Discovery of antibodies by memory b cell recall

Abstract

The invention relates to discovery of antibodies by memory B cell recall. Specifically provided is a method of identifying an antibody specific for a target, the method comprising: (a) immunizing an animal with a target antigen one or more rounds, (b) reducing serum antibody titer of the animal by two or more times, (c) immunizing the animal with a recalled antigen having an antibody binding region of at least 30% identity to the target antigen, (d) generating an antibody sequence from the B cells of the animal 1-20 days after immunization in step (c), and (e) selecting an antibody from the antibody sequence by one or more priority factors.

Description

Technical Field

[0001] This application relates to the discovery of monoclonal antibodies. Specifically, this application relates to a method for directly discovering antibodies from B cells using high-throughput sequencing without experimental enrichment of target-specific B cells / antibodies. Background Art

[0002] Monoclonal antibodies have been widely used in the therapeutic field. Since the first drug approval in 1986, the FDA has approved more than 100 antibody-based drugs. In addition, monoclonal antibodies are also widely used as research / diagnostic reagents. To discover monoclonal antibodies, a variety of techniques have been developed. The hybridoma technique was invented by Georges Kohler and Cesar Milstein in 1975. This is a method for producing large amounts of monoclonal antibodies that can be screened and produced. Phage display technology was developed by George P Smith and Sir Gregory P Winter in the early 1990s. This is an in vitro display and screening technique that has accelerated the discovery and engineering of monoclonal antibodies. Recently, to address some of the drawbacks of phage display technology, several single B cell isolation and culture techniques have been developed to develop antibodies. Generally, the proportion of antigen-specific B cells in the repertoire is very low, so various methods, such as B cell plate sorting, sorting, phage plate sorting, etc., are needed to enrich antigen-specific B cells / antibodies before screening.

[0003] B cells produce antibodies. There are two main types of B cells responsible for antibody-mediated humoral memory responses: plasma cells (PCs) that secrete antibodies and memory B cells (MBCs) that have membrane-attached antibodies (B cell receptors). However, only MBCs are involved in the recall response. The MBC recall response describes a process in which antigen-specific MBCs encounter the same antigen again, and they clonally expand and produce large amounts of antigen-specific antibodies ( Figure 1)。This is the natural response of the immune system against pathogens and other invaders. Detailed studies have shown that during the recall process, when new antigen stimulation occurs through membrane-bound B cell receptors, MBCs may differentiate into short-lived PCs or plasmablasts, proliferate through clonal expansion, or enter the germinal center (GC) for further affinity maturation and repertoire diversification to gain better protection with higher affinity for antigen variants and broader epitope coverage. There are three factors in B cell fate determination: the affinity for the antigen, the duration of interaction with the antigen and cognate T cells, and the survival signals provided by the microenvironment. The general situation seems to be that B cells with higher affinity BCRs are more likely to differentiate into plasma cells. In fact, compared with IgM+ MBCs, IgG+ MBCs have been shown to preferentially differentiate into short-lived PCs upon secondary encounter with the same antigen. PCs are antibody-secreting cells with extremely high rates of antibody gene transcription, translation, and antibody secretion. Based on single-cell expression data from the website www.proteinatlas.org, when B cells differentiate into plasma cells, the expression of 4 human IGHG genes (IGHG1, IGHG2, IGHG3, and IGHG4) increases by 5.0, 10.7, 4.1, and 0.4-fold, respectively.

[0004] A recent study has highlighted how early MBCs (eMBCs) are formed, as early as 2.5 days after antigen encounter. These cells do not undergo somatic hypermutation (SHM) and mostly do not undergo class switch, thus showing a different transcriptional program compared to GC-dependent counterparts. In addition, it was found that the number of vaccine-specific plasma cells increased as early as day 7 after boost immunization together with clonal sequence expansion, and these expansions led to increased mutations, and the reduction in diversity and CDR3 sequence length in the repertoire may indicate that these vaccine-specific plasma cells may have passed through the germinal center or remain short-lived plasma cells, as somatic hypermutation (SHM) and affinity maturation can occur in the germinal center and extrafollicular B cell responses. The type of animal used, the duration between two boosters, and the nature or structure of the immunogen all affect the quality and quantity of B cell recall, including the percentage composition of naive B cells, short (GC-independent) or long-lived MBCs, plasmablasts, and short (GC-independent) or long-lived PCs ( Figure 1 C).

[0005] High-throughput sequencing technologies such as next-generation sequencing (NGS) techniques have been widely used as effective sampling methods for analyzing the immune repertoire. These technologies can sequence millions of B cells to obtain a profile of the B cell repertoire 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. Summary of the Invention

[0006] The present application describes a method for effectively discovering antibodies, which is based on the MBC recall response captured by high-throughput sequencing technology, skips the experimental selection / enrichment steps, and identifies antibodies from the immune repertoire.

[0007] On the one hand, a method for generating antibodies specific to a target is provided, the method comprising:

[0008] (a) immunizing an animal with a target antigen one or more times,

[0009] (b) reducing the serum antibody titer of the animal by 2-fold or more compared to after the previous immunization,

[0010] (c) immunizing the animal with a recall antigen, the antibody-binding region of which has at least 30% identity with the target antigen,

[0011] (d) generating antibody sequences from the B cells of the animal 1-20 days after the immunization in step (c),

[0012] (e) selecting antibodies from the antibody sequences by one or more prioritization factors,

[0013] Optionally, f) repeating steps (b) to (e).

[0014] In one or more embodiments, the animal is selected from murine, rabbit, feline, canine, simian, camelid animals.

[0015] In one or more embodiments, the animal is a camelid animal, including but not limited to alpaca (Vicugna pacos), Bactrian camel (Camelus bactrianus), guanaco (Lama guanicoe).

[0016] In one or more embodiments, step (b) comprises: interrupting the immunization of the animal until the serum antibody titer is reduced by 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold or more. A 2-fold reduction in serum antibody titer means that the serum antibody titer is 1 / 2 of the serum antibody titer after the previous immunization. The serum antibody titer after the previous immunization can be measured several days after the previous immunization, for example, 1-20 days, preferably 1-14 days, more preferably 4-11 days, and further preferably 4-7 days.

[0017] In one or more embodiments, step (b) includes: subjecting the animal to an immune interruption for 21 days or longer, such as 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days (6 weeks), 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks or longer.

[0018] In one or more embodiments, in step (d): generating antibody sequences from B cells of the animal at 1 - 14 days (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days), preferably 4 - 11 days, and more preferably 4 - 7 days after immunization in step (c).

[0019] In one or more embodiments, the method further includes collecting B cells of the animal after immunization in step (c) at 1 - 14 days (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days), preferably 4 - 11 days, and more preferably 4 - 7 days after immunization in step (c).

[0020] In one or more embodiments, the animal is a llama, and in step (d): generating antibody sequences from B cells of the animal at 1 - 14 days (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days), preferably 4 - 11 days, and more preferably 4 - 7 days after immunization in step (c).

[0021] In one or more embodiments, the animal is a Bactrian camel, and in step (d): generating antibody sequences from B cells of the animal at 1 - 14 days (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days), preferably 7 - 14 days after immunization in step (c).

[0022] In one or more embodiments, the animal is a guanaco, and in step (d): generating antibody sequences from B cells of the animal at 1 - 14 days (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days), preferably 4 - 14 days after immunization in step (c).

[0023] In one or more embodiments, step (e) includes: grouping the antibody sequences according to antibody characteristics, where the antibody characteristics are selected from one or more of the following: one or more or all CDR sequences, VH sequences, VL sequences, VHH sequences, antibody sequences, lineages, and clusters, and selecting groups through one or more priority factors.

[0024] In one or more embodiments, the antibody sequence described in step (d) is a VHH sequence, a VH sequence, a VL sequence, a VH and VL sequence, or a full-length antibody sequence.

[0025] In one or more embodiments, the priority factors are selected from: the abundance or frequency of the antibody sequence or group from high to low, the growth rate of the abundance or frequency of the antibody sequence or group from high to low, the change in the abundance or frequency of the antibody sequence or group during the immune process, antibody affinity maturation, sharing the same naive B cell origin among VHHs, avoiding sequences with poor developability, and combinations thereof.

[0026] In one or more embodiments, the growth rate is the growth rate of the abundance or frequency of the antibody sequence or group of the B cells of the animal 1 - 20 days after immunization in step (c) compared to the B cells of the animal after step (b).

[0027] In one or more embodiments, the priority factors are selected from any one or any combination of any two or three of the following: (A) the top 10 - 20, 21 - 30, 31 - 40, 41 - 50, 51 - 60, 61 - 70, 71 - 80, 81 - 90, 91 - 100, 101 - 200, 201 - 300, 301 - 400, 401 - 500, 501 - 600, 601 - 700, 701 - 800, 801 - 900, 901 - 1000, 1001 - 1100, 1101 - 1200, 1201 - 1300, 1301 - 1400, 1401 - 1500, 1501 - 1600, 1601 - 1700, 1701 - 1800, 1801 - 1900 or 1901 - 2000 or lower in terms of the abundance or frequency of the antibody sequence or group, (B) the growth rate of the abundance or frequency of the antibody sequence or group reaches 5 - fold (e.g., 5.5 - fold, 6 - fold, 6.5 - fold, 7 - fold, 7.5 - fold, 8 - fold, 8.5 - fold, 9 - fold, 9.5 - fold, 10 - fold) or higher, and (C) the mutation value of the antibody sequence or group has an increasing trend compared to before, preferably, the mutation value of the antibody sequence or group satisfies the following relationship: after immunization in step (c) for 1 - 20 days > after step (b) > after step (a).

[0028] In one or more embodiments, the mutation value of the antibody sequence or group is measured by a mismatch score or a bit score.

[0029] In one or more embodiments, the priority factors are selected from: the abundance or frequency of the antibody sequence or group is in the top 500 (such as top 400, top 300, top 200, top 100, top 90, top 80, top 70, top 60, top 50, top 40, top 30, top 20, top 10) or higher, and / or, the growth rate of the abundance or frequency of the antibody sequence or group reaches 5-fold (such as 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold) or higher.

[0030] In one or more embodiments, the method further includes: determining the growth rate of the abundance or frequency of the antibody characteristics of the B cells of the animal 1 - 20 days after immunization in step (c) compared to the B cells of the animal after step (b).

[0031] In one or more embodiments, if the growth rate of 1, 2, 3, or 4 selected from antibody sequences, CDR groups, lineages, and clusters is less than 2, the antibodies selected in (e) are excluded.

[0032] In one or more embodiments, if the antibodies selected in (e) have at least one factor of poor developability, the antibodies are excluded, where the at least one developability is immunogenicity, expression, homogeneity, solubility, stability, viscosity, or formulation.

[0033] In one or more embodiments, the antibody characteristics are 1, 2, or 3 of the heavy chain CDRs or the CDRs of the heavy chain antibody. In one or more embodiments, the antibody characteristics are 1, 2, or 3 of the light chain CDRs.

[0034] In one or more embodiments, the antibodies selected in (e) are within the antibody sequences or groups ranked in the top 100, top 200, top 300, or top 500 in terms of abundance or frequency. In one or more embodiments, the antibodies selected in (e) are within the CDR groups ranked in the top 100, top 200, top 300, or top 500 in terms of abundance or frequency. In one or more embodiments, the antibodies selected in (e) are within the top 100, top 200, top 300, or top 500 lineages ranked in terms of abundance or frequency. In one or more embodiments, the antibodies selected in (d) are within the top 100, top 200, top 300, or top 500 clusters ranked in terms of abundance or frequency.

[0035] In one or more embodiments, the antibody is a heavy chain antibody, and the antibody characteristics are 1, 2, or 3 of CDR1, CDR2, and CDR3 of the heavy chain antibody.

[0036] In one or more embodiments, the priority factors are: antibody sequences with abundances or frequencies ranked in the top 10 - 20, 21 - 30, 31 - 40, 41 - 50, 51 - 60, 61 - 70, 71 - 80, 81 - 90, 91 - 100, 101 - 200, 201 - 300, 301 - 400, 401 - 500, 501 - 600, 601 - 700, 701 - 800, 801 - 900, 901 - 1000, 1001 - 1100, 1101 - 1200, 1201 - 1300, 1301 - 1400, 1401 - 1500, 1501 - 1600, 1601 - 1700, 1701 - 1800, 1801 - 1900, or 1901 - 2000 or lower, and / or, antibody sequences with growth rates of abundances or frequencies reaching 5 - fold (e.g., 5.5 - fold, 6 - fold, 6.5 - fold, 7 - fold, 7.5 - fold, 8 - fold, 8.5 - fold, 9 - fold, 9.5 - fold, 10 - fold) or more.

