added at 50 pl/well to the plate and incubated for about 1 hr (± 15 mins). 8873 antibody was diluted at 0.5 pg/mL in 1% Amersham, added to the plate at 50 pl/well, and incubated for about Ihr (± 15 mins). The plate was then read on an MSD plate reader using MSD read buffer. Table 32 below provides results of the cell lysate assays for Cas9 from different suppliers.

Table 32. Results of cell lysate assays using 8810/8873 antibody pair

[0288] FIG. 30A depicts plots of standard curves for Supplier A Cas9 cell lysate assays (left panel) and Supplier B Cas9 cell lysate assays (right panel) using the 8810/8873 antibody pair. FIG. 30B depicts plots of observed concentrations for Supplier A Cas9 cell lysate assays and Supplier B Cas9 cell lysate assays using the 8810/8873 antibody pair. The observed LOQ for both Cas9 samples is 12.35 ng/mL.

Terminology

[0289] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0290] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated.

[0291] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0292] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0293] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0294] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. An antibody or fragment thereof, wherein the antibody or fragment thereof has specificity to a Cas9 protein and comprises:

2. The antibody or fragment thereof of claim 1, comprising

3. The antibody or fragment thereof of claim 1, comprising: a VL CDR1 of SEQ ID NO: 1 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 1; a VL CDR2 of SEQ ID NO: 14 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 14; a VL CDR3 of SEQ ID NO: 27 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 27; a VH CDR1 of SEQ ID NO: 40 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 40; a VH CDR2 of SEQ ID NO: 53 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 53; and a VH CDR3 of SEQ ID NO: 66 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 66.

4. The antibody or fragment thereof of claim 1, comprising: a VL CDR1 of SEQ ID NO: 4 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 4; a VL CDR2 of SEQ ID NO: 17 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 17; a VL CDR3 of SEQ ID NO: 30 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 30; a VH CDR1 of SEQ ID NO: 43 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 43; a VH CDR2 of SEQ ID NO: 56 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 56; and a VH CDR3 of SEQ ID NO: 69 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 69.

5. The antibody or fragment thereof of claim 1, comprising: a VL CDR1 of SEQ ID NO: 7 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 7; a VL CDR2 of SEQ ID NO: 20 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 20; a VL CDR3 of SEQ ID NO: 33 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 33; a VH CDR1 of SEQ ID NO: 46 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 46; a VH CDR2 of SEQ ID NO: 59 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 59; and a VH CDR3 of SEQ ID NO: 72 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 72.

6. The antibody or fragment thereof of claim 1, comprising: a VL CDR1 of SEQ ID NO: 8 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 8; a VL CDR2 of SEQ ID NO: 21 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 21; a VL CDR3 of SEQ ID NO: 34 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 34; a VH CDR1 of SEQ ID NO: 47 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 47; a VH CDR2 of SEQ ID NO: 60 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 60; and a VH CDR3 of SEQ ID NO: 73 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 73.

7. The antibody or fragment thereof of any one of claims 1 -6, comprising a light chain variable region comprising (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-91, (ii) an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-91, or (iii) an amino acid sequence having one, two or three mismatches relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-91.

8. The antibody or fragment thereof of any one of claims 1-7, comprising a heavy chain variable region comprising (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-104, (ii) an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-104, (iii) an amino acid sequence having one, two or three mismatches relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-104.

9. The antibody or fragment thereof of any one of claims 1 -8, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 79 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 79, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 92 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 92.

10. The antibody or fragment thereof of any one of claims 1 -8, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 82 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 82, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 95.

11. The antibody or fragment thereof of any one of claims 1-8, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 85 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 85, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 98 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 98.

12. The antibody or fragment thereof of any one of claims 1-8, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 86 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 86, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 99 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 99.

13. The antibody or fragment thereof of any one of claims 1-12, wherein the antibody or fragment thereof comprises a Fc domain.

14. The antibody or fragment thereof of any one of claims 1-12, wherein the antibody or fragment thereof is a single-chain variable fragment (scFv), a single-domain antibody, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a Fv fragment, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual specific antibody, an anti -idiotypic antibody, a bispecific antibody, or a functionally active epitope-binding fragment thereof.

15. A composition, comprising one or more of the antibodies or fragments thereof of any one of claims 1-14; and a buffer.

16. The composition of claim 15, comprising a Cas9 capable of specific binding to the one or more of the antibodies or fragments thereof of any one of claims 1-14.

