Discovery of antibodies by longitudinal lineage tracking

Abstract

A method of producing an antibody specific for a target antigen, the method comprising (a) immunizing an animal one or more rounds with the target antigen; (b) collecting a first sample from B cells of the animal; (c) identifying one or more first antibodies specifically binding to the antigen from the B cells in the first sample, and determining a genetic element characterizing the B cell lineage of the B cells in the first sample; (d) performing one or more additional rounds of immunization on the animal with the target antigen; (e) collecting a second sample from the B cells of the animal; (f) identifying one or more second samples from the B cells of the second sample, the B cells of the second sample being from the same cell lineage as the B cells from the first sample; and (g) testing the specific binding of the second antibody to a target antigen. Optionally, in certain embodiments, steps (d) to (g) are repeated.

Description

Cross - Reference to Related Applications

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 382,105, filed on November 2, 2022, the entire content of which is incorporated herein by reference. Background Art (1) Technical Field

[0002] This application generally relates to antibody discovery. More specifically, the application aims to discover antibodies within the same lineage. (2) Description of the related art

[0003] Antibodies have been used in many applications, where high affinity and specificity are key properties for their practical use. In the immune system, antibodies are produced by B cells. After encountering an antigen, naive B cells expand and proliferate and undergo multiple rounds of somatic hypermutation (SHM) and / or gene conversion and positive / negative selection in the germinal center to generate a diverse set of B cells, thus producing antibodies with higher affinity and specificity for the target antigen compared to antibodies earlier in the B - cell lineage ( Figure 1 , Küppers R., 2005). This in - vivo affinity maturation process generally increases antibody activity as it accumulates an increasing number of mutations in the antibody sequence. See, for example, Jiang et al., 2013; Kepler et al., 2014; Zhang et al., 2016; Pan et al., 2023; U.S. Patent No. 7117096B2; U.S. Patent No. 7432063B2; U.S. Patent No. 10101333B2; PCT Patent Publication No. WO2020 / 176815. However, the main cellular and molecular mechanisms of this process remain poorly characterized.

[0004] In addition, in - vivo affinity maturation and other unbiased screening methods can reduce the developability issues that are often caused by conventional phage - display screening processes (Daniela Bumbaca et al., 2011).

[0005] High - throughput sequencing technologies such as next - generation sequencing (NGS) technologies have been widely used as effective sampling methods for analyzing the immune repertoire. These technologies can sequence millions of B cells to obtain a snapshot of the B - cell repertoire data at a specific time point, thus providing an effective and cost - effective sampling method for studying the dynamics of the immune repertoire and tracking longitudinal lineage changes (DeKosky et al., 2013; Phad et al., 2022).

[0006] The present invention utilizes the affinity maturation process captured by high-throughput sequencing technology in immunized animals to obtain high-affinity antibodies against antigens from a B-cell clone lineage that produced antigen-binding antibodies earlier in the lineage. Summary of the Invention

[0007] Provided herein is a method for generating antibodies by immunizing an animal with an antigen over time, repeatedly isolating B cells from the animal, and identifying antibodies in the later-isolated B cells that are of the same lineage as the earlier-isolated B cells, wherein the earlier-isolated B cells produce antibodies that bind the antigen.

[0008] Thus, in some embodiments, provided herein is a method for generating antibodies specific for a target antigen. The method comprises: (a) immunizing an animal with one or more rounds of the target antigen; (b) collecting a first sample of B cells from the animal; (c) identifying one or more first antibodies that specifically bind the antigen from the B cells in the first sample and determining genetic elements that characterize the B-cell lineage of the B cells in the first sample; (d) immunizing the animal with one or more additional rounds of the target antigen; (e) collecting a second sample of B cells from the animal; (f) identifying one or more second antibodies from the B cells of the second sample, wherein the B cells of the second sample are from the same cell lineage as the B cells from the first sample; and (g) testing the specific binding of the second antibody to the target antigen.

[0009] Steps (d) through (g), the additional rounds of immunization, collection of B cells, and identification and testing of antibodies can be repeated one or more times. Brief Description of the Drawings

[0010] Figure 1 is a schematic diagram of a germinal center and B-cell development therein from Küppers R., 2005.

[0011] Figure 2 is a schematic diagram of an exemplary timeline and steps for performing the method described herein.

[0012] Figure 3 is an exemplary flowchart for processing and analyzing NGS data generated from a library of PBMC samples from an animal immunized multiple times with a target antigen.

[0013] Figure 4It is an exemplary flowchart for processing and selecting antibodies for testing.

[0014] Figure 5 It shows an exemplary map of antibody domains, covering part of the CDR3 sequence of the antibody with PCR primers (arrows) to enrich specific lineage groups.

[0015] Figure 6 It is a schematic diagram of lineage group and clone mapping data from the NGS dataset (ALP07VHH_PB_PAN) after the 7th immunization.

[0016] Figure 7A and 7B It shows a schematic diagram where the mismatch score increases with further immunization. Figure 7A It is from the NGS dataset (ALP07VHH_PB_PAN) generated after the 7th immunization. The average mismatch score of the dataset is 8.49. Figure 7B It is from the NGS dataset (NBL504 - A27L1P1L - R2) generated after the 24th immunization. The average mismatch score of the dataset is 15.524. The difference in mismatch scores between these two datasets is significant (p < 0.001, T - test).

[0017] Figure 8 It is a schematic diagram of lineage group mapping between NGS datasets. ALP07VHH_PB_PAN maps to NBL504 - A27L1P1L - R2. There are 28 shared lineage groups between these two NGS datasets, with 2518 and 2341 lineage groups respectively.

[0018] Figure 9 It shows the immunization and blood collection schedule of alpaca A050, as well as 7 PBMC samples used in the analysis.

[0019] Figure 10 It shows the antibody titers of 7 PBMC samples.

[0020] Figure 11 It is a schematic diagram of the average mismatch score of the NGS library constructed using 7 PBMC samples.

[0021] Figure 12 It shows the average mismatch score of shared lineages in the immunoglobulin repertoire of 7 PBMC samples.

[0022] Figure 13 It shows the dynamic changes in mismatch scores of three antigen - specific lineages.

[0023] Figure 14 It is a correlation plot of the mismatch score and ELISA value of the conjugate from alpaca A050.

[0024] Figure 15 The correlation graph of the mismatch score and ELISA value of the llama-derived conjugate. Detailed implementation Definition

[0025] The term "plurality" means more than 1, such as more than 2, more than 5, more than 10, more than 20, more than 50, more than 100, more than 200, more than 500, more than 1000, more than 2000, more than 5000, more than 10000, more than 20000, more than 50000, more than 100000, and generally not exceeding about 200000. "Group" means containing a plurality of items.

[0026] As used herein, the term "epitope" may include any protein determinant capable of specifically binding to an immunoglobulin or T cell receptor. Epitope determinants are usually composed of chemically active molecular surface groups, such as amino acids or sugar side chains, and usually have specific three-dimensional structural features as well as specific charge features. An antibody is said to specifically bind to an antigen when the equilibrium dissociation constant ≤ 1 μM, preferably ≤ 100 nM, more preferably ≤ 10 nM.

[0027] The term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction.

[0028] The term "immune response" as used herein refers to the selective damage, destruction or elimination of an organism against an invading pathogen, an infected pathogen cell or tissue, a cancer cell or (in the case of autoimmunity or pathological inflammation) a normal organic cell or tissue, caused by the action of cells or the liver such as lymphocytes, antigen-presenting cells, phagocytes, granulocytes and soluble macromolecules (including antibodies, cytokines and complements).