[0037] In one or more embodiments, the priority factors are: antibody sequences with abundances or frequencies in the top 500 (e.g., top 400, top 300, top 200, top 100, top 90, top 80, top 70, top 60, top 50, top 40, top 30, top 20, top 10) or higher, and / or, antibody sequences with growth rates of abundances or frequencies reaching 5 - fold (e.g., 5.5 - fold, 6 - fold, 6.5 - fold, 7 - fold, 7.5 - fold, 8 - fold, 8.5 - fold, 9 - fold, 9.5 - fold, 10 - fold) or more.

[0038] In one or more embodiments, the priority factors are: groups (CDR groups) with abundances or frequencies ranked in the top 10 - 20, 21 - 30, 31 - 40, 41 - 50, 51 - 60, 61 - 70, 71 - 80, 81 - 90, 91 - 100, 101 - 200, 201 - 300, 301 - 400, 401 - 500, 501 - 600, 601 - 700, 701 - 800, 801 - 900, 901 - 1000, 1001 - 1100, 1101 - 1200, 1201 - 1300, 1301 - 1400, 1401 - 1500, 1501 - 1600, 1601 - 1700, 1701 - 1800, 1801 - 1900, or 1901 - 2000 or lower when grouped by CDR sequences (i.e., antibody sequences in the group have the same CDR), and / or, groups (CDR groups) with growth rates of abundances or frequencies reaching 5 - fold (e.g., 5.5 - fold, 6 - fold, 6.5 - fold, 7 - fold, 7.5 - fold, 8 - fold, 8.5 - fold, 9 - fold, 9.5 - fold, 10 - fold) or more.

[0039] In one or more embodiments, the priority factors are: when grouped by CDR sequences, groups (CDR groups) with an abundance or frequency in the top 500 (e.g., top 400, top 300, top 200, top 100, top 90, top 80, top 70, top 60, top 50, top 40, top 30, top 20, top 10) or higher, and / or, groups (CDR groups) with a growth rate of abundance or frequency reaching 5-fold (e.g., 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold) or more.

[0040] In one or more embodiments, the antibody is a heavy chain antibody, the antibody is characterized by VHH, and the priority factors are: groups with a sequence abundance or frequency ranked in the top 10 - 20, 21 - 30, 31 - 40, 41 - 50, 51 - 60, 61 - 70, 71 - 80, 81 - 90, 91 - 100, 101 - 200, 201 - 300, 301 - 400, 401 - 500, 501 - 600, 601 - 700, 701 - 800, 801 - 900, 901 - 1000, 1001 - 1100, 1101 - 1200, 1201 - 1300, 1301 - 1400, 1401 - 1500, 1501 - 1600, 1601 - 1700, 1701 - 1800, 1801 - 1900 or 1901 - 2000 or lower, and / or, groups with a growth rate of sequence abundance or frequency reaching 5-fold (e.g., 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold) or more.

[0041] In one or more embodiments, the antibody is a single domain antibody, the antibody is characterized by a lineage, and the antibodies in a lineage map to the same V and J germline genes and have a maximum distance of a specific CDR3 equal to or less than 1 between the two closest CDR3s in the lineage, where all CDR3s have the same length.

[0042] In one or more embodiments, the priority factors are: groups (lineages) ranked in the top 10 - 20, 21 - 30, 31 - 40, 41 - 50, 51 - 60, 61 - 70, 71 - 80, 81 - 90, 91 - 100, 101 - 200, 201 - 300, 301 - 400, 401 - 500, 501 - 600, 601 - 700, 701 - 800, 801 - 900, 901 - 1000, 1001 - 1100, 1101 - 1200, 1201 - 1300, 1301 - 1400, 1401 - 1500, 1501 - 1600, 1601 - 1700, 1701 - 1800, 1801 - 1900 or 1901 - 2000 or lower in terms of abundance or frequency, and / or, groups (lineages) with a growth rate of the abundance or frequency reaching 5 - fold (e.g., 5.5 - fold, 6 - fold, 6.5 - fold, 7 - fold, 7.5 - fold, 8 - fold, 8.5 - fold, 9 - fold, 9.5 - fold, 10 - fold) or more.

[0043] In one or more embodiments, the priority factors are: groups (lineages) in the top 500 (e.g., top 400, top 300, top 200, top 100, top 90, top 80, top 70, top 60, top 50, top 40, top 30, top 20, top 10) or higher in terms of abundance or frequency, and / or, groups (lineages) with a growth rate of the abundance or frequency reaching 5 - fold (e.g., 5.5 - fold, 6 - fold, 6.5 - fold, 7 - fold, 7.5 - fold, 8 - fold, 8.5 - fold, 9 - fold, 9.5 - fold, 10 - fold) or more.

[0044] In one or more embodiments, the antibody is a single - domain antibody, the antibody is characterized by a cluster, and the antibodies in the cluster have the same CDR3 length and have a CDR3 identity of greater than or equal to 80% between the two closest CDR3s.

[0045] In one or more embodiments, the priority factors are: groups (clusters) ranked in the top 10 - 20, 21 - 30, 31 - 40, 41 - 50, 51 - 60, 61 - 70, 71 - 80, 81 - 90, 91 - 100, 101 - 200, 201 - 300, 301 - 400, 401 - 500, 501 - 600, 601 - 700, 701 - 800, 801 - 900, 901 - 1000, 1001 - 1100, 1101 - 1200, 1201 - 1300, 1301 - 1400, 1401 - 1500, 1501 - 1600, 1601 - 1700, 1701 - 1800, 1801 - 1900 or 1901 - 2000 or lower in terms of abundance or frequency, and / or, groups (clusters) with a growth rate of the abundance or frequency reaching 5 - fold (e.g., 5.5 - fold, 6 - fold, 6.5 - fold, 7 - fold, 7.5 - fold, 8 - fold, 8.5 - fold, 9 - fold, 9.5 - fold, 10 - fold) or more.

[0046] In one or more embodiments, the priority factors are: groups (clusters) with an abundance or frequency in the top 500 (e.g., top 400, top 300, top 200, top 100, top 90, top 80, top 70, top 60, top 50, top 40, top 30, top 20, top 10) or higher, and / or, groups (clusters) with a growth rate of abundance or frequency reaching 5-fold (e.g., 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold) or more.

[0047] In one or more embodiments, the antibody is an immunoglobulin, and the sequences of VH and / or VL of one lineage are the same.

[0048] In one or more embodiments, the antibody is an immunoglobulin, and the antibodies in one lineage map to the same V and J germline genes, and have a maximum distance of a specific HCDR3 equal to or less than 1 between the two closest HCDR3s in the lineage, where all HCDR3s have the same length.

[0049] In one or more embodiments, the antibody is an immunoglobulin, and the antibodies in the cluster have the same HCDR3 length and have a CDR3 identity of greater than or equal to 80% between the two closest CDR3s in the cluster.

[0050] In one or more embodiments, the antibody is an immunoglobulin, and the antibodies in one CDR group contain the same LCDR1, LCDR2, and LCDR3 sequences, and / or the same HCDR1, HCDR2, and HCDR3 sequences.

[0051] In one or more embodiments, after the groups are selected, antibodies can be further selected from the selected groups according to the priority factors. The priority factors are, for example, the ranking of the abundance or frequency of the antibody sequences in the group and / or avoiding sequences with poor developability.

[0052] In one or more embodiments, the method further includes differentiating lineages by VHH characteristics, and the VHH characteristics are selected from one or more of the following:

[0053] i) FR2 hydrophilic region,

[0054] ii) Extended CDR1,

[0055] iii) Additional disulfide bonds between CDR1-CDR3 or FR2-CDR3,

[0056] iv) Additional disulfide bonds within CDR3,

[0057] v) Long CDR3 (≥15aa),

[0058] vi) An additional disulfide bond within CDR1,

[0059] vii) A non-classical VHH having the same V and J germlines as conventional IgG1,

[0060] viii) A non-classical VHH having a predetermined sequence signature,

[0061] ix) A new classical binding loop structure, and

[0062] x) A convergent motif or sequence signature.

[0063] In one or more embodiments, the sequences are generated by sequencing techniques. Preferably, the sequences are generated by NGS or single-cell sequencing techniques.

[0064] In one or more embodiments, the target is a peptide, protein, hapten (e.g., conjugated to a carrier molecule), mRNA, DNA, a viral vector permitting expression of a target antigen, or a cell.

[0065] In one or more embodiments, the target comprises one or more selected from the group consisting of mesothelin, PDL1, UPAR.

[0066] In one or more embodiments, the target antigen is a peptide, protein, hapten (e.g., conjugated to a carrier molecule), mRNA, DNA, a viral vector permitting expression of a target antigen, or a cell.

[0067] In one or more embodiments, the target antigen is the target or a fragment of the target comprising one or more of its functional domains. In one or more embodiments, the recall antigen is the target antigen or a fragment of the target antigen comprising one or more of its functional domains.

[0068] In one or more embodiments, the target antigen comprises one or more or all of the epitopes of the target. In one or more embodiments, the target antigen comprises or consists of a fragment of the target that is an epitope.

[0069] In one or more embodiments, the recall antigen comprises one or more or all of the epitopes of the target antigen. In one or more embodiments, the recall antigen comprises or consists of a fragment of the target antigen that is an epitope.

[0070] In one or more embodiments, the recall antigen is the same as the target antigen.

[0071] In one or more embodiments, the target antigen is a fragment of mesothelin comprising or consisting of its domains 2 and 3, and the recall antigen is a fragment of mesothelin comprising or consisting of its domains 2 and 3.

[0072] In one or more embodiments, the antibody is expressed by prokaryotic or eukaryotic cells.

[0073] In one or more embodiments, the antibody is a single-domain antibody or an immunoglobulin.

[0074] In one or more embodiments, step (e) further comprises testing the specific binding of the selected antibody to the target. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 is the MBC recall flowchart.

[0076] Figure 2 is the schematic flowchart of an embodiment of the present invention.

[0077] Figure 3 Shows Figure 2 an exemplary process of step g) in

[0078] Figure 4 shows the detailed immunization process (A) and simplified diagram (B) of immunizing alpaca A30 with mesothelin antigen.

[0079] Figure 5 Shows as Figure 3 shown the titers of each blood sample. After the interruption of immunization, the titers (PRE-IM11 and PRE-IM13) decreased. Subsequent immunizations rapidly increased the titers (IM11, IM13-D7, IM13-D11).

[0080] Figure 6 Shows that the top-ranked lineages with a frequency >= 0.1% in the IM11 sample (MBC recall sample) showed rapid growth compared to the samples 7 days before IM11 (PRE-IM11) and 14 days after IM12 (A). Growth rate of the top 30 lineages (B).

[0081] Figure 7 Shows the lineage frequency dynamics of the top-ranked lineages based on a frequency >= 0.1% in the IM11 sample (A) and the IM13-D4 sample (B) among 7 samples. The MBC recall process can be initiated multiple times, and similar rapid growth is observed.

[0082] Figure 8 Shows the Morisita-Horn overlap index of the lineages among 7 samples based on the mapping results.

[0083] Figure 9 Shows the cluster frequency dynamics of the top-ranked clusters based on a frequency >= 0.1% in the IM11 sample (A) and the IM13-D4 sample (B) among 7 samples.

[0084] Figure 10 Shows the Morisita - Horn overlap index of clusters among 7 samples based on the mapping results.

[0085] Figure 11 Shows the CDR - group frequency dynamics of the top - ranked CDR groups in sample IM11 (A) and sample IM13 - D4 (B) among 7 samples based on a frequency ≥ 0.1%.

[0086] Figure 12 Shows the Morisita - Horn overlap index of CDR groups among 7 samples based on the mapping results.

[0087] Figure 13 Shows the sequence - frequency dynamics of the top - ranked sequences in sample IM11 (A) and sample IM13 - D4 (B) among 7 samples based on a frequency ≥ 0.1%.

[0088] Figure 14 Shows the Morisita - Horn overlap index of sequences among 7 samples based on the mapping results.

[0089] Figure 15 Shows the ELISA binding results of sequences selected from IM11 and IM12. Detailed implementation mode

[0090] Through the following detailed description, other objects, features and advantages of the present invention will be obvious. It should be understood that within the scope of the present invention, the above - mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form preferred technical solutions.

[0091] Unless otherwise specified, the techniques used in the present invention are standard methods well - known to those skilled in the art. These techniques are described and explained throughout the literature, such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et, al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors).

[0092] The present invention utilizes the MBC recall process, aiming to maximize the ratio of antigen-specific MBCs and PCs through extrafollicular activation and skip the traditional antigen-specific B cell enrichment step, and capture these antibodies using high-throughput sequencing technology.

[0093] Generally, the memory B cell repertoire is dominated by large IgM, IgA, and IgG2 clone families, while the IgG1 family, including those specific to the recalled antigen, is smaller in size (Phad, G.E. et al., 2022). The present invention designs specific B cell recall strategies and protocols to reduce the "interference" of non-related immune B cell sequences and significantly increase the frequency of antigen-specific B cell sequences.

