Abstract: According to a first aspect of the invention, a method for the generation of a cell line is provided, comprising the steps of (a) providing a plurality of mammalian B cells, wherein each of the plurality of B cells comprises a transgenic genomic DNA sequence encoding a marker protein inserted into an endogenous immunoglobulin locus comprised in said B cell, and wherein the transgenic genomic DNA sequence is amenable to cleavage by a site directed nuclease, particularly Cas9: (b) replacing the transgenic genomic DNA sequence encoding a marker protein with a second transgenic DNA sequence encoding a protein of interest; (c) sorting B cells based on the presence or absence of the marker protein; and (d) collecting B cells in which the marker protein is absent.

Abstract: According to a first aspect of the invention, a method for the generation of a cell line is provided, comprising the steps of (a) providing a plurality of mammalian B cells, wherein each of the plurality of B cells comprises a transgenic genomic DNA sequence encoding a marker protein inserted into an endogenous immunoglobulin locus comprised in said B cell, and wherein the transgenic genomic DNA sequence is amenable to cleavage by a site directed nuclease, particularly Cas9; (b) replacing the transgenic genomic DNA sequence encoding a marker protein with a second transgenic DNA sequence encoding a protein of interest; (c) sorting B cells based on the presence or absence of the marker protein; and (d) collecting B cells in which the marker protein is absent.

Abstract: Provided herein, in some embodiments, are engineered nucleic acid targeting vectors that include a sequence of interest flanked by homology arms, each homology arm comprising a sequence homologous to a sequence in a safe harbor site in the human genome in any one of the following loci: 1q31, 3p24, 7q35, and Xq21. Also provided herein are methods of using and compositions the comprising engineered nucleic acid targeting vectors.

Abstract: According to a first aspect of the invention, a method for the generation of a cell line is provided, comprising the steps of (a) providing a plurality of mammalian B cells, wherein each of the plurality of B cells comprises a transgenic genomic DNA sequence encoding a marker protein inserted into an endogenous immunoglobulin locus comprised in said B cell, and wherein the transgenic genomic DNA sequence is amenable to cleavage by a site directed nuclease, particularly Cas9; (b) replacing the transgenic genomic DNA sequence encoding a marker protein with a second transgenic DNA sequence encoding a protein of interest; (c) sorting B cells based on the presence or absence of the marker protein; and (d) collecting B cells in which the marker protein is absent.

Abstract: According to a first aspect of the invention, a method for the generation of a cell line is provided, comprising the steps of (a) providing a plurality of mammalian B cells, wherein each of the plurality of B cells comprises a transgenic genomic DNA sequence encoding a marker protein inserted into an endogenous immunoglobulin locus comprised in said B cell, and wherein the transgenic genomic DNA sequence is amenable to cleavage by a site directed nuclease, particularly Cas9; (b) replacing the transgenic genomic DNA sequence encoding a marker protein with a second transgenic DNA sequence encoding a protein of interest; (c) sorting B cells based on the presence or absence of the marker protein; and (d) collecting B cells in which the marker protein is absent.

Abstract: The present methods use a variational autoencoder (VAE) and deep generative modelling to learn meaningful representations from the immune repertoires. The system can map input sequences into a lower-dimensional latent space, which reveals a large amount of convergent sequence patterns. The system can identify patterns present in convergent clusters that are highly predictive for antigen exposure and/or antigen specificity. The system can generate, from the latent space, novel functional antibody sequence variants in-silico.

Abstract: The present disclosure describes systems and methods to make predictions classifying one or more properties of a binding protein such as an antibody, for example, antibody affinity or specificity for an antigen. The system can include one or more machine learning models that can extrapolate complex relationships between amino acid sequence and function. The system can be trained on high-quality training data generated through a two-step single-site and combinatorial deep mutational scanning approach. The trained models can then make predictions on novel variant sequences generated in silico. The present disclosure describes amino acid sequences generated by the systems and methods provided, and uses of the generated sequences to produce proteins for therapeutic and diagnostic use.

Abstract: According to a first aspect of the invention, a method for the generation of a cell line is provided, comprising the steps of (a) providing a plurality of mammalian B cells, wherein each of the plurality of B cells comprises a transgenic genomic DNA sequence encoding a marker protein inserted into an endogenous immunoglobulin locus comprised in said B cell, and wherein the transgenic genomic DNA sequence is amenable to cleavage by a site directed nuclease, particularly Cas9; (b) replacing the transgenic genomic DNA sequence encoding a marker protein with a second transgenic DNA sequence encoding a protein of interest; (c) sorting B cells based on the presence or absence of the marker protein; and (d) collecting B cells in which the marker protein is absent.