[0029] As used herein, the term "antibody" refers to (a) a full immunoglobulin, (b) a monoclonal or polyclonal antigen-binding fragment having an Fc (crystallizable fragment) region or an FcRn-binding fragment of the Fc region ("Fc fragment" or "Fc region"), (c) a nanobody (including naturally occurring camelid nanobodies and single heavy chain ("VHH") antibodies), or (d) IgNAR antibodies found in sharks and other cartilaginous fish. The antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of full antibodies. Antigen-binding fragments include, in particular, Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), single-domain antibodies, chimeric antibodies, diabodies, and polypeptides that contain at least a portion of an immunoglobulin sufficient to confer specific antigen-binding to the polypeptide. The Fc region includes portions of two heavy chains and constitutes two or three classes of antibodies. The Fc region can be produced by recombinant DNA techniques or by enzymatic (e.g., papain cleavage) or chemical cleavage of full antibodies.

[0030] As used herein, the term "antibody fragment" refers to a protein fragment that contains only a portion of a full antibody and generally includes the antigen-binding site of the full antibody and thus retains the ability to bind antigen. Examples of antibody fragments included in this definition are: (i) Fab fragments having VL, CL, VH, and CH1 regions; (ii) Fab' fragments, which are Fab fragments having one or more cysteine residues at the C-terminus of the CH1 region; (iii) Fd fragments having VH and CH1 regions; (iv) Fd' fragments having VH and CH1 regions and one or more cysteine residues at the C-terminus of the CH1 region; (v) Fv fragments having VL and VH domains of an antibody single arm; (vi) dAb fragments consisting of a VH region (Ward et al., 1989); (vii) isolated CDR regions; (viii) F(ab')2 fragments, a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge in the hinge region; (ix) single-chain antibody molecules (e.g., single-chain Fv; scFv) (Bird et al., 1988; Huston et al., 1988); (x) "diabodies" having two antigen-binding sites, which include a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP404097; WO93 / 11161; Hollinger et al., 1993); (xi) "linear antibodies" comprising a pair of tandem Fd fragments (VH-CH1-VH-CH1) that together with complementary light chain polypeptides form a pair of antigen-binding regions (Zapata et al., 1995; U.S. Patent No. 5,641,870).

[0031] As used herein, a "single-chain variable fragment", "single-chain antibody variable fragment", or "scFv" antibody refers to an antibody form that contains only the variable regions of the heavy chain (VH) and the light chain (VL) linked by a linker peptide. The scFv can be expressed as a single-chain polypeptide. The scFv retains the specificity of the intact antibody from which it is derived. The light and heavy chains can be in any order, e.g., VH-linker-VL or VL-linker-VH, so long as the specificity of the scFv for the target antigen is retained.

[0032] As used herein, an "isolated antibody" can refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to the TRAIL protein can be substantially free of antibodies that specifically bind to antigens other than the TRAIL protein). However, an isolated antibody that specifically binds to the human TRAIL protein may cross-react with other antigens, such as TRAIL proteins from other species. In addition, an isolated antibody can be substantially free of other cellular materials and / or chemicals.

[0033] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" can refer to a preparation of antibody molecules having a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0034] As used herein, the term "recombinant human antibody" can refer to any human antibody that is prepared, expressed, produced, or isolated by recombinant means, such as (a) an antibody isolated from or prepared from a transgenic or transchromosomic animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (as described below), (b) an antibody isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) an antibody isolated from a recombinant combinatorial human antibody library, and (d) an antibody prepared, expressed, produced, or isolated by any other means that involves splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when using a human Ig sequence transgenic animal, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant human antibody, although derived from or related to human germline VH and VL sequences, may not naturally occur within the repertoire of in vivo human antibodies.

[0035] The term "isotype" can refer to the class of antibody (such as IgM or IgG1) encoded by the heavy chain constant region gene. Antibodies can be immunoglobulin G (IgG), IgM, IgE, IgA, or IgD molecules, or derivatives thereof.

[0036] The term "VHH"2 ”, “VHH 3 ” and “VH 1 ” respectively represent the heavy chains of three camel IgG isotypes IgG2, IgG3, and IgG1. VL 1 represents the light chain of camel IgG1. Camel VL 1 includes but is not limited to Vκ and Vλ.

[0037] As used herein, the terms "corresponding positioned amino acid" and "corresponding amino acid" are used interchangeably herein and refer to amino acid residues that are in the same position (i.e., they are opposite each other) when aligning two or more amino acid sequences. Methods for aligning and numbering antibody sequences are well known in the art.

[0038] The term "native" antibody refers to an antibody in which the heavy and light chains of the antibody have been made and paired by the immune system of a multicellular organism. The spleen, lymph nodes, bone marrow, blood, and other lymphoid tissues are examples of tissues containing cells that produce native antibodies. For example, the antibodies produced by B cells isolated from the first animal immunized with an antigen are native antibodies. Native antibodies contain naturally paired light and heavy chains.

[0039] The term "naturally paired" refers to heavy and light chain sequences that have been paired by the immune system of a multicellular organism.

[0040] As used herein, the term "mixture" refers to a combination of elements, e.g., cells that are dispersed and in no particular order. A mixture is homogeneous and does not separate spatially into distinct components. Exemplary mixtures of elements include many different cells that are present in the same aqueous solution in a spatially undressed manner.

[0041] The term "evaluate" includes any form of measurement and includes the presence of a particular element. The terms "determine", "measure", "evaluate", "assess", and "assay" are used interchangeably herein and include quantitative and / or qualitative determinations. An evaluation can be relative or absolute. "Evaluate the presence" includes determining the amount of something present and / or determining whether it is present.

[0042] The term "enriched" refers to a component of a composition (e.g., a particular type of cell) that is more concentrated (e.g., at least 2x, at least 5x, at least 10x, at least 50x, at least 100x, at least 1000x) relative to other components (e.g., other cells) in a sample. In some cases, the enriched object may represent a large portion of the sample in which it is present (e.g., more than 2%, more than 5%, more than 10%, more than 20%, more than 50% or more, typically up to about 90%-100%).

[0043] The term "enrichment" refers to any means of obtaining antigen - specific cells from a larger population of B cells. As described in more detail below, enrichment can be accomplished, for example, by panning, using beads, or cell sorting.

[0044] In the context of obtaining an element (e.g., a cell or a sequence), the term "obtain" refers to including receiving the element and physically generating the element.

[0045] The term "peripheral blood mononuclear cell" or "PBMC" refers to blood cells with a single approximately round nucleus (as opposed to a lobed nucleus), including lymphocytes (T cells, B cells, and NK cells), monocytes, and macrophages. PBMCs can be enriched from whole blood using a Ficoll gradient.

[0046] The term "antigen - specific B cell" refers to memory B cells and their progenitors that have antibodies that specifically bind surface antigens.

[0047] A cell "derives from" a host if the cell or its progeny are obtained from the host cell. The progeny of a progenitor cell derive from the progenitor cell.

[0048] The term "panning" is used to refer to a method of applying B cells to a container (e.g., a plate) having one or more surfaces coated with an antigen or a portion thereof. Unbound cells can be removed by washing the surface after the cells are applied to the surface.