[0094] Definitions

[0095] Unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" as used in this specification and the appended claims include plural referents. Similarly, the terms a (or an), one (or more) and at least one can be used interchangeably herein.

[0096] When these terms appear in the specification and claims, the terms "comprising" and its variants do not have a limiting meaning. Thus, the terms "comprising" and "including" are used interchangeably.

[0097] "Optional" or "optionally" means that the subsequent described event, situation, or component may or may not occur, and the description includes instances where the event, circumstance, or component occurs and instances where it does not occur.

[0098] In this document, ranges may be expressed as from "about" one specific value and / or to "about" another specific value. When expressing such a range, examples include starting from a specific value and / or ending at another specific value. Similarly, when a numerical value is approximated by use of the antecedent "about", it should be understood that the specific value constitutes another aspect. It should also be understood that each endpoint of each range is significant both in relation to the other endpoint and independently of the other endpoint.

[0099] Unless otherwise clearly stated, any method described herein should not be construed as requiring that its steps be performed in a particular order. Thus, when a method claim does not actually state that its steps follow a certain order or when it is not otherwise specifically indicated in the claims or the specification that the steps are limited to a specific order, no particular order is intended to be implied. Any single or multiple features or aspects recited in any one claim may be combined with any other feature or aspect recited in any one or more other claims or may be permuted with any other feature or aspect recited in any one or more other claims.

[0100] The term "titer" refers to the maximum dilution of the original serum with a detectable signal (ELISA OD450 value of 0.3).

[0101] As used herein, animals include mammals such as murine, rabbit, feline, canine, simian, camelid, etc. that can produce an immune response. Camelids such as alpaca (Vicugna pacos), Bactrian camel (Camelus bactrianus), llama (Lama guanicoe).

[0102] As used herein, "frequency" refers to the proportion of the count (abundance) of an event (such as an antibody sequence) in the library sequence.

[0103] In this Chinese text, the term "expansion ratio" refers to the ratio of the frequency in one situation divided by the frequency in another situation.

[0104] As used herein, "non-related immune B cell sequences" or "non-related immune B cell sequence interference" is triggered by many body self-defense stimuli, non-related pathogens, and long-lived PCs. In order to reduce these "interference" B cell responses to an "almost" background level, sufficient interruption is necessary. As used herein, "immune interruption" refers to the time period after target antigen immunization and before recall antigen immunization.

[0105] As used herein, the terms "homology" and "identity" are used interchangeably. To determine the percentage identity or homology of two sequences, the sequences can be aligned for optimal comparison. Then the nucleotides or amino acids are compared at the corresponding nucleotide or amino acid positions of the two sequences. For example, when the same nucleotide or amino acid is at the corresponding position in the second sequence, the nucleotide or amino acid in the first sequence is considered identical to the second sequence. The percentage identity is calculated by determining the number of identical positions divided by the total number of positions (i.e., the overlapping positions) multiplied by 100. As defined herein, the term "homology" means at least 30%, in another aspect at least 40%, in another aspect at least 50%, in another aspect at least 60%, in another aspect at least 70%, in another aspect at least 75%, in another aspect at least 80%, in another aspect at least 85% identity, in another aspect at least 90% identity, in another aspect at least 95% identity, in another aspect at least 99% identity, and in another aspect complete identity.

[0106] As used herein, the term "antibody feature" refers to any feature associated with an antibody that groups antibody sequences by certain similar or identical characteristics. These features can be one or more CDR sequences, VH and / or VL sequences, full antibody sequences, lineages, and / or clusters. For example, if the CDR1, CDR2, and CDR3 sequences of an antibody sequence are the same, the antibody sequences can be grouped by CDR sequence (i.e., into CDR groups); if the sequences map to the same V / J germline genes and have the same length of CDR3, the antibody sequences can be grouped by lineage, where when the CDR3 length is greater than 4, the maximum amino acid sequence difference does not exceed 1, and when the CDR3 length is equal to or less than 4, the amino acid sequence difference is 0; if the sequences have the same length of CDR3 and have 80% or more identity in the CDR3 sequence, the antibody sequences can be grouped by cluster.

[0107] According to the description of the present application, an immunized animal can be boosted with a "recall antigen". The recall antigen can be the same as the original antigen or a part of the original antigen. In principle, any antigen containing a similar epitope in the original immunizing antigen can be used as a recall antigen.

[0108] As used herein, the term "epitope" can include any protein determinant capable of specifically binding to an immunoglobulin or T cell receptor. Epitope determinants usually consist 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 an antigen when the equilibrium dissociation constant ≤ 1 μM, preferably ≤ 100 nM, more preferably ≤ 10 nM.

[0109] As used herein, an epitope can include a functional domain of a protein. The term "functional domain" can include a domain or a combination of domains with similar or combined functions. For example, the functional domains of mesothelin include domain 1, domain 2, and / or domain 3, the functional domain of PDL1 includes a domain with a distal Ig variable (V) region and a proximal Ig constant (C) region, and the functional domain of UPAR includes an extracellular region.

[0110] The term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction.

[0111] As used herein, the term "immune response" refers to the selective impairment, destruction, or elimination of an organism against an invading pathogen, an infected pathogen's 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 lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver. As used herein, "immunizing" an animal with an antigen refers to the process of exposing the animal to the antigen to an extent that elicits an immune response. The immunization can be carried out using immunization methods known in the art.

[0112] As used herein, "antigen-specific T cell response" can refer to a T cell response induced by antigen stimulation specific for T cells. Non-limiting examples of the response of T cells to antigen-specific stimulation include proliferation and cytokine production (e.g., IL-2 production).

[0113] As used herein, the term "antibody" refers to (a) a complete immunoglobulin, (b) a monoclonal or polyclonal antigen-binding fragment with or without 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 its antigen-binding fragment, or (d) IgNAR antibodies found in sharks and other cartilaginous fish. The antigen-binding fragment can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody. 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 technology or by enzymatic (e.g., papain cleavage) or chemical cleavage of a complete antibody.

[0114] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of an antibody heavy chain or light chain. The variable domains of the heavy and light chains can be referred to as "V H " and "V L ", respectively. These domains are generally the most variable (relative to other antibodies of the same class) parts of an antibody and contain the antigen-binding site.

[0115] As used herein, the "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 light chain (VL) connected 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 chain and heavy chain can be in any order, e.g., VH-linker-VL or VL-linker-VH, as long as the specificity of the scFv for the antigen is retained.

[0116] The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers 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.

[0117] The "heavy chain antibody" described herein is an antibody derived from camelids or sharks. Compared with the above-mentioned 4-chain antibody, the heavy chain antibody lacks the light chain and the heavy chain constant region 1 (CH1), and contains only two heavy chains consisting of a variable region (VHH) and other constant regions, where the variable region is connected to the constant region by a hinge region-like structure. Each heavy chain of a camel heavy chain antibody contains a variable region (VHH) and two constant regions (CH2 and CH3), and each heavy chain of a shark heavy chain antibody contains a variable region and five constant regions (CH1-CH5). The antigen-binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies. Heavy chain antibodies can have the CH2 and CH3 of human IgG-Fc by fusing with the constant region of human IgG-Fc.

[0118] As used herein, the terms "single-domain antibody", "variable domain of the heavy chain of a heavy chain antibody", "VHH", and "nanobody" are used interchangeably and all refer to a single-domain antibody that specifically recognizes and binds an antigen. A single-domain antibody is the variable region of a heavy chain antibody. Generally, a single-domain antibody contains three CDRs and four FRs. A single-domain antibody is the smallest functional antigen-binding fragment.

[0119] The term "isotype" can refer to the antibody class encoded by the heavy chain constant region gene (such as IgM or IgG1). Antibodies can be immunoglobulin G (IgG), IgM, IgE, IgA, or IgD molecules, or derivatives thereof.

[0120] The term "VHH 2 ", "VHH 3 ", and "VH 1 " represent the heavy chains of camelid IgG isotypes IgG2, IgG3, and IgG1, respectively. VL 1 represents the light chain of camel IgG1. Camel VL 1 includes but is not limited to Vκ and Vλ.

[0121] The term "evaluate" includes any form of measurement, and includes the presence of specific elements. 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 quantity of something present, and / or determining whether it is present.

[0122] The term "peripheral blood mononuclear cell" or "PBMC" refers to blood cells having 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 density gradient.

[0123] The term "hypergrowth" means that the frequency of an event (such as a sequence or a group consisting of sequences) is higher than 1%. Hypergrowth can be used to describe sequences and groups that are highly amplified in an immune repertoire.

[0124] "Rank", "antibody sequence rank", or "group rank" refers to the order in which antibody sequences or groups are arranged according to priority factors. Priority factors include, but are not limited to, the abundance, frequency, growth rate of antibody sequences or groups, the dynamic changes in the abundance and frequency of antibody sequences or groups during an immune process, antibody affinity maturation, antibody sequences having the same naive B cell origin between VHH and VH, avoiding poorly developable sequences, and combinations thereof.

[0125] The term "Hamming distance" refers to the number of positions at which corresponding symbols are different between two sequences of equal length.

[0126] As used herein, the terms "antibodies grouped by antibody characteristics", "antibodies related to antibody characteristics", and "antibodies associated with antibody characteristics" and their grammatical equivalents are antibodies produced by cells having a common B cell ancestor. Antibodies related to antibody characteristics bind to the same epitope of an antigen, and they are usually very similar in sequence, particularly in the light and heavy chain CDR3s. The heavy and light chain CDR3s of antibodies related to antibody characteristics 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). When an antibody characteristic includes CDR3, 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 having a rearranged light chain VIC region and a rearranged heavy chain VDJ region and produces antibodies that have not yet undergone affinity maturation.

[0127] As used herein, the term "at least CDR3" or "at least CDR3 sequence" refers only to a CDR3 sequence, a sequence comprising a CDR3 sequence together with CDR1 and / or CDR2 sequences, or a sequence of at least 50 contiguous amino acids of a variable domain up to the entire length of the variable domain that includes CDR3.

[0128] In the present application, the term "CDR" also referred to as "complementary determining region" generally refers to a region in the variable domain of an antibody, the sequence of which is highly variable and / or forms structurally defined loops. Typically, an antibody includes six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3). Heavy chain antibodies consist only of heavy chains (referred to as VHH). Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, AbM, Chothia, IMGT, considering Kabat / Chothia together, etc. These coding systems are known in the art and can be found, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum.

[0129] As used herein, the term "lineage" refers to the theoretical descent. A "lineage" is a "group", and sometimes a group of antibodies 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.

[0130] As used herein, the term "sub-subgroup" refers to a further grouping of sequences within a lineage based on unique features or characteristics. A "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 VHH. Applying VHH sequence features can help in better selection / narrowing down of test lineages (representative sequences), which may result in better biological function / biological activity outcomes.

[0131] 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 lineage tree.

[0132] 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 technique.

[0133] 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 a nucleic acid sequence can be translated into an amino acid sequence on a computer.

[0134] As used herein, the term "most abundantly expressed" refers to the most abundant protein sequence in a sample. The abundance of a protein can be determined by counting the sequence reads encoding that protein. The protein encoded by the most sequence reads is the most abundant protein.

[0135] The phrases "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".

[0136] The term "specifically binds" refers to the ability of an antibody to preferentially bind to a particular 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. Specific binding can be detected using any technique known in the art.

[0137] As used herein, the term "substantially does not bind" to a protein or cell can mean that it does not bind or does not bind with high affinity to the protein or cell, i.e., binds with a KD of 2x10 -6 M or greater, more preferably 1x10 -5 M or greater, more preferably 1x10 -4 M or greater, more preferably 1x10 -3 M or greater, even more preferably 1x10 -2 M or greater to the protein or cell.

[0138] "High affinity" for an IgG antibody can refer to an antibody having a KD of 1x10 -6 M or less, preferably 1x10 -7 M or less, more preferably 1x10 -8 M or less, even more preferably 1x10 -9 M or less, even more preferably 1x10 -10 M or less for the antigen. However, "high affinity" binding may vary for other antibody isotypes.

[0139] A "CDR-grafted antibody" is an antibody that contains one or more CDRs derived from a particular species or isotype and a framework of another antibody of the same or a different species or isotype.

[0140] Differ from antibody sequences derived from 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 make humanized antibodies can be found in U.S.Pat.Nos.6054297, 5886152, and 5877293.

[0141] 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 also possible.

[0142] The term "bispecific antibody" refers to an antibody that binds to two non-overlapping epitopes of an antigen. In some embodiments, the bispecific antibody includes a VHH that contains only a heavy chain and no light chain. In some embodiments, the bispecific antibody contains a VHH of only a heavy chain and a conventional VH 1 / VL 1 Yes. In some embodiments, the bispecific antibody contains two conventional VHs 1 / VL 1 Yes. In some embodiments, the bispecific antibody has a first heavy chain and a first light chain from a monoclonal antibody targeting one epitope, and heavy and light chains from another antibody targeting another epitope. In some examples, the other light or heavy chain can be different from the first light or heavy chain.