17. The composition of claim 15, comprising a Cas9 or a variant or derivative thereof.

18. The composition of any one of claims 16-16, wherein the Cas9 is selected from the group consisting of Streptococcus pyogenes Cas9, Streptococcus aureus Cas9, Staphylococcus lugdunensis Cas9, Neisseria meningitides Cas9, Streptococcus thermophilus CRISPR1 Cas9, Streptococcus thermophilus CRISPR3 Cas9, Treponema denticola Cas9, Campylobacter jejuni Cas9, Lachnospiraceae bacterium ND2006 Cpfl and Acidaminococcus sp. BV3L6 Cpfl.

19. The composition of any one of claims 15-18, wherein one of the one or more of the antibodies or fragments thereof is associated with a detectable label.

20. The composition of claim 19, wherein the detectable label is selected from the group consisting of: a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, an electrochemiluminescent label, and a combination thereof.

21. The composition of claim 20, wherein the electrochemiluminescent label is a sulfotag.

22. The composition of any one of claims 15-21, comprising a biological sample.

23. The composition of any one of claims 15-21, comprising cells, cell supernatant, cell lysate, cell extract, or a combination thereof; and optionally the cell supernatant is post-thaw cell supernatant.

24. The composition of claim 23, wherein the cells comprise liver cells, immune cells, blood cells, stem cells, peripheral blood mononuclear cells (PBMCs), or a combination thereof.

25. The composition of claim 24, wherein the immune cells comprise T cells, NK cells, or a combination thereof; and optionally the T cells comprise CAR-T cells and/or the NK cells comprise CAR-NK cells.

26. The composition of claim 24, wherein the stem cells comprise hemopoietic stem cells (HSCs), hemopoietic progenitor cells (HPCs), induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells, mesenchymal stem cells (MSCs), tissue-specific stem cells, or a combination thereof.

27. The composition of any one of claims 15-26, comprising two of the antibodies or fragments thereof of any one of claims 1-14.

28. The composition of claim 27, wherein one of the antibody or fragment thereof is biotinylated, and optionally the other antibody or fragment thereof is sulfo-tagged.

29. A polynucleotide encoding the antibody or fragment thereof of any one of claims 1-14.

30. A kit for detecting Cas9, comprising one or more of the antibodies or fragments thereof of any one of claims 1-14.

31. The kit of claim 30, wherein one of the one or more of the antibodies or fragments thereof is associated with a detectable label.

32. The kit of claim 31, wherein the detectable label is selected from the group consisting of: a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, an electrochemiluminescent label, and a combination thereof.

33. The kit of claim 32, wherein the electrochemiluminescent label is a sulfo-tag.

34. The kit of any one of claims 30-33, comprising two of the antibody or fragment thereof of any one of claims 1-14, wherein the two of the antibody or fragment thereof are different from one another.

35. The kit of claim 34, wherein one of the antibody or fragment thereof is biotinylated, and optionally the other antibody or fragment thereof is sulfo-tagged.

36. The kit of claim 34, wherein one of the antibody or fragment thereof is sulfotagged, and optionally the other antibody or fragment thereof is biotinylated.

37. The kit of any one of claims 34-36, wherein a first antibody or fragment thereof of the two of the antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 1 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 1, a VL CDR2 of SEQ ID NO: 14 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 14, a VL CDR3 of SEQ ID NO: 27 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 27, a VH CDR1 of SEQ ID NO: 40 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 40, a VH CDR2 of SEQ ID NO: 53 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 53, and a VH CDR3 of SEQ ID NO: 66 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 66; and wherein a second antibody or fragment thereof of the two of the antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 4 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 17 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 17, a VL CDR3 of SEQ ID NO: 30 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 30, a VH CDR1 of SEQ ID NO: 43 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 43, a VH CDR2 of SEQ ID NO: 56 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 56, and a VH CDR3 of SEQ ID NO: 69 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 69.

38. The kit of any one of claims 34-36, wherein a first antibody or fragment thereof of the two of the antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 7 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 7, a VL CDR2 of SEQ ID NO: 20 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 20, a VL CDR3 of SEQ ID NO: 33 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 33, a VH CDR1 of SEQ ID NO: 46 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 46, a VH CDR2 of SEQ ID NO: 59 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 59, and a VH CDR3 of SEQ ID NO: 72 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 72; and wherein a second antibody or fragment thereof of the two of the antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 8 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 8, a VL CDR2 of SEQ ID NO: 21 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 21, a VL CDR3 of SEQ ID NO: 34 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 34, a VH CDR1 of SEQ ID NO: 47 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 47, a VH CDR2 of SEQ ID NO: 60 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 60, and a VH CDR3 of SEQ ID NO: 73 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 73.