[0049] The term "bead - based enrichment" is used to refer to a method of mixing B cells with beads (e.g., magnetic beads) conjugated with an antigen or a portion thereof.

[0050] The term "cell sorting" is used to refer to a method of mixing B cells with a detectable antigen (e.g., a fluorescently detectable antigen) in solution. In a cell sorting method, cells that bind the antigen are sorted from unbound cells. Fluorescence - activated cell sorting (FACS) is an example of a cell sorting method.

[0051] The term "activation" refers to stimulating a B cell to a) proliferate, b) differentiate into plasmablasts and / or plasma cells, and c) secrete antibodies. B cell activation can be accomplished by contacting the B cell with an antigen, a T cell expressing CD40L, and a cytokine, although other methods are known (see, e.g., Wykes, Imm. Cell. Biol. 2003 81:328–331).

[0052] The term "activated B cell" refers to a population of cells that includes the progeny of activated B cells. As described above, activation results in B cell proliferation, and the progeny of such cells are referred to herein as activated B cells.

[0053] The term "collection" refers to the act of separating cells in a culture medium from a substrate. For example, collection can be performed by pipetting or decanting.

[0054] The term "immunized with an antigen" and its grammatical synonyms (e.g., "immunize an animal") are intended to refer to any animal (human, rabbit, mouse, rat, sheep, cow, chicken, camel) that has been immunized against an antigen. For example, an animal can be exposed to a foreign antigen by exposure to an infectious agent, vaccination, or by administration of an antigen and an adjuvant (e.g., by injection). The term "immunized by an antigen" is also intended to include animals that have an immune response to "self" antigens, i.e., animals suffering from an autoimmune disease.

[0055] The term "lineage rank" refers to the order in which lineages are arranged according to priority factors. Priority factors include, but are not limited to, the abundance of lineage sequences, the amplification factor, the dynamic changes in lineage sequences before and after removal of certain unwanted B cells, the dynamic changes in the abundance of lineage sequences during the immune process, lineages with the same naïve B cell origin between VHH and VH, avoiding sequences with development risks, and combinations thereof.

[0056] The term "Hamming distance" refers to the number of positions at which corresponding symbols are different between two sequences of equal length.

[0057] As used herein, the terms "lineage-grouped antibodies", "lineage-related antibodies", and "antibodies related to a lineage" and their grammatically equivalent variants are antibodies produced by cells having a common B cell ancestor. Lineage-related antibodies bind to the same epitope of an antigen, and they are typically very similar in sequence, especially in the light-chain and heavy-chain CDR3s. The heavy-chain and light-chain CDR3s of lineage-related antibodies can have the same length and almost identical sequences (i.e., differing by at most 5, i.e., 0, 1, 2, 3, 4, or 5 residues). In a group of CDR3s from a lineage, the minimum CDR3 distance for a particular CDR3 is the minimum Hamming distance of that CDR3 compared to all other CDR3s of the same length. In some embodiments, the minimum CDR3 distance is equal to or less than 1. In certain cases, the B cell ancestor contains a genome with a rearranged light-chain VIC region and a rearranged heavy-chain VDJ region and produces antibodies that have not yet undergone affinity maturation. Naïve or "virgin" B cells present in spleen tissue are exemplary common B cell ancestors.

[0058] Related antibodies are related through a common progenitor antibody, such as an antibody produced in a naïve B cell ancestor. The term "lineage-related antibodies" is intended to describe a group of antibodies produced by cells from the same B cell ancestor. A "lineage group" contains a group of antibodies that are interrelated by lineage.

[0059] As used herein, the term "at least CDR3" or "at least CDR3 sequence" refers only to the CDR3 sequence, the CDR3 sequence together with the CDR1 and / or CDR2 sequences, or a sequence comprising CDR3 of at least 50 contiguous amino acids up to the entire length of the variable domain.

[0060] As used herein, the term "phylogenetic tree" refers to a diagram generated by phylogenetic analysis that depicts the hypothesized branching sequence of an individual species of interest. The branch points within the phylogenetic tree are called nodes.

[0061] As used herein, the term "lineage" refers to theoretical descent. "Lineage" is used interchangeably with "group", and sometimes an antibody group related to a lineage is referred to as a "lineage group". The terms "group" or "lineage" are exclusive, as a sequence can only belong to one group or lineage.

[0062] As used herein, the term "sub-subgroup" refers to a further grouping of sequences within a lineage based on unique features or characteristics. "Subgroup" is not exclusive, meaning that a sequence can be in different subgroups. For example, a sequence can have two, three, four, five, or six unique features simultaneously. "Sub-subgroup" only applies to VHHs. Applying VHH sequence features can help better select / narrow down the test lineage (representative sequences), which may result in better biological function / biological activity outcomes.

[0063] As used herein, the term "lineage analysis" refers to the analysis of the theoretical lineage of an antibody, which is typically done by analyzing a phylogenetic tree.

[0064] As used herein, the term "sequence read" refers to a nucleotide sequence determined by a sequencer, such as by base calling software associated with the technology.

[0065] As used herein, the term "obtaining an amino acid sequence" refers to obtaining a file containing an amino acid sequence. It is well known that nucleic acid sequences can be translated into amino acid sequences on a computer.

[0066] The terms "monoclonal antibody that recognizes an epitope", "antibody that recognizes an antigen", and "antigen-specific antibody" are used interchangeably herein with the term "antibody that specifically binds to an antigen" or its grammatical equivalents.

[0067] The term "specifically binds" refers to the ability of an antibody to preferentially bind to a specific antigen present in a homogeneous mixture of different molecules. In certain embodiments, the specific binding interaction can distinguish between desired and undesired molecules in a sample, in some embodiments by more than about 10 to 100-fold, such as more than about 1000- or 10,000-fold.

[0068] As used herein, the term "substantially non-binding" to a protein or cell can mean that it does not bind or does not bind to the protein or cell with high affinity, i.e., with a KD of 2 x 10 -6 M or greater, more preferably 1 x 10 -5 M or greater, more preferably 1 x 10 -4 M or greater, more preferably 1 x 10 -3 M or greater, even more preferably 1 x 10 -2 M or greater binds to the protein or cell.

[0069] "High affinity" for an IgG antibody can mean an antibody having a KD of 1 x 10 -6 M or less, preferably 1 x 10 -7 M or less, more preferably 1 x 10 -8 M or less, even more preferably 1 x 10 -9 M or less, even more preferably 1 x 10 -10 M or less for the target antigen. However, "high affinity" binding may vary for other antibody isotypes.

[0070] The term "rarity score" can refer to a measure of similarity to the human germline sequence. In some embodiments, the value is calculated based on the germline framework regions. Prior to calculation, a profile of the percentage of use of each residue in each of the lengths of the four framework regions is determined based on all human IGHV germlines. For each VHH sequence, the residue at each position in the four framework regions is compared to the profile of the framework regions of the same length, and the rarity score for each position in the framework region is calculated according to the percentage of use of that residue divided by the highest percentage of use at the same position. The rarity score for a sequence is the average of the rarity scores of all framework region residues.