[0143] The binding of the antibodies of the present invention to antigens can be evaluated using one or more techniques commonly used in the art. For example, 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 cell lines expressing human antigens (such as HEK293 cells). Alternatively or in addition, the binding of the antibodies, including binding kinetics (such as K D values) etc. can be tested in BIAcore binding assays, Octet Red96 (Pall), etc.

[0144] 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.

[0145] 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. Before calculation, a profile of the percentage of use of each residue in each length of the 4 framework regions is determined based on all human IGHV germlines. For each VHH sequence, the residues at each position in the 4 framework regions are 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 the residue divided by the highest percentage of use at the same position. The rarity score of a sequence is the average of the rarity scores of all framework region residues.

[0146] 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.

[0147] The term "bit score" is an output value of the sequence local alignment search tool BLAST, which is used to describe the overall quality of the alignment. The larger the numerical value of the bit score, the higher the sequence similarity.

[0148] The term "pharmaceutical preparation" refers to a preparation in a form that allows the active ingredient contained therein to be biologically effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is administered.

[0149] A "therapeutically effective amount" of an agent (such as a pharmaceutical preparation or a cell) refers to an amount that effectively achieves the desired therapeutic outcome (such as for treating a disease, disorder, or condition) and / or the pharmacokinetic or pharmacodynamic effects of the treatment within a dosage and a necessary time period. The therapeutically effective amount can vary depending on factors such as the disease state, age, gender, and weight of the subject, as well as the cell population administered. In some embodiments, the provided methods include administering cells and / or compositions in an effective amount (such as a therapeutically effective amount).

[0150] Method

[0151] The present application provides a method for effectively discovering antibodies, which can identify antibodies from an immune repertoire without experimental selection / enrichment steps. Specifically, a method for generating antibodies specific to a target is provided, which includes: (a) immunizing an animal with a target antigen for one or more rounds, (b) reducing the serum antibody titer of the animal by 2-fold or more, (c) immunizing the animal with a recall antigen (such as one round of immunization), the antibody-binding region of which has at least 30% identity with the target antigen, (d) generating antibody sequences from the B cells of the animal 1-20 days after immunization in step (c), and (e) selecting antibodies from the antibody sequences by one or more prioritization factors. In one embodiment, more antibodies can be selected by repeating steps (b) to (e).

[0152] The inventors have found that reducing the serum antibody titer of an animal by more than 2-fold compared to after the last immunization is crucial for the effectiveness of the antibodies after immunization with the recall antigen. Of course, reducing by a greater multiple has a better effect (such as reducing by 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold or more).

[0153] In one or more embodiments, the serum antibody titer of the animal is reduced by at least 2-fold compared to 1-20 days after the previous immunization. In one or more embodiments, the serum antibody titer of the animal is reduced by at least 2-fold compared to 1-14 days, 4-11 days, or 4-7 days after the previous immunization.

[0154] The method for reducing the serum antibody titer of an animal is not limited, and this effect can generally be obtained by interrupting immunization for a certain period of time. Step (b) can include: subjecting the animal to an immunization interruption of 21 days or longer, such as 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days (6 weeks) or longer.

[0155] In one or more embodiments, the method further includes collecting B cells of an animal within 1 - 20 days (preferably 1 - 14 days, more preferably 4 - 11 days, still more preferably 4 - 7 days) after step (c) immunization for subsequent antibody sequence grouping.

[0156] In one or more embodiments, the method includes steps b.1): collecting the first antigen - specific B cells of the animal, and b.2): generating an antibody NGS library containing VHH 2 、VHH 3 and VH 1 and VL 1 chain sequences after step b) and before step c). In one embodiment, b.2) may include 1) preparing cDNA from the antigen - specific B cells; 2) sequencing the cDNA to obtain a plurality of VHH 2 、VHH 3 、VH 1 heavy - chain sequences and a plurality of VL 1 (Vκ and Vλ) light - chain sequences to generate camel IgG2 (HcAb), IgG3 (HcAb) and IgG1 (conventional Ab) libraries. In one embodiment, the camel antibodies produced include IgG2. In one embodiment, the camel antibodies produced include IgG3.

[0157] In one or more embodiments, step d) includes: step d.1): collecting the second antigen - specific B cells of the animal 1 - 20 days after immunization, and step d.2): generating an antibody NGS library containing VHH 2 、VHH 3 and VH 1 and VL 1 chain sequences from the antigen - specific B cells. In one embodiment, d.2) may include 1) preparing cDNA from the antigen - specific B cells; 2) sequencing the cDNA to obtain a plurality of VHH 2 、VHH 3 、VH 1 heavy - chain sequences and a plurality of VL 1 (Vκ and Vλ) light - chain sequences to generate camel IgG2 (HcAb), IgG3 (HcAb) and IgG1 (conventional Ab) libraries. In one embodiment, the antigen contains multiple epitopes. In one embodiment, the camel antibodies produced include IgG2. In one embodiment, the camel antibodies produced include IgG3.

[0158] In one or more embodiments, step e) includes: e.1) grouping the antibody sequences according to antibody characteristics, and e.2) ranking the VHH heavy - chains (VHH 2 、VHH 3) Sort the groups and select the groups with higher rankings; e.3) Select representative sequences from the selected groups according to the priority factors within the groups. The antibody features are selected from one or more of the following: CDR sequences, VH sequences, VL sequences, VHH sequences, antibody sequences, lineages, and clusters. In one or more embodiments, step e.2) includes further selecting affinity-matured groups from the groups with higher rankings. Affinity maturation can be judged in the following way: the mutation value of the antibody sequence or group compared to the germline gene has an increasing trend compared to before. For example, the mutation value of the antibody sequence or group compared to the germline gene satisfies the following relationship: 1 - 20 days after immunization in step (c) > after step (b) > after step (a). The mutation value can be measured by the mismatch score or the bit score. The larger the mismatch score, the larger the mutation value. The smaller the bit score, the larger the mutation value.

[0159] The antibodies produced by the present invention can be humanized by replacing the amino acids at one or more replaceable positions of the original antibody with the amino acids at the corresponding positions in a human antibody. In one or more embodiments, the method further includes replacing the amino acids at one or more replaceable positions in the original VHH or VH or VL with the amino acids at the corresponding positions in a human antibody. In one embodiment, the replaceable positions are in the CDR region. In one embodiment, the replaceable positions are in the FR region.

[0160] In this text, the priority factors include the abundance or frequency of antibody sequences or groups from high to low, the growth rate of the abundance or frequency of antibody sequences or groups from high to low, the change in the abundance or frequency of antibody sequences or groups during the immune process, antibody affinity maturation, antibody sequences or groups sharing the same naïve B cell origin among VHHs, avoiding sequences with poor developability, and combinations thereof. In one or more embodiments, the priority factors are selected from: antibody sequences or groups ranked in the top 10 - 20, 21 - 30, 31 - 40, 41 - 50, 51 - 60, 61 - 70, 71 - 80, 81 - 90, 91 - 100, 101 - 200, 201 - 300, 301 - 400, 401 - 500, 501 - 600, 601 - 700, 701 - 800, 801 - 900, 901 - 1000, 1001 - 1100, 1101 - 1200, 1201 - 1300, 1301 - 1400, 1401 - 1500, 1501 - 1600, 1601 - 1700, 1701 - 1800, 1801 - 1900, or 1901 - 2000 or lower in terms of abundance or frequency, and / or, antibody sequences or groups with a growth rate of the abundance or frequency reaching 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold or higher. In some embodiments, the method further includes repeating the steps (b) to (e) above to identify antibodies, wherein the representative sequences are selected from groups ranked in the top 2,000 to 10,000 in terms of abundance or frequency.

[0161] In this text, the abundance or frequency of a group is the sum of the abundances or frequencies of all antibody sequences (such as VH, VL, VHH, or full-length antibody sequences) in a certain group. For example, the abundance or frequency of a lineage is the sum of the abundances or frequencies of all antibody sequences in a certain lineage group. The abundance or frequency of a cluster is the sum of the abundances or frequencies of all antibody sequences in a certain cluster group. The abundance or frequency of a CDR group is the sum of the abundances or frequencies of all antibody sequences in the group when antibody sequences are grouped according to CDR sequences (i.e., the antibody sequences in the group have the same CDR). The growth rate of the abundance or frequency of a group refers to the ratio of the abundance or frequency of a group in a certain situation to the abundance or frequency of the mapped group in another situation. The mutation value of a group refers to the average of the mutation values of all antibody sequences in the group.

[0162] In one or more embodiments, if the developability is poor, the antibody is excluded from further development. The developability can be judged by analyzing the sequence characteristics of the antibody sequence. Non-limiting examples of poor developability include unpaired cysteine, N-linked glycosylation, methionine oxidation, tryptophan oxidation, asparagine deamidation, aspartic acid isomerization, lysine glycosylation, N-terminal glutamate, integrin binding, CD11c, fragmentation, immunogenicity, expression, homogeneity, solubility, stability, viscosity, and / or formulatability.

[0163] In the present disclosure, B cells can be collected by any method known in the art, such as from blood, spleen, lymph nodes, or bone marrow. In a preferred embodiment, B cells are collected from blood.

[0164] In the present disclosure, sequencing can be performed by any method known now or discovered later. In some embodiments, sequencing is performed by next-generation sequencing (NGS). Any NGS method can be used in these examples. See, for example, Slatko et al. (2018) for an overview of NGS methods.

[0165] 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 has the same V / J germline gene as the lineage 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.

[0166] Similarly, a group from one NGS data set can be mapped to a group from another NGS data set. If the CDR sequence group of one NGS data set shares the same CDR1 / CDR2 / CDR3 sequence as the CDR sequence group of another NGS data set, they can be mapped to the CDR sequence group of the other NGS data set. If the lineage group of one NGS data set has the same V / J germline gene as the lineage group of another NGS data set and shares a common CDR3, the lineage group of the one NGS data set will be mapped to the lineage group of the other NGS data set. If the cluster group of one NGS data set shares a common CDR3 with the cluster group of another NGS data set, the cluster group of the one NGS data set will be mapped to the cluster group of the other NGS data set.

[0167] In some embodiments, the growth rate of each sequence is generated by comparing the frequencies of antibody characteristic sequences between the first and second antigen-specific B cells. The sequences can be grouped into CDR groups, for example, if their CDR1, CDR2, and CDR3 sequences are the same. In addition, the 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 a maximum of 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 cluster groups if they have CDR3s of the same length and 80% or more identity in the CDR3 sequence. The growth rate of the groups is also calculated. In some embodiments, the growth rate of a group refers to the frequency ratio between the group in the sequence library (NGS dataset) of the second antigen-specific B cells and the group mapped to it in the sequence library (NGS dataset) of the first antigen-specific B cells. In a specific embodiment, the "growth rate" of a sequence or group refers to the ratio of the frequency of the sequence or group with a specific antibody characteristic in the antibody sequences generated by B cells after immunization with the recall antigen to the frequency of the sequence or group with the same antibody characteristic in the antibody sequences generated by B cells before immunization with the recall antigen.

[0168] As used herein, a "target" can be a peptide, protein, hapten (e.g., conjugated to a carrier molecule), mRNA, DNA, viral vector allowing expression of the target antigen, or a cell. In some embodiments, the target comprises one or more of the following: mesothelin, PDL1, UPAR.

[0169] As used herein, a "target antigen" comprises one or more or all of the epitopes of the said target. A "target antigen" can be a peptide, protein, hapten (e.g., conjugated to a carrier molecule), mRNA, DNA, viral vector allowing expression of the target antigen, or a cell or a part thereof comprising an epitope.

[0170] In some embodiments herein, the target antigen comprises a part (e.g., a sequence fragment) of the target as an epitope or consists of the epitope. For example, the target antigen is the target or a part (e.g., a sequence fragment) of the target comprising one or more of its functional domains. In some other embodiments herein, the target comprises a part (e.g., a sequence fragment) of the target antigen as an epitope or consists of the epitope. In some embodiments, the target is the target antigen or a part (e.g., a sequence fragment) of the target antigen comprising one or more of its functional domains.

[0171] As used herein, a "recall antigen" comprises one or more or all of the epitopes of the said target antigen. A "recall antigen" can be a peptide, protein, hapten (e.g., conjugated to a carrier molecule), mRNA, DNA, viral vector allowing expression of the target antigen, or a cell or a part thereof comprising an epitope.

[0172] In some embodiments herein, the recall antigen comprises or consists of a part (e.g., a sequence fragment) of the target as an epitope. For example, the recall antigen is the target or a part (e.g., a sequence fragment) of the target that contains one or more of its functional domains.