Abstract: Methods and compositions for identification of candicate antigen-specific variable regions as well as generation of antibodies or antigen-binding fragments that could have desired antigen specificity are provided. For example, in certain aspects methods for determining amino acid sequences of serum antibody CDR and abundancy level are described. In some aspects, methods for determining nucleic acid sequences of antibody variable region sequences and frequency are provided. Furthermore, the invention provides methods for identification and generation of antibody or antigen-binding fragments that comprise highly-represented CDR.

Abstract: Methods and compositions for identification of candidate antigen-specific variable regions as well as generation of antibodies or antigen-binding fragments that could have desired antigen specificity are provided. For example, in certain aspects methods for determining amino acid sequences of serum antibody CDR and abundancy level are described. In some aspects, methods for determining nucleic acid sequences of antibody variable region sequences and frequency are provided. Furthermore, the invention provides methods for identification and generation of antibody or antigen-binding fragments that comprise highly-represented CDR.

Abstract: Methods and compositions involving polypeptides having an aglycosylated antibody Fc domain. In certain embodiments, polypeptides have an aglycosylated Fc domain that contains one or more substitutions compared to a native Fc domain. Additionally, some embodiments involve an Fc domain that is binds some Fc receptors but not others. For example, polypeptides are provided with an aglycosylated Fc domain that selectively binds Fc?RI at a level within 2-fold of a glycosylated Fc domain, but that is significantly reduced for binding to other Fc receptors. Furthermore, methods and compositions are provided for promoting antibody-dependent cell-mediated toxicity (ADCC) using a polypeptide having a modified aglycosylated Fc domain and a second non-Fc binding domain, which can be an antigen binding region of an antibody or a non-antigen binding region. Some embodiments concern antibodies with such polypeptides, which may have the same or different non-Fc binding domain.

Abstract: Methods and compositions involving polypeptides having an aglycosylated antibody Fc domain. In certain embodiments, polypeptides have an aglycosylated Fc domain that contains one or more substitutions compared to a native Fc domain. Additionally, some embodiments involve an Fc domain that is binds some Fc receptors but not others. For example, polypeptides are provided with an aglycosylated Fe domain that selectively binds Fc?RI at a level within 2-fold of a glycosylated Fc domain, but that is significantly reduced for binding to other Fc receptors. Furthermore, methods and compositions are provided for promoting antibody-dependent cell-mediated toxicity (ADCC) using a polypeptide having a modified aglycosylated Fc domain and a second non-Fc binding domain, which can be an antigen binding region of an antibody or a non-antigen binding region. Some embodiments concern antibodies with such polypeptides, which may have the same or different non-Fc binding domain.

Abstract: Methods and compositions for identification of candidate antigen-specific variable regions as well as generation of antibodies or antigen-binding fragments that could have desired antigen specificity are provided. For example, in certain aspects methods for determining amino acid sequences of serum antibody CDR and abundancy level are described. In some aspects, methods for determining nucleic acid sequences of antibody variable region sequences and frequency are provided. Furthermore, the invention provides methods for identification and generation of antibody or antigen-binding fragments that comprise highly-represented CDR.

Abstract: Methods and compositions involving polypeptides having an aglycosylated antibody Fc domain. In certain embodiments, polypeptides have an aglycosylated Fc domain that contains one or more substitutions compared to a native Fc domain. Additionally, some embodiments involve an Fc domain that is binds some Fc receptors but not others. For example, polypeptides are provided with an aglycosylated Fe domain that selectively binds Fc?RI at a level within 2-fold of a glycosylated Fc domain, but that is significantly reduced for binding to other Fc receptors. Furthermore, methods and compositions are provided for promoting antibody-dependent cell-mediated toxicity (ADCC) using a polypeptide having a modified aglycosylated Fc domain and a second non-Fc binding domain, which can be an antigen binding region of an antibody or a non-antigen binding region. Some embodiments concern antibodies with such polypeptides, which may have the same or different non-Fc binding domain.

Abstract: Nanoparticles that activate complement in the absence of biological molecules are described. The nanoparticles are shown to specifically target antigen presenting cells in specifically in lymph nodes, without the use of a biological molecule for targeting. These particles are useful vehicles for delivering immunotherapeutics.