[0071] A "humanized antibody" has a sequence that is different from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions. Thus, compared to non-human antibodies, humanized antibodies are less likely to induce an immune response and / or induce a less severe immune response when administered to a subject. In one embodiment, certain amino acids in the framework and constant regions of the heavy and / or light chains of a non-human species antibody are mutated to produce a humanized antibody. In another embodiment, the constant region from a human antibody is fused with the variable region of a non-human species. In another embodiment, a humanized antibody is a CDR-grafted antibody that contains one or more CDRs derived from a particular species or isotype and a human antibody framework. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are altered to reduce the potential immunogenicity when the non-human antibody is administered to a human subject, where the altered amino acid residues are not critical for the immunospecific binding of the antibody to its antigen, or the changes made to the amino acid sequence are conservative changes such that the binding of the humanized antibody to the antigen is not worse than the binding of the non-human antibody to the antigen. Examples of how to prepare humanized antibodies can be found in U.S. Pat. Nos. 6,054,297, 5,886,152, and 5,877,293.

[0072] The term "chimeric antibody" refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, one or more CDRs are derived from a human antibody. In another embodiment, all CDRs are derived from a human antibody. In another embodiment, CDRs from more than one human antibody are mixed and matched in the chimeric antibody. For example, a chimeric antibody can contain CDR1 from the light chain of a first human antibody, CDR2 and CDR3 from the light chain of a second human antibody, and CDRs from the heavy chain of a third antibody. Other combinations are possible.

[0073] The term "bispecific antibody" refers to an antibody that binds to two non-overlapping epitopes of an antigen. In some embodiments, a bispecific antibody includes a VHH that contains only a heavy chain and no light chain. In some embodiments, a bispecific antibody contains a VHH of only the heavy chain and a conventional VH1 / VL1 pair. In some embodiments, a bispecific antibody contains two conventional VH1 / VL1 pairs. In some embodiments, a bispecific antibody has a first heavy chain and a first light chain from a monoclonal antibody targeting one epitope, and a heavy chain and a light chain of another antibody targeting another epitope. In some examples, the other light chain or heavy chain can be different from the first light chain or heavy chain.

[0074] The term SAbDab refers to the Structural Antibody Database at opig.stats.ox.ac.uk / webapps / sabdab.

[0075] The binding of the antibodies of the present invention to an antigen can be evaluated using one or more techniques commonly used in the art. For example, in some embodiments, the antibodies can be tested by ELISA assays, such as using recombinant antigen proteins. Other suitable binding assays include, but are not limited to, flow cytometry assays, in which the antibodies are reacted with a cell line expressing the human antigen, such as HEK293 cells. Alternatively or additionally, the binding of the antibodies, including binding kinetics (such as KD values), etc., can be tested in BIAcore binding assays, Octet Red96 (Pall), etc.

[0076] The term "single B cell sorting" refers to the sorting of individual and isolated single B cells based on antigen specificity. Techniques for single cell isolation and sorting include, but are not limited to: FACS (fluorescence-activated cell sorting, e.g., using fluorescently labeled antigens to isolate antigen-binding cells), ISAAC (immunospot array analysis on chip), LCM (laser capture microdissection), microengraving, and droplet microfluidics.

[0077] The term "mismatch score" refers to a measure for determining the SHM rate. It is calculated as the average number of mismatches in a 100 bp alignment with the best-matching germline gene.

[0078] The method claimed in the present invention relates to the identification of an antibody that binds to an antigen, which is generated by affinity maturation in the same B cell lineage as the B cell that produced an antigen-binding antibody earlier in the lineage.

[0079] Thus, in some embodiments, a method for generating an antibody specific for a target antigen is provided. The method comprises (a) immunizing an animal with the target antigen one or more rounds; (b) collecting a first sample from the B cells of the animal; (c) identifying one or more first antibodies that specifically bind to the antigen from the B cells of the first sample, and determining the genetic elements characterizing the B cell lineage of the B cells in the first sample; (d) immunizing the animal with the target antigen one or more rounds; (e) collecting a second sample from the B cells of the animal; (f) identifying one or more second antibodies from the B cells in the second sample that are from the same B cell lineage as the B cells in the first sample; and (g) testing the specific binding of the second antibody to the target antigen.

[0080] In certain embodiments, the method further comprises repeating steps (d) to (g).

[0081] In these methods, antibodies can be generated against any antigen, such as peptides, proteins, haptens (e.g., conjugated to a carrier molecule), mRNA, DNA, viral vectors that allow expression of the target antigen, or cells.

[0082] Figure 2 An exemplary timeline and steps of using the claimed method are shown. The animal is immunized multiple times with the target antigen. After the primary immunization, serum PBMCs are collected and the serum titer against the target antigen is measured. If the titer is sufficient, the PBMCs can be used to generate antibodies specific to the target antigen using methods such as phage display, B cell panning, hybridoma development, NGS, etc. The animal is then further immunized and PBMCs are collected again. NGS data are generated from these PBMC samples, and the data are analyzed to calculate the mismatch score and identify groups, as Figure 3 shown. Sequences that are in the same group as the previously generated first antibody and have a higher mismatch score than the previously generated first antibody are selected for testing ( Figure 4 ).

[0083] B cells in these methods can be collected by any means known in the art, such as from blood, spleen, lymph nodes, or bone marrow.

[0084] The identification and sequencing in (c) can be performed by any means known now or later discovered. In some embodiments, the sequencing is performed by NGS. Any NGS method can be used in these examples. See, for example, Slatko et al. (2018) for an overview of NGS methods.

[0085] In various embodiments, prior to constructing the NGS library and generating NGS data, the B cell sample is enriched for the target antigen by any means known now or later discovered, such as using phage display, B cell panning, FACS sorting, or other techniques.

[0086] B cells in a particular lineage can be identified by any means known now or later discovered (e.g., in (f)). In some embodiments, the identification in (f) is by sequencing a portion of the variable region from the B cells and identifying genetic elements that characterize the B cell lineage. In other embodiments, for example, in cases where the lineage is too rare to be identified by NGS, the identification is performed by polymerase chain reaction (PCR) of genetic elements that characterize the B cell lineage. In some of these embodiments, the genetic element is part of the CDR3 sequence of the first antibody heavy and / or light chain. See, for example, Yaari et al. (2015), Briney et al. (2016), and Kepler et al. (2014) for exemplary methods of identifying and isolating B cell lineages.

[0087] NGS technology has been widely used as an effective sampling method for capturing the immune repertoire. Using NGS, millions of B cells can be sequenced cost-effectively to obtain a good profile of the B cell repertoire at a specific time point, and tens of thousands of different antibody sequences can be generated from a typical NGS library. It is an effective immune repertoire sampling method that has been used to study the dynamics of the immune repertoire and its lineage changes. In addition, by using NGS and primer amplification of antibody sequences with specific sequences, such as in CDR3, the lineage groups of interest from the immune repertoire can be enriched and their changes can be traced. Using the DNA sequences of antibodies derived from NGS, the SHM rate of sequence accumulation can be estimated by comparing the sequences with germline sequences, and antibodies with higher activity than the original antibodies can be discovered by selecting and testing sequences with a high SHM rate from the same lineage group.

[0088] In some embodiments, an NDS library is constructed using PCR primers that cover partial CDR3 sequences of the antibodies identified in step (c) to enrich the lineage groups of these binders ( Figure 5 ). Because the immune system is very active, some lineage groups may become very small after a period of time. To capture these lineages, specially designed PCR primers may be required to enrich these lineages.

[0089] To map a sequence to a group from NGS data, the following criteria can be used: if the sequence has the same CDR1 / CDR2 / CDR3 sequence as one of the sequences in the CDR sequence group, the sequence is mapped to the CDR sequence group; if the sequence maps to the same V / J germline gene and has the same CDR3 sequence as one of the sequences in the lineage, the sequence is mapped to the lineage group; if the sequence has the same CDR3 sequence as one of the sequences in the cluster, the sequence is mapped to the cluster group.