39. The kit of any one of claims 34-36, wherein a first antibody or fragment thereof of the two of the antibody or fragment thereof comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 85 or a peptide having about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 85, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 98 or a peptide having about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 98; and wherein a second antibody or fragment thereof of the two of the antibody or fragment thereof comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 86 or a peptide having about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 86, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 99 or a peptide having about 85%, 90%, 95%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 99.

40. A method of using the antibody or fragment thereof of any one of claims 1-14, comprising: contacting a biological sample with the antibody or fragment thereof of any one of the claims 1-14, thereby detecting the presence or absence of Cas9 in the biological sample.

41. A method of detecting Cas9 in a biological sample, comprising: contacting a biological sample with one or more of the antibodies or fragments thereof of any one of claims 1-14 under conditions allowing the one or more antibodies or fragments thereof to bind to Cas9, wherein the biological sample comprises Cas9 or is suspected of comprising Cas9, and detecting the binding of the one or more antibodies or fragments thereof to Cas9, wherein the detection of the binding is indicative of the presence of Cas9 in the biological sample.

42. The method of claim 41, comprising determining the amount of Cas9 in the biological sample.

43. The method of any one of claims 41-42, wherein the biological sample comprises Cas9-gRNA ribonucleoprotein (RNP).

44. The method of any one of claims 41-43, wherein the biological sample comprises free Cas9 protein or a fragment thereof, optionally the free Cas9 protein is ApoCas9.

45. The method of any one of claims 41-44, wherein the Cas9 is a Cas9 having endogenous nuclease activity, a small Cas9, a dead Cas9 (dCas9), a fusion protein comprising Cas9, or a variant thereof; and optionally the fusion protein is a Cas9 base editor.

46. The method of any one of claims 41-44, wherein the Cas9 is fused with or linked with one or more polypeptide domains, and optionally the Cas9 is fused with or linked with one polypeptide domain or two polypeptide domains.

47. The method of claim 46, wherein the polypeptide domain comprises a polypeptide domain having an activity, and optionally the activity is an enzymatic activity.

48. The method of claim 47, wherein the activity is a deaminase activity, reverse transcriptase activity, transcription activation activity, a transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, methylase activity, demethylase activity, protease activity, transposase activity, recombinase activity, nuclease, or a combination thereof.

49. The method of any one of claims 46-48, wherein the polypeptide domain comprises a tag, a label, a nucleic acid binding domain, a helix-tum-helix domain, a zinc finger domain, or a combination thereof.

50. The method of any one of claims 41-49, wherein the biological sample comprises cell lysate, cell supernatant, cell extract, or a combination thereof; and optionally the cell supernatant is post-thaw cell supernatant.

51. The method of claim 50, wherein the cells comprise liver cells, immune cells, blood cells, stem cells, peripheral blood mononuclear cells (PBMCs), or a combination thereof.

52. The method of claim 51, wherein the immune cells comprise T cells, NK cells, or a combination thereof; and optionally the T cells comprise CAR-T cells and/or the NK cells comprise CAR-NK cells.

53. The method of claim 51, wherein the stem cells comprise hemopoietic stem cells (HSCs), hemopoietic progenitor cells (HPCs), induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells, mesenchymal stem cells (MSCs), tissue-specific stem cells, or a combination thereof.

54. The method of any one of claims 41-53, wherein the detecting the binding of the antibody or fragment thereof to the Cas9 is performed using an immunodetection assay.

55. The method of claim 54, wherein the immunodetection assay is selected from the group consisting of: western blotting, enzyme-linked immunosorbent assay (ELISA), and beadbased luminescent amplification assay; and optionally the ELISA assay is a electro chemiluminescent (ELC) ELISA assay, and the bead-based luminescent amplification assay is an ALPHALisa Assay.

56. The method of any one of claims 41-55, wherein the contacting comprises: contacting the biological sample with a first antibody or fragment thereof of any one of claims 1-14 under a condition allowing binding of the first antibody or fragment thereof to Cas9 to form a first antibody-Cas9 complex; contacting the first antibody-Cas9 complex with a second antibody or fragment thereof of any one of claims 1-14 under a condition allowing binding of the second antibody or fragment thereof to the first antibody-Cas9 complex; and detecting the binding of the second antibody or fragment thereof to the first antibody-Cas9 complex.