[0173] In some embodiments herein, the recall antigen comprises or consists of a part (e.g., a sequence fragment) of the target as an epitope. For example, the recall antigen is the target or a part (e.g., a sequence fragment) of the target that contains one or more of its functional domains. In some other embodiments herein, the target comprises or consists of a part (e.g., a sequence fragment) of the recall antigen as an epitope. In some embodiments, the target is the recall antigen or a part (e.g., a sequence fragment) of the recall antigen that contains one or more of its functional domains.

[0174] In some embodiments herein, the recall antigen comprises or consists of a part (e.g., a sequence fragment) of the target antigen as an epitope. For example, the recall antigen is the target antigen or a part (e.g., a sequence fragment) of the target antigen that contains one or more of its functional domains. In some other embodiments herein, the target antigen comprises or consists of a part (e.g., a sequence fragment) of the recall antigen as an epitope. In some embodiments, the target antigen is the recall antigen or a part (e.g., a sequence fragment) of the recall antigen that contains one or more of its functional domains. In one or more embodiments, the recall antigen is the same as the target antigen.

[0175] In some embodiments, the target antigen is a fragment of mesothelin that contains domains 2 and 3 or consists thereof, and the recall antigen is a fragment of mesothelin that contains domains 2 and 3 or consists thereof.

[0176] In one or more embodiments, the target antigen is as shown in any one or more of SEQ ID NO:1-3, and the recall antigen is as shown in any one or more of SEQ ID NO:1-3. In one or more embodiments, the target antigen is as shown in SEQ ID NO:2, and the recall antigen is as shown in SEQ ID NO:2. SEQ ID NO:1 is the full-length human MSLN protein, SEQ ID NO:2 is domains 2 and 3 of the human MSLN protein, and SEQ ID NO:3 is domains 2 and 3 of the cynomolgus monkey MSLN protein.

[0177] In one or more embodiments, the target antigen is as shown in SEQ ID NO:4, the recall antigen is as shown in SEQ ID NO:4, and SEQ ID NO:4 is the full length of human PD-L1. In one or more embodiments, the target antigen is as shown in SEQ ID NO:5, the recall antigen is as shown in SEQ ID NO:5, and SEQ ID NO:5 is the full length of human uPAR.

[0178] In one or more embodiments, in the method for generating antibodies specific to a target described herein, step (a) includes immunizing an animal with the target antigen in multiple rounds, wherein the target antigen is a homologous sequence in different species or a fragment thereof containing one or more functional domains. For example, the target antigen can be mesothelin in different species or a fragment thereof containing functional domains (such as 1, 2, or 3 of domain 1, domain 2, and domain 3). Exemplarily, the target antigens are as shown in SEQ ID NO:2 and 3, and the recall antigen is as shown in SEQ ID NO:1 or 2. The different species are preferably different species within the same order, such as animals of the order Primates, including but not limited to humans and cynomolgus macaques. In one or more embodiments, step (a) includes immunizing 1 - 4 times with the antigen shown in SEQ ID NO:2, then immunizing 1 - 2 times with the antigen shown in SEQ ID NO:3, and then immunizing 1 - 4 times with the antigen shown in SEQ ID NO:2.

[0179] The method includes selecting more antibodies by repeating steps (b) to (e). When repeating steps (b) to (e), the recall antigen can be the same as or different from the previous one, as long as the recall antigen contains one or more or all of the epitopes of the target antigen. Exemplarily, the target antigens are as shown in SEQ ID NO:2 and 3, the first recall antigen is as shown in SEQ ID NO:2, and the second recall antigen is as shown in SEQ ID NO:1.

[0180] In the art, antigens for immunization may have modifications to enhance their immunogenicity, and such modifications are common knowledge in the art. For example, an immunoglobulin Fc (such as the Fc of IgG1 or IgG2b) or a His tag is added to the C-terminus of the antigen. The Fc may be derived from a desired species, such as human, mouse, rat, rabbit, dog, monkey, or camelid. The human IgG1 Fc sequence is shown in SEQ ID NO:50, and the IgG2b Fc sequence of Llama is shown in SEQ ID NO:51. In some embodiments, the target antigen may or may not contain modifications, and the recall antigen does not contain modifications. In one or more embodiments, the target antigen may or may not contain modifications, and the recall antigen contains modifications. In one or more embodiments, the target antigen contains or does not contain an immunoglobulin Fc at the C-terminus, and the recall antigen does not contain an immunoglobulin Fc. In one or more embodiments, the target antigen contains or does not contain an immunoglobulin Fc at the C-terminus, and the recall antigen contains an immunoglobulin Fc at the C-terminus. In one or more embodiments, the target antigen contains or does not contain a His tag at the C-terminus, and the recall antigen contains or does not contain a His tag at the C-terminus.

[0181] In one or more embodiments, the target antigen is SEQ ID NO:2 with human IgG1 Fc at the C-terminus, and the recall antigen is SEQ ID NO:2 without modification at the C-terminus. In one or more embodiments, the target antigen is SEQ ID NO:2 with human IgG1 Fc at the C-terminus, the first recall antigen is SEQ ID NO:2 without modification at the C-terminus, and the second recall antigen is SEQ ID NO:1 with a His tag at the C-terminus.

[0182] In one or more embodiments, the target antigen is SEQ ID NO:4 with Llama IgG2b Fc tag at the C-terminus, and the recall antigen is SEQ ID NO:4 without modification at the C-terminus. In one or more embodiments, the target antigen is SEQ ID NO:5 with a His tag at the C-terminus, and the recall antigen is SEQ ID NO:5 without modification at the C-terminus.

[0183] A schematic diagram of an embodiment of the present application is as Figure 2 shown. This embodiment includes: 1) performing one or more rounds of standard immunization using a protein, peptide, DNA, mRNA, or cellular antigen or immunogen (such as DNA, a small molecule conjugated to a carrier protein, a peptide conjugated to a carrier protein, a protein, or an antigen complex such as a cell or tissue), 2) monitoring the antibody titer of the serum, which can reach 256k or higher ( Figure 5) 3) Immunological interruption for more than 6 weeks. The repertoire is very dynamic, and the titers usually decline after a few weeks of immunological interruption. 4) Collect PBMC samples before boosting (i.e., immunizing with recall antigen) as the baseline for analyzing B cell antibody characteristics. 5) Boost the animals with the recall antigen. 6) Collect PBMC samples from the boosted animals between 3 and 11 days after boosting to maximize the opportunity to capture GC-independent and antigen-specific MBCs and PCs. 7) Prepare NGS libraries from these PBMC samples using standard procedures and generate antibody sequences using a sequencer or other sequencing technologies (methods known in the art). 8) Select sequences from the NGS library. 9) Obtain antibodies with the selected sequences and test to determine whether the selected antibodies have biological functions related to the antigen. In some embodiments, the biological function related to the antigen is specific binding or neutralization.

[0184] An exemplary process for step 8) of selecting sequences from the NGS library is as Figure 3 shown. First, process the sequences generated from these PBMC samples to identify the CDR regions and germline sequences of each sequence. Calculate the count (abundance), frequency, growth rate, mutation value, etc. of the sequences. For example, calculate the growth rate of each sequence by comparing it with the corresponding sequence in the baseline sample (step 4)) collected before boosting. The mutation value refers to the mutation value of the antibody sequence compared to the germline sequence. As described above, the mutation value can be measured by methods well known in the art such as mismatch score, bit score, etc.

[0185] The sequences are further analyzed and grouped. If their CDR1, CDR2, and CDR3 sequences are the same, the sequences are grouped into CDR sequences. If the sequences map to the same V / J germline gene and have CDR3s of the same length, where CDR3s with a length greater than 4 have a maximum of 1 aa difference and CDR3s with a length equal to or shorter than 4 have 0 differences, the sequences are further grouped into lineages, and if the sequences have CDR3s of the same length and the CDR3 sequences have 80% or more identity, they are grouped into clusters.

[0186] Then, select groups according to priority factors. In some embodiments, select groups with a high abundance or frequency ranking (e.g., top 30, 60, 90, 120, 150), and / or a growth rate of 5-fold or more.

[0187] In some embodiments, the sequence data is processed as follows: i. Group the nanobody sequences in the library by CDR sequence (CDR group), lineage, and cluster; and ii. Generate the growth rates of the sequences, CDR groups, lineages, and / or clusters by comparing the sequences in the NGS data after and before boosting.

[0188] In some embodiments, the top 100 counts and / or sequences or groups with a growth rate of 5-fold or more are selected for testing according to sequences, CDR groups, lineages, or clusters.

[0189] By analyzing sequence features, antibodies can be selected or excluded based on sequence features. For example, if an antibody sequence has sequence features associated with poor developability, it is excluded.

[0190] As Figure 2 shown, the recall antigen can be reused and PBMC samples can be collected to discover more antibodies.

[0191] The present invention also provides a method for generating a target-specific antibody, the method comprising the steps of: (i) obtaining an identified antibody according to the method for identifying an antibody in any embodiment of the present specification, (ii) testing the specific binding of the antibody obtained in (i) to the target. In one embodiment, (ii) may include 1) synthesizing DNA of the selected representative sequence, 2) constructing a vector containing the DNA sequence, 3) expressing the vector in cells, 4) testing the affinity and biological activity of the antibody. In some embodiments, step 4) includes determining the binding affinity, specificity, and / or neutralizing ability of the antibody against the antigen.

[0192] Hereinafter, the present invention is further described by way of the accompanying drawings and examples. The scope of the present invention is not limited to the specific embodiments described herein, and the examples are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly included within the scope of the present invention. Any embodiment of the present invention will be modified as necessary to be applicable to any other embodiment of the present invention, unless otherwise clearly stated.

[0193] It should be understood that these examples are only for illustrating the present invention and not for limiting the scope of the present invention. The experimental methods without specific conditions noted in the following examples are generally carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 2002), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0194] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred methods and materials described herein are for illustrative purposes only.

[0195] Examples

[0196] Example 1. Memory B cell recall discovers multiple binders against MSLN

[0197] I. Immunization

[0198] MSLN (Mesothelin) is a well-known oncology target suitable for CAR-T therapy. In the development of antibodies against this target, domains 2 and 3 of human MSLN were used to immunize alpacas. The specific immunization method has the following steps: 1. Take out the protein to be immunized. According to the immunization plan, when immunizing for the first time, aspirate a dose of 0.5 mg into a 5 mL EP tube and dilute it to a volume of 1 mL with DPBS. When boosting, the immunogen dose is halved, and at the same time, it is emulsified with Freund's incomplete adjuvant; 2. Use a handheld homogenizer emulsifier, adjust to gear 4, and slowly emulsify the protein adjuvant mixed solution until it is completely emulsified; 3. Slowly aspirate the emulsified antigen solution into a 1 mL syringe for standby; 4. Bind the alpaca. When immunizing, select about ten points on the alpaca's neck at the junction with the front legs, the inguinal area of the hind legs, and the back as immunization points. The distance between points is more than 5 cm. Use a wool razor to shave the small area of the immunization point to expose the epidermis; 5. Use iodophor / alcohol cotton balls to wipe and disinfect the immunization points; 6. Take out the antigen emulsion emulsified in step 4, and use the intradermal injection immunization method to inject and immunize the selected immunization points, with an immunization dose of 200 μL per point; 7. Observe whether the immunization site bleeds, and gently wipe and disinfect it again with iodophor / alcohol.

[0199] The detailed immunization schedule is as Figure 4 shown. After 10 rounds of immunization, there was an immunization interruption of 156 days and a recall antigen immunization (IM11 on July 14, 2022), and then there was another immunization interruption of 132 days and a recall antigen immunization (IM13 on December 7, 2022). The detailed titer data of some collected blood is as Figure 5 shown. The sequence of NBL501-AgD23 is shown in SEQ ID NO:2. The sequence of NBL501-cynoAgD23 is shown in SEQ ID NO:3.

[0200] The titer detection method has the following steps: 1. Antigen coating: Dilute the corresponding antigen with coating buffer to 1 μg / mL, add 100 μL per well, and coat overnight at 4°C; 2. Washing: Add 250 μL of PBST per well and wash 3 times; 3. Blocking: After washing, add 200 μL of 3% BSA blocking solution per well and incubate statically at 37°C for 2 hours; 4. Washing: Add 250 μL of PBST per well and wash 3 times; 5. Sample addition and incubation: Take out the alpaca serum to be tested, dilute it 11 gradients by two-fold ratio such as 1 / 1000, 1 / 2000... with 1% BSA, add them to the 96-well ELISA plate coated with antigen respectively, 100 μL per well, and incubate statically at room temperature for 1 hour; 6. Plate washing: Add 250 μL of PBST per well and wash 3 times; 7. Add enzyme-labeled antibody and incubate: Take out the secondary antibody Gaot pAb to Llama IgG H&L (HRP) (ab112786), after dilution, add 100 μL per well to the ELISA plate wells and incubate statically at room temperature for 0.5 h; 8. Color development: Add 250 μL of PBST per well and wash 3 times, add 100 μL of TMB per well and develop color for about 5 minutes; 9. Termination and plate reading: Add 50 μL of hydrochloric acid to each well to terminate the reaction, and read the plate at a double wavelength of 450 / 650.