[0090] Similarly, a group from one NGS dataset can be mapped to a group from another NGS dataset. If the CDR sequence group of one NGS dataset shares the same CDR1 / CDR2 / CDR3 sequence as the CDR sequence group of another NGS dataset, they can be mapped to the CDR sequence group of the other NGS dataset. If the lineage group of one NGS dataset maps to the same V / J germline gene and shares a common CDR3 with the lineage group of another NGS dataset, the lineage group of the one NGS dataset will be mapped to the lineage group of the other NGS dataset. If the cluster group of one NGS dataset shares a common CDR3 with the cluster group of another NGS dataset, the cluster group of the one NGS dataset will be mapped to the cluster group of the other NGS dataset.

[0091] The methods of the present invention are not limited to any particular immunization schedule or time between collection in (b) and collection in (e). In some embodiments, the collection in (b) is at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 18 months, or 24 months before the collection in (e).

[0092] Any type of antibody can be collected using these methods. In some embodiments, the antibody is an immunoglobulin or fragment having two light chains and two heavy chains, or one light chain and one heavy chain. In other embodiments, the antibody is a nanobody.

[0093] In some embodiments, additional rounds of immunization are performed, B cells are collected, and B cells are identified from lineages that produce antibodies that bind to the previously collected target antigen. For clarity, the term "second antibody" should be broadly interpreted to refer to antibodies identified after these additional rounds of immunization.

[0094] Any number of first antibodies and B cell lineages can be identified in these methods. In some embodiments, more than 1, 2, 5, 10, 25, 50, 100, 150, or 200 first antibodies are identified and sequenced in (c).

[0095] In some embodiments, the binding affinity, specificity, and / or neutralizing ability of the first antibody or second antibody against the antigen are determined.

[0096] In various embodiments, a B cell sequence library is created and sequenced from the antibody sequences in B cells before immunization, for example to help determine lineages and to help identify CDR regions.

[0097] In additional embodiments, a mismatch score is generated for each first antibody and / or second antibody by aligning the sequence encoding each first antibody and / or second antibody with the germline sequence of the animal.

[0098] Any antibody identified by the methods claimed in the present invention can be further evaluated to determine its beneficial properties.

[0099] Any antibody identified by the methods of the present invention can be further evaluated to identify favorable features. In various embodiments, the first antibodies are grouped by CDR sequence, lineage, and cluster.

[0100] In some embodiments, an enrichment score for each sequence is generated by comparing the sequence frequencies between two samples. Sequences can be grouped into CDR sequences, for example, if their CDR1, CDR2, and CDR3 sequences are the same. Additionally, sequences can be further grouped into lineages, for example, if the sequences map to the same V / J germline genes and have CDR3s of the same length, where CDR3s with a length greater than 4 have at most 1 aa difference and CDR3s with a length equal to or shorter than 4 have 0 aa differences; sequences can also be further grouped into clusters if they have CDR3s of the same length and have 80% or more identity in the CDR3 sequence. Similar enrichment scores for the groups are also calculated. Clones in the sequences and groups that do not show any enrichment can be filtered for testing.

[0101] In additional embodiments that consider lineages or lineage rankings, the lineage prioritization factors are one or more of the lineages with sequence abundances from high to low, lineages with amplification coefficients from high to low, lineages with changes in sequence abundances during the immune process, lineages with changes in sequence abundances before and after depleting certain unwanted B cells, lineages that share the same naïve B cell origin between VHH and VH1, or lineages that avoid sequences with poor developability (liability).

[0102] In further embodiments, the developability of the first, second, or third antibody is evaluated, and if the developability is poor, it is excluded from further development. Non-limiting examples of poor developability include unpaired cysteines, N-linked glycosylation, methionine oxidation, tryptophan oxidation, asparagine deamidation, aspartic acid isomerization, lysine glycosylation, N-terminal glutamic acid, integrin binding, CD11c, fragmentation, immunogenicity, expression, homogeneity, solubility, stability, viscosity, and / or formulatability.

[0103] In other embodiments, the antibody is a nanobody. In some of these embodiments, the characteristics that characterize an effective nanobody are evaluated by a model. See specifically WO 2020 / 176815 and the PCT patent application titled "Selection of Nanobodies Using Sequence Features" (the entire content of which is incorporated herein by reference) filed by the applicant on November 2, 2023. The characteristics include: (a) A hydrophilic region in FR2; (b) An extended CDR1; (c) An additional disulfide bond between CDR1-CDR3 or FR2-CDR3; (d) An additional disulfide bond within CDR3; (f) A long CDR3 (≥15 aa); (g) An additional disulfide bond within CDR1; (h) A non-classical VHH having the same V and J germlines as conventional IgG1; (i) A non-classical VHH having predetermined sequence characteristics; (j) A new classical binding loop structure; (k) A convergent motif or sequence characteristic; (l) Phenylalanine (F) at position 42 (IMGT numbering); (l) A short hinge; (m) Two or more cysteines in the nanobody sequence; (n) Glutamine (Q) at position 123 (IMGT numbering); (o) A low immunogenicity index; (p) A non-classical VHH derived from the germline IGHV3; (q) A non-classical VHH derived from the germline IGHV4; (r) Histidine (H), aspartic acid (D), or glutamic acid (E) in the CDR region; (s) Histidine (H), aspartic acid (D), or glutamic acid (E) in the first sixteen amino acid residues of the FR3 region, the FR2 region, and the first three amino acid residues of the nanobody sequence; (t) Tyrosine (Y) at position 42 (IMGT numbering), and a nanobody having a cyclic concave paratope structure configuration; or (u) Phenylalanine (F) at position 42 (IMGT numbering), and a nanobody having a convex paratope structure configuration.

[0104] In these methods, any antibody can be expressed by any means known in the art. In some embodiments, the selected antibody is expressed in prokaryotic cells. In other embodiments, the selected antibody is expressed in eukaryotic cells.

[0105] Preferred embodiments are described in the examples below. By considering the specification or practice of the present invention disclosed herein, other embodiments of the present invention within the scope of the claims herein will be apparent to those skilled in the art. This specification, including the examples, should be considered merely exemplary, and the true scope and concept of the present invention are defined by the appended claims.

[0106] Example 1: Discovery of High-Affinity Nanobodies Against PD1 by Longitudinal Lineage Tracing

[0107] Using the PD1 recombinant protein as an immunogen, a llama was immunized multiple times. After the seventh immunization, the PBMCs of the animal were enriched, and multiple nanobodies (in the form of VHH) were identified from the PBMCs using phage display technology. Three of these binders (Table 1) showed multiple binding activities belonging to different lineage groups.

[0108] Table 1. Three binders against PD1

[0109] Multiple NGS datasets were generated from PBMC samples collected after the 7th, 20th, and 24th immunizations. Figure 6 The lineage groups and clone mapped groups in the NGS data after the 7th immunization are shown. In this particular case, 85 clones identified by phage display technology were mapped to 26 lineage groups (dark-colored dots).