57. The method of claim 56, wherein one or both of the first and second antibodies or fragments thereof is associated with a detectable label.

58. The method of claim 57, wherein the detectable label is selected from the group consisting of: a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, an electrochemiluminescent label, and a combination thereof.

59. The method of claim 58, wherein the electrochemiluminescent label is a sulfo-tag.

60. The method of any one of claims 56-59, wherein the first antibody or fragment thereof is biotinylated, and the second antibody or fragment thereof is sulfo-tagged.

61. The method of any one of claims 56-60, wherein the first antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 1 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 1, a VL CDR2 of SEQ ID NO: 14 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 14, a VL CDR3 of SEQ ID NO: 27 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 27, a VH CDR1 of SEQ ID NO: 40 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 40, a VH CDR2 of SEQ ID NO: 53 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 53, and a VH CDR3 of SEQ ID NO: 66 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 66; and wherein the second antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 4 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 17 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 17, a VL CDR3 of SEQ ID NO: 30 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 30, a VH CDR1 of SEQ ID NO: 43 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 43, a VH CDR2 of SEQ ID NO: 56 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 56, and a VH CDR3 of SEQ ID NO: 69 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 69.

62. The method of claim 61, wherein the biological sample comprises a cell supernatant.

63. The method of any one of claims 56-60, wherein the first antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 7 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 7, a VL CDR2 of SEQ ID NO: 20 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 20, a VL CDR3 of SEQ ID NO: 33 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 33, a VH CDR1 of SEQ ID NO: 46 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 46, a VH CDR2 of SEQ ID NO: 59 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 59, and a VH CDR3 of SEQ ID NO: 72 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 72; and wherein the second antibody or fragment thereof comprises: a VL CDR1 of SEQ ID NO: 8 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 8, a VL CDR2 of SEQ ID NO: 21 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 21, a VL CDR3 of SEQ ID NO: 34 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 34, a VH CDR1 of SEQ ID NO: 47 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 47, a VH CDR2 of SEQ ID NO: 60 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 60, and a VH CDR3 of SEQ ID NO: 73 or a variant thereof having a single substitution, deletion or insertion of SEQ ID NO: 73.

64. The method of claim 63, wherein the biological sample comprises a cell lysate.

65. The method of any one of claims 56-64, wherein the first antibody or fragment thereof is immobilized on a solid phase.

66. The method of any one of claims 56-65, comprising washing away unbound Cas9 after the contacting the biological sample with the first antibody or fragment thereof and prior to the contacting the first antibody-Cas9 complex with the second antibody or fragment thereof.

67. The method of any one of claims 41 to 66, wherein the biological sample is a cell supernatant comprising Cas9 at a concentration in the range of 0.34-0.8 ng/mL.

68. The method of any one of claims 41 to 66, wherein the biological sample is a cell lysate comprising Cas9 at a concentration in the range of 1-34 ng/mL.

69. A method of detecting Cas9 in a biological sample, comprising: contacting a biological sample with one or more of antibodies or fragments thereof having specificity to a Cas9 protein under conditions allowing the one or more antibodies or fragments thereof to bind to the Cas9, wherein the biological sample comprises Cas9 at a concentration of no more than 0.01 pg/mL; and detecting the binding of the one or more antibodies or fragments thereof to the Cas9, wherein the detection of the binding is indicative of the presence of Cas9 in the biological sample.

70. The method of claim 69, comprising determining the amount of Cas9 in the biological sample.

71. The method of any one of claims 69-70, wherein the biological sample is a cell supernatant comprising Cas9 at a concentration of no more than 0.8 ng/mL.

72. The method of any one of claims 69-70, wherein the biological sample is cell lysate.

73. The method of any one of claims 69-70, wherein the biological sample is derived from a pharmaceutical composition comprising cells.

74. The method of claim 73, wherein the cells comprise liver cells, immune cells, stem cells, peripheral blood mononuclear cells (PBMCs), or a combination thereof.

75. The method of claim 74, wherein the immune cells comprise T cells, NK cells or a combination thereof; and optionally the T cells comprise CAR-T cells and/or the NK cells comprise CAR-NK cells.

76. The method of claim 74, wherein the stem cells comprise hemopoietic stem cells (HSCs), hemopoietic progenitor cells (HPCs), induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells, mesenchymal stem cells (MSCs), tissue-specific stem cells, or a combination thereof.