[0201] As Figure 5 shown, after three rounds of immunization, the titer is the highest (1024K). The titer of the last immunization (IM10) before the interruption of immunization is 128K. On July 14, 2022, 156 days after the interruption of immunization, the titer before the booster immunization (Pre-IM11) is 64K, far lower than the peak titer and 1 / 2 of the last immunization (IM10). After the booster immunization, the titer increases to 128K after 7 days and reaches 512K after 14 days. To show whether recall requires interruption of immunization, a second immunization is carried out 14 days after the first booster. The titer (IM12 collected 7 days after the second immunization) does not increase further after the second immunization ( Figure 5 shown).

[0202] II. Sequencing and library construction

[0203] Three PBMC samples (Pre-IM11: before the first booster immunization; IM11: 7 days after the first booster immunization; IM12: 7 days after the second booster immunization, that is, 21 days after the first booster immunization) are sequenced using NGS technology to obtain three NGS sequence data (sequence libraries).

[0204] The method for collecting PBMC is as follows: 1) Take fresh anticoagulated whole blood, add 1× dilution washing solution at a ratio of 1:1, dilute the blood (to reduce blood viscosity), gently mix well, and set aside; 2) Add an appropriate amount of mononuclear cell separation solution into a sterile centrifuge tube, lay the diluted blood sample flat above the liquid surface of the separation solution (separation solution: diluted whole blood = 1:2), and keep the interface of the two liquid surfaces clear; 3) Centrifuge at 800g at room temperature for 20 min - 30 min (note: set a slower acceleration and deceleration); 4) After centrifugation, aspirate and discard the plasma layer, carefully aspirate the PBMC layer (i.e., the buffy coat) and transfer it to a 15 mL centrifuge tube. After centrifugation, the liquid level in the tube from top to bottom is the diluted plasma layer, PBMC layer, separation solution layer, and red blood cell layer in sequence; 5) Add 10 mL of 1× dilution washing solution to the centrifuge tube to resuspend the cells, centrifuge at 250g at room temperature for 10 min, and discard the supernatant. Repeat this step 1 - 2 times, and it can be used for subsequent experiments.

[0205] The specific steps for NGS library construction and sequencing are as follows:

[0206] 1. RNA extraction by Trizol method:

[0207] (1) Take PBMC immersed in Trizol from the -80 °C refrigerator;

[0208] (2) Add chloroform at 20% of the total volume of Trizol, invert up and down repeatedly for 15 times until the solution turns milky white, mix well and let it stand at room temperature for 3 min;

[0209] (3) Centrifuge at 4 °C and 12000g in a refrigerated centrifuge for 15 min, take the upper aqueous phase to a new centrifuge tube;

[0210] (4) Estimate the total volume of the supernatant in the previous step, add an equal volume of isopropanol, mix well up and down, and let it stand at room temperature for 10 min;

[0211] (5) Centrifuge at 4 °C and 12000g in a refrigerated centrifuge for 10 min to precipitate total RNA;

[0212] (6) Remove the supernatant, add 500 μL of pre-cooled 75% alcohol, gently shake the centrifuge tube to suspend the precipitate, centrifuge at 4 °C and 7500 rpm for 1 min, remove the supernatant, and centrifuge again at 4 °C and 7500 rpm for 1 min to remove all ethanol;

[0213] (7) Open the lid and let it stand at room temperature to dry, add about 200 μL of RNase-Free Water to dissolve RNA.

[0214] 2. Reverse transcription: According to Takara's PrimeScript TMPerform reverse transcription reaction using the II 1st Strand cDNA Synthesis Kit (Cat: 6210A).

[0215] 3. Amplification of target fragment: Using the reverse-transcribed cDNA as a template, perform PCR reaction with upstream and downstream primers specific for alpaca, and excise and recover the target fragment for NGS sequencing. These upstream and downstream primers can specifically amplify the nanobody gene of B cells

[0216] Forward primer (SEQ ID NO: 45 - 47): LeaderMix:

[0217] PME207-LPVH1 gcagtggctgcaggtgtccactcg

[0218] PME207-LPVH4 gcaggtccccaaggtgtcctgtcc

[0219] PME207-LPVH3 ggtggtcctggctgctct

[0220] Reverse primer (SEQ ID NO: 48 - 49): HingMix:

[0221] PME207-Hig2b-R gtttttgttcctggcctcccgggccactagtttgtggttttggtgtcttggg

[0222] PME207-Hig2c-R gtttttgttcctggcctcccgggccactagtggggtcttcgctgtggtgcgc

[0223] 4. Library construction and sequencing: Use Vazyme's VAHTS TM Universal DNA Library Prep Kit for V3 kit for library construction; use Illumina Miseq for sequencing.

[0224] III. Sequence grouping and sorting

[0225] Identify the CDR regions in the sequences in the sequence library, and calculate the sequence abundance, frequency, growth rate, and mutation value. The sequence growth rate is the ratio of the sequence frequency after enhancement to the sequence frequency before enhancement.

[0226] The sequences are grouped, and there are specifically three grouping methods: ① Grouping by CDR: The antibody sequences in the CDR group have the same CDR (3 CDRs for single-domain antibodies); ② Grouping by lineage: The antibodies in the lineage are mapped to the same V and J germline genes, and the maximum distance of a specific CDR3 between the two closest CDR3s in the lineage is equal to or less than 1, where all CDR3s have the same length; ③ Grouping by cluster: The antibodies in the cluster have the same CDR3 length, and the CDR3 identity between the two closest CDR3s is greater than or equal to 80%.

[0227] For each group, calculate the group abundance, group frequency, group growth rate, and group mutation value (the mutation value in this example is the mismatch score). As described above, the group abundance is the sum of the abundances of all sequences in the group, the group frequency is the sum of the frequencies of all sequences in the group, the group growth rate is the ratio of the enhanced group frequency to the group frequency mapped to this group before enhancement, and the group mismatch score is the average of the mismatch scores of all sequences in the group.

[0228] The summary of the NGS data processing of the three samples is shown in Table 1.

[0229] Table 1

[0230]

[0231] Select the groups with the top counts or a growth rate of 5 times or more according to the sequences, CDR groups, lineages, or clusters.

[0232] Select a representative sequence from the group. Generally, select the sequence with the highest abundance. If there are issues such as developability with this sequence, then select the sequence with the second highest abundance, and so on. Synthesize and test the affinity of the selected sequences. The affinity test uses methods well-known in the art such as the ELISA method.

[0233] IV. Mapping using the identified binders

[0234] For this project, during the first 10 rounds of immunization, hundreds of MSLN binders have been identified from the blood of the animal using traditional screening methods (phage display method). If memory B cell recall works, we expect some of these binders to reappear. To determine possible such cases, we map the MSLN binders to the lineages of the three samples according to the following criteria: The binder and the lineage share the same V and J genes, and the CDR3 sequence of the binder is the same as a sequence in the lineage. The results of the three samples are summarized in Table 2. In Table 2, A30 represents the animal number, NBL501 represents the project number, and the values in the table are the number of lineages covered when the identified MSLN binders are mapped to the top 30, top 60, top 90, top 120, and top 150 lineages.

[0235] Table 2

[0236]

[0237] Based on previous experience, PBMCs usually contain 0.1 - 5% antigen - specific B cells. Without antigen - specific enrichment, it is usually impossible for us to observe conjugates in the top - ranked lineages, probably because these B cells are induced by various pathogens or non - related immunogens and other body self - defense stimuli, while long - lived PCs usually dominate. However, as shown in Table 1, the sample on day 7 after boost (IM11) contains 25 conjugate - mapped lineages among the top 150 lineages analyzed, indicating that many of the top - ranked lineages in the IM11 sample are conjugates. In particular, among the top 30 lineages, there are 11 conjugate - mapped lineages, with a proportion as high as 36.7%.

[0238] We also mapped the above - identified MSLN conjugates to the clusters of the three samples according to the following criteria: one of the sequences in the conjugate and the cluster has the same CDR3 sequence. The results are summarized in Table 3, and the values in the table are the number of clusters covered when the identified MSLN conjugates are mapped to the top 30, top 60, top 90, top 120, and top 150 clusters.

[0239] Table 3

[0240]

[0241] As shown in Table 3, the sample on day 7 after boost (IM11) contains 23 conjugate - mapped lineages among the top 150 clusters analyzed, indicating that many of the top - ranked clusters in the IM11 sample are conjugates.

[0242] We also mapped the MSLN conjugates to the CDR groups of the three samples according to the following criteria: the conjugate has the same CDR1, CDR2, and CDR3 sequences as one of the sequences in the CDR group. The results are summarized in Table 4, and the values in the table are the number of CDR groups covered when the identified MSLN conjugates are mapped to the top 30, top 60, top 90, top 120, and top 150 CDR groups.

[0243] Table 4

[0244]

[0245] As shown in Table 4, the sample on day 7 after boost (IM11) contains 15 conjugate - mapped lineages among the top 150 CDR groups analyzed, indicating that many of the top - ranked CDR groups in the IM11 sample are conjugates.

[0246] To check whether the top-ranked lineages without conjugate mapping in IM11 contain conjugates, sequences were selected from the above lineages for synthesis and testing. The sequence selection method is as follows: select the sequence with the highest abundance. If there are issues such as developability with this sequence, then select the sequence with the second-highest abundance, and so on. Specifically, from the 19 lineages among the top 30 that did not have the previously discovered conjugate mapping, one sequence was selected for each to be tested (SEQ ID NO:6 - 24, NBL501#283–NBL501#301). Among them, 6 sequences failed to express. Using an OD value greater than 0.5 at 100 nM as the binding criterion, 12 out of the remaining 13 bound to the antigen (as Figure 15 shown), with a positive rate of 92% (as shown in Table 5). For control, we also tested clones from the top-ranked lineages in IM12. Specifically, from the top 20 lineages, one sequence was selected for each to be tested (SEQ ID NO:25 - 44, NBL501#302 - NBL501#321, and their amino acid sequences are shown at the end of the text). 2 sequences failed to express. Using an OD value greater than 0.5 at 100 nM as the binding criterion, 3 out of the remaining 18 bound to the antigen (as Figure 15 shown), with a positive rate of only 17% (as shown in Table 5). Since the IM12 sample was collected 7 days after the second boost and there was no increase in titer after the boost, this result indicates that to successfully recall memory B cells, immune interruption is required to allow the titer to decline. Such a result is in line with our expectations because a large amount of antibodies (high titer) in the serum will bind to the boost antigen and prevent them from activating memory B cells.

[0247] Table 5

[0248]

[0249] To analyze the repertoire dynamics during the memory B cell recall process, we compared the frequency changes of the top-ranked lineages with a frequency ≥0.1% in the IM11 sample among the three samples ( Figure 6 , A). The results showed that during the B cell recall process, the processes of lineage expansion and contraction were very dynamic: most of these top-ranked lineages expanded after the boost immunization and rapidly contracted to low levels in the IM12 sample (i.e., 21 days after the boost immunization). Figure 6 , B shows the growth rates of the top 30 lineages in IM11 compared to the corresponding lineages in the PRE-IM11 sample. Most of them showed more than 20-fold growth, and some of these lineages were super-growing lineages with a frequency of 1% or higher. Table 6 shows the number of super-growing lineages in these three samples. IM11 had the most super-growing lineages.

[0250] Table 6

[0251] A30 (NBL501) Number of hyper-growth lineages PRE-IM11 1 IM11 12 IM12 5

[0252] V. Secondary Recall

[0253] On December 7, 2022, 132 days after the previous immunization, a second memory B cell recall boost immunization was performed. Four PBMC samples (IM13-D4, IM13-D7, IM13-D11, and IM13-D14) were collected on the 4th, 7th, 11th, and 14th days respectively after the secondary recall boost immunization. Figure 5 , B shows the titers of these 4 samples as well as the titer of the sample before boost (PRE-IM13). Compared with the titer after IM12 immunization, 132 days after the immunization interruption, the titer decreased from 512K to 32K, a decrease of more than 10-fold. The boost immunization restored the titer to 512K within 11 days ( Figure 5 , B). Mapping the existing MSLN conjugates to these samples showed that IM13-D4 and IM13-D7 had most of the mapped conjugates in the top-ranked lineages (Table 7, the values in the table are the number of lineages covered when the identified MSLN conjugates are mapped to the top 30, top 60, top 90, top 120, and top 150 lineages), indicating that many of the top-ranked lineages in these two samples are conjugates. These two samples also had the most hyper-proliferative lineages (Table 7).