[0110] Three lineage groups mapped by three clones in Table 1 were labeled with the corresponding clone names. With further immunization, we verified whether, as expected, the antibody sequences accumulated more and more mutations. By comparing the NGS datasets generated after the 7th immunization with those after the 24th immunization, a significant increase in mismatch scores was observed (Figure 7). For longitudinal lineage tracing, we also performed a mapping analysis on the two NGS datasets. Figure 8 An example of such mapping results is shown. As expected, the immune repertoire is highly dynamic, with many lineages disappearing while many new lineages appear. Table 2 shows the frequency changes of some shared lineages after further immunization.

[0111] Table 2. Figure 8 Frequency changes of some shared lineage populations. Shared pedigree Frequency in ALP07VHH_PB_PAN Frequency in NBL504-A27L1P1L-R1 1 14.1％ 19.3％ 2 10.8％ 5.2％ 3 10.7％ 0.01％ 4 1.5％ 8.1％ 5 0.69％ 0.09％ 6 0.26％ 14.1％ 7 0.17％ 0.003％ 8 0.09％ 0.04％

[0112] To discover antibodies that might be better than the three antibodies in Table 1, we mapped these three clones into groups of various NGS datasets and selected and tested clones with high mismatch scores. The results are shown in Table 3. The SPR results are shown in Table 4. In all three cases, based on the KD values, an approximately 5-fold increase in affinity was observed. According to the IC50 values, improved blocking activity was also observed in two cases.

[0113] Table 3. Clone selection of three binders

[0114] Table 4. SPR and blocking results of selected clones

[0115] Example 2: Observation of mismatch score changes and their correlation with affinity through longitudinal analysis

[0116] To discover binders against the PD-L1 target (NBL518 project), a llama (A050) was immunized with human PD-L1 immunogen. Figure 9 The detailed immunization and blood collection schedule for this animal is shown. To avoid immunoglobulin repertoire bias caused by experimental screening / enrichment steps, PBMC samples from some of the blood collection samples ( Figure 9 ) were directly used to construct NGS libraries. As the number of immunizations increased, the antibody titers of these PBMC samples gradually increased ( Figure 10 ). The average mismatch score for each library was calculated based on the NGS sequences. Figure 11 It was shown that the average mismatch scores of all samples after immunization were higher than those of the pre-immunization samples and showed a gradually increasing trend as the number of immunizations increased. To analyze the changes in mismatch scores within the lineage, we focused on the shared lineages among these samples. A total of 206 shared lineages were found, and their average mismatch scores gradually increased ( Figure 12 ). To focus on antigen-specific lineages (e.g., lineages containing target-binding antibodies), we identified three such lineages shared among the post-immunization samples. Figure 13 The dynamic changes in mismatch scores for these three lineages are shown. Overall, as the number of immunizations increased, the mismatch scores gradually increased, consistent with the results of Figure 11 and Figure 12 .

[0117] Through multiple discovery experiments, various binders with different affinities for the PD-L1 target were identified from this animal. A low but statistically significant correlation (P<0.05) was observed between the mismatch scores of these antibodies and the ELISA values ( Figure 14 ). Similar results were also observed for the binders identified from llamas ( Figure 15 ). These results again indicate that affinity increases with the accumulation of somatic mutations. Embodiments:

[0118] Embodiment 1. A method for generating an antibody specific for a target antigen, the method comprising: (a) immunizing an animal with the target antigen for one or more rounds; (b) collecting a first sample from the B cells of the animal; (c) Identify one or more first antibodies that specifically bind to the antigen from the B cells in the first sample, and determine genetic elements that characterize the B cell lineage of the B cells in the first sample; (d) Immunize the animal with the target antigen for one or more additional rounds; (e) Collect a second sample from the B cells of the animal; (f) Identify one or more second samples from the B cells of the second sample, where the B cells of the second sample are from the same cell lineage as the B cells from the first sample; and (g) Test the specific binding of the second antibody to the target antigen.

[0119] Embodiment 2. The method according to Embodiment 1, further comprising: (h) Repeat steps (d) to (g).

[0120] Embodiment 3. The method according to Embodiment 1 or 2, wherein the identification in (c) comprises sequencing the first antibody by NGS.

[0121] Embodiment 4. The method according to any one of Embodiments 1-3, wherein the identification in (c) comprises B cell panning and / or phage display.

[0122] Embodiment 5. The method according to any one of Embodiments 1-4, wherein the B cells in the second sample in (f) recognize the genetic elements that characterize the B cell lineage.

[0123] Embodiment 6. The method according to Embodiment 5, wherein the genetic element is a part of the variable region of the first antibody.

[0124] Embodiment 7. The method according to Embodiment 6, wherein the genetic element is a part of the CDR3 sequence of the heavy chain and / or light chain of the first antibody.

[0125] Embodiment 8. The method according to any one of Embodiments 1-4, wherein the identification in (f) comprises sequencing the second antibody by NGS.

[0126] Embodiment 9. The method according to Embodiment 1, wherein the identification in (f) comprises polymerase chain reaction (PCR) of the genetic elements that characterize the B cell lineage.

[0127] Embodiment 10. The method according to Embodiment 9, wherein the genetic element is a part of the variable region of the first antibody.

[0128] Embodiment 11. The method according to embodiment 10, wherein the genetic element is part of the CDR3 sequence of the first antibody heavy chain and / or light chain.

[0129] Embodiment 12. The method according to any one of embodiments 1-11, wherein the collection in (b) is at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 18 months or 24 months before the collection in (e).

[0130] Embodiment 13. The method according to any one of embodiments 1-12, wherein the first antibody or the second antibody is an immunoglobulin or a nanobody.

[0131] Embodiment 14. The method according to any one of embodiments 1-13, wherein the animal is a mammal.

[0132] Embodiment 15. The method according to embodiment 14, wherein the mammal is a camel.

[0133] Embodiment 16. The method according to any one of embodiments 1-15, wherein two or more first antibodies are identified in (c).

[0134] Embodiment 17. The method according to embodiment 16, wherein the first antibodies are grouped by CDR sequence, lineage and cluster.

[0135] Embodiment 18. The method according to embodiment 16 or 17, wherein a mismatch score is generated for each first antibody and / or second antibody by aligning the sequence encoding each first antibody and / or second antibody with the germline sequence of the animal.

[0136] Embodiment 19. The method according to embodiment 18, 19. wherein in step (g), second antibodies with higher mismatch scores are preferentially tested.

[0137] Embodiment 20. The method according to any one of embodiments 17-19, wherein sequences within the same CDR group have the same CDR1, CDR2 and CDR3 sequences.

[0138] Embodiment 21. The method according to any one of embodiments 17-19, wherein sequences in the lineage map to the same V and J germline genes and have a maximum distance of specific CDR3 equal to or less than 1 aa between the two closest CDR3s in the lineage, wherein all CDR3s have the same length.

[0139] Embodiment 22. The method according to any one of embodiments 17-19, wherein the sequences in the cluster have the same CDR3 length, and the CDR3 identity between the two closest CDRs3 in the cluster is greater than 80%.

[0140] Embodiment 23. The method according to any one of embodiments 17-22, wherein the first antibody or the second antibody is a nanobody.