[0254] Table 7

[0255]

[0256] To analyze the kinetics of repertoire changes during the two memory B cell recalls, we compared the frequency changes of the top-ranked lineages with a frequency ≥ 0.1% in the IM11 sample ( Figure 7 , A) and the top-ranked lineages with a frequency ≥ 0.1% in the IM13-D4 sample ( Figure 7 , B). The results showed that some lineages grew during both recalls, while others grew only during one of the two recalls. Given the dynamic nature of the repertoire, such results may not be surprising. However, these results do demonstrate the value of multiple recalls in identifying more conjugates. To quantitatively evaluate the similarity of these seven repertoires, we calculated the Morisita-Horn lineage overlap index in these samples (Rempala and Seweryn, 2013)( Figure 8)。The overlap indices between IM11, IM12, and PRE-IM11 were all less than 10%, indicating that the repertoire changed rapidly during the memory B cell recall process. IM13-D4 and D7 showed high similarity with an overlap index of 81.32%, while IM13-D11 and D14 showed high similarity with an overlap rate of 72.47%. The similarity between IM13-D7 and IM13-D11 was not high, with an overlap index of only 11.79%, indicating that the repertoire changed rapidly between D7 and D11. Interestingly, the sample collection intervals for IM11 and IM13-D4 / D7 were approximately 5 months, but their repertoires showed reasonably good similarity, with overlap indices of 48.21% and 27.98% respectively. These results demonstrate the robustness of the memory B cell recall method.

[0257] Based on clusters( Figure 9 、 10 ), CDR groups( Figure 11 、 12 ) and full-length sequences( Figure 13 、 14 ) similar dynamic changes were found.

[0258] Example 2. Memory B cell recall was performed on more animals immunized with different antigens

[0259] To further confirm the reproducibility of this method, we performed memory B cell recall on more animals immunized with different antigens. Table 8 summarizes the PBMC sample information, titers, corresponding conjugate mapping data, and the number of hyper-growing lineages from these experiments. Among them, NBL501: antigen MSLN, NBL518: antigen PDL1, NBL521: antigen UPAR.

[0260] The antigen sources used in the examples are as follows:

[0261] Human Mesothelin(C-6His); Cat: CP51-1mg; Novoprotein / Nearest Shore;

[0262] hPD-L1, Llama IgG2b Fc; Cat: H211118604; Huakang Biologics;

[0263] Human uPAR, His Tag; Cat: #UPR-H5226; Acro.

[0264] The antigen sequences are as follows:

[0265] >Human Mesothelin(MSLN);

[0266] EVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMN GSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLD(SEQ ID NO:1)

[0267] >hPD-L1;

[0268] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER(SEQ ID NO:4)

[0269] >Human uPAR;

[0270] LRCMQCKTNGDCRVEECALGQDLCRTTIVRLWEEGEELELVEKSCTHSEKTNRTLSYRTGLKITSLTEVVCGLDLCNQGNSGRAVTYSRSRYLECISCGSSDMSCERGRHQSLQCRSPEEQCLDVVTHWIQEGEEGRPKDDRHLRGCGYLPGCPGSNGFHNNDTFHFLKCCNTTKCNEGPILELENLPQNGRQCYSCKGNSTHGCSSEETFLIDCRGPMNQCLVATGTHEPKNQSYMVRGCATASMCQHAHLGDAFSMNHIDVSCCTKSGCNHPDLDVQYR(SEQ ID NO:5)

[0271] Table 8

[0272]

[0273]

[0274] As shown in Table 8, memory B cell recall is effective for different antigens and animals. Specifically, samples taken several days after boost contain multiple conjugate-mapped lineages among the top-ranked lineages analyzed, indicating that many of the top-ranked lineages in these samples are conjugates. Overall, samples with more conjugates among the top-ranked lineages also have more hyper-growing lineages, suggesting rapid growth of antigen-specific clones and lineages during memory B cell recall.

[0275] To test whether top-ranked lineages not mapped by any conjugate are conjugates, we selected sequences from some of these lineages for testing. Table 9 summarizes the results of these experiments. Overall, samples on Day 4 / 7 have a high conjugate positivity rate among the top-ranked lineages, consistent with the conjugate mapping data.

[0276] Table 9

[0277]

[0278] To study the relationship between lineage growth rate, high abundance, and conjugates, we summarized the number of lineages with growth rates higher than 5-fold and lower than 5-fold and the corresponding number of conjugate-mapped lineages among the top 30, top 60, and top 90 lineages in terms of abundance (Table 10). The results show that compared with the lineages of pre-boost samples, the number of lineages with growth rates higher than 5-fold is greater than the number of lineages with growth rates lower than 5-fold, and most of the conjugate-mapped lineages are among the lineages with growth rates higher than 5-fold. Comparing with the sum of all D7, top 90 lineage data, conjugate-mapped lineages are 27% among the lineages with growth rates higher than 5-fold and 3% among the lineages with growth rates lower than 5-fold. These data show the value of selecting lineages with a 5-fold growth rate.

[0279] Table 10

[0280]

[0281] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims. At the same time, all the documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference.

[0282] SEQ ID NO:1

[0283] EVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLD

[0284] SEQ ID NO:2>NBL501-AgD23

[0285] CSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLD

[0286] SEQ ID NO:3>NBL501-cynoAgD23

[0287] SLETLKALLKVSKGHEMSAQVATLIDRVVVGRGQLDKDTADTLTAFCPGCLCSLSPERLSSVPPSIIGAVRPQDLDTCGPRQLDVLYPKARLAFQNMSGSEYFVKIRPFLGGAPTEDLKALSQQNVSMDLATFMKLRREAVLPLSVAEVQKLLGPHVEGLKVEEQHSPVRDWILKQRQDDLDTLGLGLQGGIPNGYLILDLSVREALS

[0288] SEQ ID NO:4

[0289] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER

[0290] SEQ ID NO:5

[0291] LRCMQCKTNGDCRVEECALGQDLCRTTIVRLWEEGEELELVEKSCTHSEKTNRTLSYRTGLKITSLTEVVCGLDLCNQGNSGRAVTYSRSRYLECISCGSSDMSCERGRHQSLQCRSPEEQCLDVVTHWIQEGEEGRPKDDRHLRGCGYLPGCPGSNGFHNNDTFHFLKCCNTTKCNEGPILELENLPQNGRQCYSCKGNSTHGCSSEETFLIDCRGPMNQCLVATGTHEPKNQSYMVRGCATASMCQHAHLGDAFSMNHIDVSCCTKSGCNHPDLDVQYR

[0292] SEQ ID NO:6>NBL501#283

[0293] QVQLVESGGGLVHTGGSLRLSCAAIGRTFRWFRQAPGKAREYIAAISWAGDSTYYADSVKGRFTVSRDNDKNMGYLQMDNLKPEDTAVYYCNVDFVGDLTSVPQYDRWGQGTQVTVSP

[0294] SEQ ID NO:7>NBL501#284

[0295] QVQLVESGGGLVQAGGSLRLSCAVSGLTFSTYAIGWYRQAPGKEREFVATIGWRDEGQTYSNYADSVKGRFTISRDSVKNTVYLQMNSLKPEDTAIYYCHGVDSGSDWGRGTQVTVSS

[0296] SEQ ID NO:8>NBL501#285

[0297] QLQLVASGGGLVQPGGSLRLSCALSGFTFDDYTVGWFRQAPGKEREGVSCINNRDGDTYYGDSVKGRFTISKDNAKNMVYLQMSSLKPEDTAVYYCAADEPWGGIKVCSPARIGNWGQGTQVTVSS

[0298] SEQ ID NO:9>NBL501#286

[0299] EVYLVESGGGSAQPGGSLRLSCAASGIFFNRYTMGWFRQAPGKQRDLVATIKYDGTTRFADSVKGRFTIFRDNDERTVDLQMNSLKPEDTATYYCHGEAVGLPAGHDIWGQGTQVTVSS

[0300] SEQ ID NO:10>NBL501#287

[0301] QVQLVESGGGLVQPGGSLRLSCAASGSFWSIYRMGWYRQAPGKQRELVAFVTNGRSTPEYAESVKGRFTISRENGKNAVNLQMNSLKPEDTAVYYCNVVVGVTDYWGQGTQVTVSS

[0302] SEQ ID NO:11>NBL501#288

[0303] QVQVAESGGGLVQAGGSLRLSCAASGGTPALSGWWRQRPGAEREFLARIRWSDGVAWYGESVEGRFTISRDSAKNTAYLQMNALKPEDTAIYYCNANTLTSKLSWGQGTPVTVSS

[0304] SEQ ID NO:12>NBL501#289

[0305] QVQLVESGGGLVQPGGSLTLSCAASGLIFSTTQMRWYRQAPGEEREWVASSYRDGTRSYSDSVQGRFTISRDNANNMVSLQMNSLKPEDTAVYYCYALRFGDARPEWGQGTQVTVSS

[0306] SEQ ID NO:13>NBL501#290

[0307] QVRLVESGGGLVQPGGSLRLTCLTSETIVYAPMGWYRQAPGKQREVVALVYNTGLADYANFARGRFVISRDNAKNMIDLQMNNLKSEDTAVYYCNVFRDGNDYWGHGTQVTVSA

[0308] SEQ ID NO:14>NBL501#291

[0309] QVQLVESGGGLVQPGGSLRLSCVASGFIIGTTPMFWYRQAPGKEREMVARVSSAFGNSPGFTSYSESVKGRFTISRDNTKNMLFLQMNSLQPDDTGVYYCASSDYWGQGTQVTVSS

[0310] SEQ ID NO:15>NBL501#292

[0311] QFQVIESGGGLVPPGGSLSLSCTTSGRTLSNWNMAWYSQVPGKERELVAAIDWSGRFTYYQDSVKGRFTISRDKEKNTVSLQMNNLKPDDTATYYCNRQAGLESFTHFDYWGQGTQVTVSS

[0312] SEQ ID NO:16>NBL501#293

[0313] QLQLVESGGGLVQPGGSLRLSCTGSGLDWYSIGWFRLRPGKEREGLACISSADDSTYYVDSVKGRFTVSRGDDRKTVFLQLNDLKPEDTGVYYCAAEASYVCWDTHIRETGYGNWGQGTQVTVAS

[0314] SEQ ID NO:17>NBL501#294

[0315] QVQFVESGGGLVQPGGSLKLSCAASGFDFENWYMSWFRQAPGKEREIVATIPNDGGIPIYEDFVKGRFTISRDNAKDTLYLQMNNLKSEDTAVYFCNGRRGTWTWGQGTRVTVSS

[0316] SEQ ID NO:18>NBL501#295

[0317] QLQLVESGGGLVQPGESLRLSCAGSGFSLKYLAVGWFRQVPGKEREGVSCIGPSDDTTYSADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAADHGEKFRLCRTGPSNSGSWGQGTQVTVSS

[0318] SEQ ID NO:19>NBL501#296

[0319] QVQLVESGGGLVQPGGSLRLSCEARGFIFGSSPMGWYRQAEGKEREMVARISSVFGNSPGFTSYAESVKGRFTISRDNAKTTLYLQMNNLKPEDTAVYYCTTFDSWGQGTQVTVSS

[0320] SEQ ID NO:20>NBL501#297

[0321] QVHLVESGGGLAQPGGSLRLSCAASGFRFSEWPMTWARQLPGQRLEWLSAISENGRGTNYVRDVEGRFIISRDNAKNTLYLQMDNLKPEDTGLYVCARGFGAESTLETRGQGTRVTVSS

[0322] SEQ ID NO:21>NBL501#298

[0323] QIQFVESGGGLVQPGGSLSLSCTAPGVVFSIFAVAWYRQAPGKERELVAALTNGYATQYADSVKGRFTVSRDTDTNTAHLQMNNLKPEDTAIYFCNVDGPFGTHREWPPYDYWGRGTQVTVSP

[0324] SEQ ID NO:22>NBL501#299

[0325] QVQLVESGGGLAQPGKSLRLSCAASGFPFSARIMSWYRQAPGRERELVAFISNEGGANYADSMRGRFTISRDNAKNTVYLEISSLRPEDTAVYYCAVRLGGTAYWGQGTQVTVSS

[0326] SEQ ID NO:23>NBL501#300

[0327] QVQLVESGGGSVQPGGSLRLSCAASNTIRQYTMGWYRQAPGKQRELVAAHYSDGRTNYPDSVRGRFAASRDNDKNTLYLQMDSLKPDDTGVYYCNIASWADPSRTLGSWGRGTQVTVSS

[0328] SEQ ID NO:24>NBL501#301

[0329] QVQLVESGGGLVQSGGSLRLSCVASGLTLSTTAVQWHRQAPGKEREWVASIYRADGSTGGSTSYADSVKGRFIISRDNAKNTAYLQMTSLTPEDTAVYYCREALWGNWGQGTQVTVSS

[0330] SEQ ID NO:25>NBL501#302

[0331] QVQLVESGGGSVQSGGSLRLSCRTSGFDFSNSFMYWYRQPPGKTRVSVATITTDGRTNYSDSVRGRFTISRDNTKRTIDLQMDSLRPEDTAVYYCFVYYQGRAYWGQGTQVTVSS

[0332] SEQ ID NO:26>NBL501#303

[0333] QVQVAESGGGLVQLGGSLRLTCVVSGTIFSINDMGWYRQAPGKQRELVATLTTGTTLRSGVTTNYADSVKGRFIVFKGDVEDTFYLQMNNLKPDDTAVYFCRARQGREYWGQGTQVTVSS

[0334] SEQ ID NO:27>NBL501#304

[0335] QVQLVESGGGLVQAGDSLRLSCAASGGGFSTYAMAWYRQAPGKEREFVARIWWNSPTTTIADSVRGRFTITKDNAKNTMYLEMKSLKPEDTAIYYCNLNTGNVAREPPGGHWGQGTQVTVSA

[0336] SEQ ID NO:28>NBL501#305

[0337] QVRFVESGGGWVHAGGSLRLSCAASGSSFSGYGMRWYRQAPGKERELVAHITSTGNTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNRLPAPLGWGQGTRVTVSS

[0338] SEQ ID NO:29>NBL501#306

[0339] AQVLVESGGGLVQAGGSLRLSCAASRSISTYDIRWYRQAPGKERDFVAAISWSGRDTYADSVKGRFTVSRDNAKNTAYLEMSSLKPEDTAIYLCNLAGYWGQGTQVTVSS

[0340] SEQ ID NO:30>NBL501#307

[0341] QVQLAESGGGLVQAGGSLRLSCEASGRTVSSPYMAWYRQAPGKERELVASVTRGGTTYYADSAKGRFTISRDNAANTVYLQMYSLKPEDTAIYYCYVDNGFGARHWGQGTQVTVSS

[0342] SEQ ID NO:31>NBL501#308

[0343] QVQFVESGGGLVQPGGSLRLSCLASGDNISIYAMGWYRQAPGKQRELVADITPAGITNYAASVKGRFTISRDNAKSTVYLQMNSLQPEDTAVYYCKRRYVADSWGQGTQVTVSS

[0344] SEQ ID NO:32>NBL501#309

[0345] QVRLVESGGGSAQPGGSLRLSCVASGLPFGNYAMRWYRQAPGKERELVATVTSGGETYADSVKGRFTISRDNAKRMVYLQMDSLKPEDTGVYYCNAPLWNYWGQGTQVTVSS

[0346] SEQ ID NO:33>NBL501#310

[0347] QVQFVESGGGLVQPGGSLTLSCIGSGFTFNRYVMGWYRQAPGQERELVTSINQYTDSVKGRFTISRHNANNTVHLQMDSLKPEDTAVYFCNAVPPRFHFGFRTDEFWGQGTQVTVSS

[0348] SEQ ID NO:34>NBL501#311

[0349] QVQLVESGGGFVQAGGSLRLSCVVSGRTSNIQTITWYRQAPGKQRREVVARIVWTGDGEEYAASVQGRFSISRDNAKNTVYLQMNNLKPEDTAIYLCSAVAGEHLDLNTYWGQGTQVTVSS

[0350] SEQ ID NO:35>NBL501#312

[0351] QVDLVESGGGLVQRGGSLRLSCAASENTLGLDSMGWYRQAPGKEREFVARLWWSGGRITYYADSVKGRFTISGDDAETTVYLSMNSLEPEDTAIYYCYSQKNFREFWGQGTQVTVYL

[0352] SEQ ID NO:36>NBL501#313

[0353] QVRLVESGGGLVRPGGSLRLSCAASGSGFGVATMAWYRQAPEKQREWVATISGGGATSYADSVKGRFTISRDNAGNTVYLQMNSLKPEDTAVYYCNAIREWWAWGQGTQVTVSS

[0354] SEQ ID NO:37>NBL501#314

[0355] QVQLVESGGGLVQAGGSLRLTCAASERTFTMAPAAWYRQAPGKEREFVASITYNAGSRDYADAVMGRFTISRDNAKNTVSLQMNSLKLEDTAVYYCNTRPPVGRYWGQGTQVTVSS

[0356] SEQ ID NO:38>NBL501#315

[0357] QVQLVESGGGVVQPGGSLRLACSASGFTLDSDVIGWFREVPGQWREGVSCITIGNRDDNIYYHDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAARRTGTVQEMCVLERVAYWGQGTQVTVSS

[0358] SEQ ID NO:39>NBL501#316

[0359] QVQLVESGGGLVQAGGSLRLSCLAAGRTFSTEDMGWYRQAPGKEREFVASVSWSGTTYVADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAALSNSWGRGTQVTVSS

[0360] SEQ ID NO:40>NBL501#317

[0361] QVQLVESGGGLVQAGGSLRLSCAASGGTFSVYTMGWYRQAPGKEREFVARITYSGFSTYDADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAPRGSSYFFNWGQGTQVTVSS

[0362] SEQ ID NO:41>NBL501#318

[0363] QVQLVESGGGLVQAGGSLRLSCAASGRTFSTYDMSWYRQAPGKERELVATISWNGRSTRYADSVKGRFTISRDDSKNTMYLQMNSLKPEDTAIYYCNNPLLNRWGQGTQVTVSS

[0364] SEQ ID NO:42>NBL501#319

[0365] QVQFVESGGGLVQPGGSLRLSCAASGFAFSSSAMVWYRQAPGKEREWVGEILPAGGGLDYRHSVRGRFTISRDNRKNVLYLQMNNLKPEDTAVYYCNARRYTFTGEYWGQGTQVTVSS

[0366] SEQ ID NO:43>NBL501#320

[0367] QVQLVESGGGLVQPGGSLRLSCAASGSFVTIGAMRWYRQAPGKQRELVATITTVATTDYADFVKGRFTISRDNAKNSVYLQMNNLEPEDTAVYFCGARFLSTTIPRTWKDYWGQGTQVTVSS

[0368] SEQ ID NO:44>NBL501#321

[0369] QVQLVESGGGLVQPGGSLRLSCAPSGSISGIYFRAWYRQAPGKQRELVASINSGGSTNYADSVQGRYTISRDDAKNTAYLQMNSLKPEDTAVYYCNVKTVWRGDYWGQGTQVTVSS

[0370] SEQ ID NO:45

[0371] GCAGTGGCTGCAGGTGTCCACTCG

[0372] SEQ ID NO:46

[0373] GCAGGTCCCCAAGGTGTCCTGTCC

[0374] SEQ ID NO:47

[0375] GGTGGTCCTGGCTGCTCT

[0376] SEQ ID NO:48

[0377] gtttttgttcctggcctcccgggccactagtTTGTGGTTTTGGTGTCTTGGG

[0378] SEQ ID NO:49

[0379] gtttttgttcctggcctcccgggccactagtGGGGTCTTCGCTGTGGTGCGC

[0380] SEQ ID NO:50>hIgG1 Fc

[0381] EPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0382] SEQ ID NO:51>Llama Fc

[0383] EPKTPKPQPQPQPQPNPTTESKCPKCPAPELLGGPSVFIFPPKPKDVLSISGRPEVTCVVVDVGQEDPEVSFNWYIDGAEVRTANTRPKEEQFNSTYRVVSVLPIQHQDWLTGKEFKCKVNNKALPAPIEKTISKAKGQTREPQVYALAPHREELAKDTVSVTCLVKGFYPPDINVEWQRNRQPEPEGTYATTPPQLDNDGTYFLYSKLSVGKNTWQRGETFTCVVMHETLHNHYTQKSISQS

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Claims

1. A method for generating antibodies specific to a target, the method comprising: (a) Immunize an animal with a target antigen for one or more rounds, (b) Reduce the serum antibody titer of the animal by 2-fold or more, (c) Immunize the animal with a recall antigen, wherein the antibody-binding region of the recall antigen has at least 30% identity to the target antigen, (d) Generate antibody sequences from the B cells of the animal 1 - 20 days after immunization in step (c), and (e) Select antibodies from the antibody sequences based on one or more prioritization factors, and Optionally, f) Repeat steps (b) to (e).

2. The method according to claim 1, wherein, (b) comprises interrupting the immunization of the animal until the serum antibody titer is reduced by 2-fold or more.

3. The method according to claim 2, wherein, (b) comprises: Subjecting the animal to an immunization interruption of 21 days or longer.

4. The method according to claim 3, wherein, (b) comprises: Subjecting the animal to an immunization interruption of 42 days or longer.

5. The method according to claim 4, wherein, (b) comprises: Subjecting the animal to an immunization interruption of 156 days or longer.

6. The method according to claim 1, wherein, (d) Generate antibody sequences from the B cells of the animal 4 - 14 days after immunization in step (c).

7. The method according to claim 6, wherein, (d) Generate antibody sequences from the B cells of the animal 4 - 11 days after immunization in step (c).

8. The method according to claim 7, wherein, (d) Generate antibody sequences from the B cells of the animal 4 - 7 days after immunization in step (c).

9. The method according to claim 1, wherein, (e) comprises: (1) Group the antibody sequences according to antibody characteristics, preferably, the antibody characteristics are selected from one or more of the following: CDR sequences, VH sequences, VL sequences, VHH sequences, lineages, and clusters; and (2) Select groups based on one or more prioritization factors.

10. The method according to claim 1 or 9, wherein, (2) The prioritization factors are selected from: the abundance or frequency of antibody sequences or groups from high to low, the growth rate of the abundance or frequency of antibody sequences or groups from high to low, the change in the abundance or frequency of antibody sequences or groups during the immunization process, antibody affinity maturation, sharing the same naive B cell origin among VHHs, avoiding sequences with poor developability, and combinations thereof.

11. The method according to claim 10, wherein, (2) The growth rate is the growth rate of the B cells of the animal 1 - 20 days after immunization in step (c) compared to the B cells of the animal after step (b).

12. The method according to claim 10 or 11, wherein, (2) The prioritization factors are selected from any one or any two or any three of the following combinations: (A) The abundance or frequency of antibody sequences or groups ranks in the top 10 - 20, 21 - 30, 31 - 40, 41 - 50, 51 - 60, 61 - 70, 71 - 80, 81 - 90, 91 - 100, 101 - 200, 201 - 300, 301 - 400, 401 - 500, 501 - 600, 601 - 700, 701 - 800, 801 - 900, 901 - 1000, 1001 - 1100, 1101 - 1200, 1201 - 1300, 1301 - 1400, 1401 - 1500, 1501 - 1600, 1601 - 1700, 1701 - 1800, 1801 - 1900, or 1901 - 2000 or lower, (B) The growth rate of the abundance or frequency of antibody sequences or groups reaches 5-fold or higher, (C) The mutation value of the antibody sequence or group has an increasing trend compared to before.

13. The method according to claim 12, wherein, The mutation values of the antibody sequence or group satisfy the following relationship: after 1 - 20 days of immunization in step (c) > after step (b) > after step (a).

14. The method according to claim 1, wherein, The recalled antigen comprises one or more or all epitopes of the target antigen.

15. The method according to claim 1, wherein,The target antigen is a peptide, protein, hapten, mRNA, DNA, viral vector allowing the expression of the target antigen, or a cell.

16. The method according to claim 9, wherein, If the growth rate of 1, 2, 3, or 4 selected from the group of antibody sequences, CDR sequence groups, lineages, and clusters is less than 2, the antibody selected in (e) is excluded.

17. The method according to claim 9, wherein, If the antibody selected in (e) has at least one factor of poor developability, the antibody is excluded, where the at least one developability is immunogenicity, expression, homogeneity, solubility, stability, viscosity, or formulation.

18. The method according to claim 1, wherein, The antibody is expressed by prokaryotic or eukaryotic cells.

19. The method according to claim 1, wherein, The antibody is a single - domain antibody or an immunoglobulin.

20. The method according to claim 9, wherein, The antibody is characterized by a lineage, and the antibodies in one lineage map to the same V and J germline genes and have a maximum distance of specific CDR3 equal to or less than 1 between the two closest CDR3s in the lineage, where all CDR3s have the same length.

21. The method according to claim 9, wherein, The antibody is characterized by a cluster, and the antibodies in the cluster have the same CDR3 length and a CDR3 identity of greater than or equal to 80% between the two closest CDR3s.

22. The method according to claim 1, wherein, The sequence is generated by sequencing technology.

23. The method according to claim 22, wherein, The sequence is generated by NGS or single - cell sequencing technology.

24. The method according to any one of claims 1 - 23, wherein, Step (e) further includes testing the specific binding of the selected antibody to the target.

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