[0141] Embodiment 24. The method according to any one of embodiments 1-23, wherein the antibodies are prioritized according to at least one of the following features: (a) FR2 hydrophilic region; (b) Extended CDR1; (c) An additional disulfide bond between CDR1-CDR3 or FR2-CDR3; (d) An additional disulfide bond within CDR3; (e) Long CDR3 (≥15aa); (f) An additional disulfide bond within CDR1; (g) A non-classical VHH having the same V and J germlines as conventional IgG1; (h) A non-classical VHH having a predetermined sequence feature; (i) A new classical binding loop structure; (j) A convergent motif or sequence feature; (k) Phenylalanine (F) at position 42 (IMGT numbering); (l) Short hinge; (m) Two or more cysteines in the nanobody sequence; (n) Glutamine (Q) at position 123 (IMGT numbering); (o) Low immunogenicity index; (p) A non-classical VHH derived from germline IGHV3; (q) A non-classical VHH derived from germline IGHV4; (r) Histidine (H), aspartic acid (D) or glutamic acid (E) in the CDR region; (s) The first sixteen amino acid residues of the FR3 region, the FR2 region, and histidine (H), aspartic acid (D) or glutamic acid (E) in the first three amino acid residues of the nanobody sequence; (t) Tyrosine (Y) at position 42 (IMGT numbering), and a nanobody having a concave paratope structure configuration; or (u) Phenylalanine (F) at position 42 (IMGT numbering), and a nanobody having a convex paratope structure configuration.

[0142] Embodiment 25. The method according to any one of embodiments 1-24, wherein if the antibody has at least one development liability, the first antibody or the second antibody is eliminated, and the at least one development liability is immunogenicity, expression, homogeneity, solubility, stability, viscosity, or formulatability.

[0143] Embodiment 26. The method according to any one of embodiments 1-24, wherein the first antibody or the second antibody is tested for broad-spectrum binding affinity.

[0144] Embodiment 27. The method according to any one of embodiments 1-26, wherein the animal is immunized with a peptide, protein, mRNA, DNA, viral vector, or cell.

[0145] Embodiment 28. The method according to any one of embodiments 1-27, wherein the binding affinity, specificity, or neutralizing ability of the first antibody or the second antibody against the antigen is determined.

[0146] Embodiment 29. The method according to any one of embodiments 1-28, wherein the first antibody or the second antibody is expressed in a prokaryotic cell.

[0147] Embodiment 30. The method according to any one of embodiments 1-28, wherein the first antibody or the second antibody is expressed in a eukaryotic cell.

[0148] In view of the above, it can be seen that several objects of the present invention have been achieved and other advantages have been obtained.

[0149] Since various changes can be made to the above methods and compositions without departing from the scope of the present invention, all the content included in the above description and shown in the drawings should be construed as illustrative rather than restrictive.

[0150] All references cited in this specification, including but not limited to patent publications and non-patent literature, and references cited therein, are hereby incorporated by reference. The discussion of references herein is only intended to summarize the assertions made by the authors and does not admit that any reference constitutes prior art. The applicant reserves the right to challenge the accuracy and relevance of the cited references.

[0151] As used herein, in certain embodiments, the term "about" or "approximately" when preceding a numerical value indicates a range of plus or minus 10% of that value. It should be understood that when a numerical range is given, unless otherwise clearly stated in the text, the values between the upper and lower limits of the range, the intermediate values to one-tenth of the unit of the lower limit, and any other marked or intermediate values of the said range are all included within the scope of the present invention. The upper and lower limits of these smaller ranges may be independently included within these smaller ranges, and they also fall within the scope of the present invention, unless the upper and lower limits of the said range are clearly excluded. When a range includes one or both of the limiting values, the present invention also includes ranges excluding one or both of the said limiting values.

[0152] In the specification and embodiments of the present application, unless there is a clear contrary indication, the indefinite article with "a" should be understood as "at least one...".

[0153] The phrase "and / or" used in the specification and embodiments should be understood to mean "one or both" of the elements so combined, i.e., elements that are present jointly in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same way, i.e., "one or more" elements so combined. Other elements other than those specifically pointed out by the "and / or" clause may optionally exist, whether or not they are related to those specifically pointed out elements. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprising", a reference to "A and / or B" may, in one embodiment, refer only to A (optionally including elements other than B); in another embodiment, it may refer only to B (optionally including elements other than A); in yet another embodiment, it may refer to both A and B (optionally including other elements); and so on.

[0154] The "or" used in this specification and embodiments should be understood to have the same meaning as the "and / or" defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one, but also including more than one, including a certain number or series of elements or components, and also including optionally unlisted items. Only terms that clearly indicate the contrary, such as "only one of... " or "exactly one of... " or when the phrase "consisting of / constituted by... " is used in the embodiments, refer to exactly including one of several elements or a series of elements. Generally speaking, as used herein, "or" can only be interpreted as indicating mutually exclusive alternative choices (i.e., "this or that but not both") when there are exclusive terms before and after it, such as "either", "(one) of which", "only (one) of which", or "exactly (one) of which". "Consisting essentially of / constituted essentially by... " should be understood to have its ordinary meaning in the field of patent law when used in embodiments.

[0155] As used herein in the specification and examples, when referring to a list of one or more elements, the phrase "at least one" shall be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows that an element may optionally be present rather than an element specifically identified in the list of elements referred to by the phrase "at least one", whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B", or, equivalently "at least one of A and / or B") can, in one embodiment, mean at least one, optionally including more than one of A, with no B present (and optionally including elements other than B); in another embodiment, it means at least one, optionally including more than one of B, with no A present (and optionally including elements other than A); in another embodiment, at least one means optionally including more than one of A, and at least one, optionally including more than one of B (and optionally including other elements); and so on. References Arbabi-Ghahroudi. Camelid Single-Domain Antibodies: Historical Perspective and Future Outlook. Front. Immunol., 20 November. Basilico et al., Four individually druggable MET hotspots mediate HGF-driven tumor progression, The Journal of Clinical Investigation, Volume 124 Number 7 July, 2014. Bird et al., Science 242:423-426 (1988). Briney et al., Scientific Reports 6:23901 (2016). Conrath KE et al Emergence and evolution of functional heavy-chain antibodies in Camelidae. Dev Comp Immunol 27:87–103, 2003. Daley LP, Clin. Vaccine Immunol. 17:239–46, 2010. Daniela Bumbaca et al., Highly specific off-target binding identified and eliminated during the humanization of an antibody against FGF receptor 4. mAbs 3:4, 376-386; July / August 2011. DeKosky, B.J. et al., High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire. Nature Biotechnology, 31(2), 166–169 (2013). Deschacht, et al A Novel Promiscuous Class of Camelid Single-Domain Antibody Contributes to the Antigen-Binding Repertoire, The Journal of Immunology. 184(10)5696-5704, May 2010. Griffin et al Analysis of heavy and light chain sequences of conventional camelid antibodies from Camelus dromedarius and Camelus bactrianus species, Journal of Immunological Methods Volume 405, Pages 35-46, March 2014. Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448(1993). Huston et al, PNAS(USA) 85:5879-5883(1988). Jiang et al, Sci. Trans. Med 5:171ra19(2013). Kepler et al, Front. Immunol. 5:170(2014). Klarenbeek, et al. Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform, mAbs 7:4, 693 - 706; 2015. Küppers R. Mechanisms of B - cell lymphoma pathogenesis. Nat Rev Cancer (2005) 5:251–262. McCoy LE, et al. Potent and broad neutralization of HIV - 1 by a llama antibody elicited by immunization. J. Exp. Med. 2012. Nguyen et al. Camel heavy - chain antibodies: diverse germline VHH and specific mechanism enlarge the antigen - binding repertoire The EMBO Journal 19 No.5 2000. Nguyen et al. Heavy - chain antibodies in Camelidae; a case of evolutionary innovation. Immunogenetics 54:39–47, 2002. Pan, X., López Acevedo, S. N., Cuziol, C., de Tavernier, E., Fahad, A. S., Longjam, P. S., Rao, S. P., Aguilera-Rodríguez, D., Rezé, M., Bricault, C. A., Gutiérrez-González, M. F., de Souza, M. O., DiNapoli, J. M., Vigne, E., Shahsavarian, M. A., & DeKosky, B. J. (2023). Large-scale antibody immune response mapping of splenic B cells and bone marrow plasma cells in a transgenic mouse model. Frontiers in Immunology, 14. Phad, G. E. et al., Clonal structure, stability and dynamics of human memory B cells and circulating plasmablasts. Nature Immunology, 23(7), 1–10(2022). Slatko et al, Curr. Protoc. Mol. Biol. 122: e59, 2018. Ward et al, Nature 341, 544 - 546(1989). Woninga, et al DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops, MABS, VOL. 8, NO. 6, 1126–1135, 2016. Yaari, G. and Kleinstein, S. Genome Med. 7: 121(2015). Zapata et al Protein Eng. 8(10): 1057 - 1062(1995). Zhang et al, Immunol. Rev. 270: 8 - 19(2016). European Patent No. 404,097. PCT Patent Publication No. WO 93 / 11161. PCT Patent Publication No. WO 2020 / 176815. PCT Patent Publication No. WO 2020 / 208555. U.S. Patent Application Publication No. 2019 / 0065677A1. U.S. Patent No. 5,641,870. U.S. Patent No. 6,054,297. U.S. Patent No. 5,886,152. U.S. Patent No. 5,877,293. US Patent 7,117,096B2. US Patent 7,432,063B2. US Patent 10,101,333B2.

Claims

1. A method for generating antibodies specific to a target antigen, the method comprising (a) immunizing an animal with the target antigen for one or more rounds; (b) collecting a first sample from the B cells of the animal; (c) identifying one or more first antibodies that specifically bind to the antigen from the B cells in the first sample, and determining genetic elements that characterize the B cell lineage of the B cells in the first sample; (d) immunizing the animal with the target antigen for one or more additional rounds; (e) collecting a second sample from the B cells of the animal; (f) identifying one or more second samples from the B cells of the second sample, the B cells of the second sample being from the same cell lineage as the B cells from the first sample; and (g) testing the specific binding of the second antibody to the target antigen.

2. The method according to claim 1, further comprising (h) repeating steps (d) to (g).

3. The method according to claim 1 or 2, wherein the identification in (c) comprises sequencing the first antibody by NGS.

4. The method according to any one of claims 1 - 3, wherein the identification in (c) comprises B cell panning and / or phage display.

5. The method according to any one of the preceding claims 1 - 4, wherein the B cells in the second sample in (f) recognize the genetic elements that characterize the B cell lineage.

6. The method according to claim 5, wherein, The genetic element is part of the variable region of the first antibody.

7. The method according to claim 6, wherein, The genetic element is part of the CDR3 sequence of the heavy chain and / or light chain of the first antibody.

8. The method according to any one of claims 1 - 4, wherein the identification in (f) comprises sequencing the second antibody by NGS.

9. The method according to claim 1, wherein the identification in (f) comprises polymerase chain reaction (PCR) of the genetic elements that characterize the B cell lineage.

10. The method according to claim 9, wherein, The genetic element is part of the variable region of the first antibody.

11. The method according to claim 10, wherein, The genetic element is part of the CDR3 sequence of the heavy chain and / or light chain of the first antibody.

12. The method according to any one of claims 1 - 11, wherein the collection in (b) is at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 18 months or 24 months before the collection in (e).

13. The method according to any one of claims 1 - 12, wherein the first antibody or the second antibody is an immunoglobulin or a nanobody.

14. The method according to any one of claims 1 - 13, wherein the animal is a mammal.

15. The method according to claim 14, wherein, The mammal is a camel.

16. The method according to any one of claims 1 - 15, wherein two or more first antibodies are identified in (c).

17. The method according to claim 16, wherein the first antibodies are grouped by CDR sequence, lineage and cluster.

18. The method according to claim 16 or 17, wherein a mismatch score is generated for each first antibody and / or second antibody by aligning the sequence encoding each first antibody and / or second antibody with the germline sequence of the animal.

19. The method according to claim 18, wherein in step (g), the second antibody with a higher mismatch score is preferentially tested.

20. The method according to any one of claims 17-19, wherein the sequences within the same CDR group have the same CDR1, CDR2, and CDR3 sequences.

21. The method according to any one of claims 17-19, wherein, Sequences in the lineage map to the same V and J germline genes and have a maximum distance of the specific CDR3 equal to or less than 1 aa between the two closest CDR3s in the lineage, where all CDR3s have the same length.

22. The method according to any one of claims 17-19, wherein the sequences in the cluster have the same CDR3 length and the CDR3 identity between the two closest CDR3s in the cluster is greater than 80%.

23. The method according to any one of claims 17-22, wherein the first antibody or the second antibody is a nanobody.

24. The method according to any one of claims 1-23, wherein the antibodies are prioritized according to at least one of the following characteristics: (a) FR2 hydrophilic region; (b) Extended CDR1; (c) Additional disulfide bonds between CDR1-CDR3 or FR2-CDR3; (d) Additional disulfide bonds within CDR3; (e) Long CDR3 (≥15 aa); (f) Additional disulfide bonds within CDR1; (g) Non-classical VHH with the same V and J germlines as conventional IgG1; (h) Non-classical VHH with a predetermined sequence feature; (i) New classical binding loop structure; (j) Convergent motif or sequence feature; (k) Phenylalanine (F) at position 42 (IMGT numbering); (l) Short hinge; (m) Two or more cysteines in the nanobody sequence; (n) Glutamine (Q) at position 123 (IMGT numbering); (o) Low immunogenicity index; (p) Non-classical VHH derived from germline IGHV3; (q) Non-classical VHH derived from germline IGHV4; (r) Histidine (H), aspartic acid (D), or glutamic acid (E) in the CDR region; (s) Histidine (H), aspartic acid (D), or glutamic acid (E) in the first sixteen amino acid residues of the FR3 region, the FR2 region, or the first three amino acid residues of the nanobody sequence; (t) Tyrosine (Y) at position 42 (IMGT numbering), and a nanobody with a cyclic concave paratope structure configuration; or (u) Phenylalanine (F) at position 42 (IMGT numbering), and a nanobody with a convex paratope structure configuration.

25. The method according to any one of claims 1-24, wherein If the antibody has at least one developability issue, the first antibody or the second antibody is eliminated, wherein the at least one developability issue is immunogenicity, expression, homogeneity, solubility, stability, viscosity, or formulatability.

26. The method according to any one of claims 1-24, wherein The first antibody or the second antibody is tested for binding at multiple different affinity levels.

27. The method according to any one of claims 1-26, wherein the animal is immunized with a peptide, protein, mRNA, DNA, viral vector, or cell.

28. The method according to any one of claims 1-27, wherein, The binding affinity, specificity, or neutralizing ability of the first antibody or the second antibody against the antigen is determined.

29. The method according to any one of claims 1-28, wherein the first antibody or the second antibody is expressed in a prokaryotic cell.

30. The method according to any one of claims 1-28, wherein the first antibody or the second antibody is expressed in a eukaryotic cell.

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