Human immunoglobulin heavy chain length CDR3 transgenic constructs and uses thereof

Abstract

A human immunoglobulin heavy chain transgene construct encoding a long CDR3 region is provided. The heavy chain transgene comprises a plurality of VH regions longer than an average value, which are operably linked to a plurality of D-D fusion segments. Transgenic animals comprising the transgene are also provided. Methods of using these transgenic animals are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 439,797, filed January 18, 2023, which is hereby incorporated by reference in its entirety for all purposes.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is electronically submitted in .XML file format and is hereby incorporated by reference in its entirety. The XML copy was created on January 12, 2024, is named ZL8017-WO-PCT_SL.xml, and is 128,639 bytes in size. Background Art

[0005] Immunotherapy has revolutionized the treatment of various diseases, including cancer and autoimmune disorders. Although therapeutic antibodies against a large number of antigens have been successfully produced, certain types of targets have been shown to be challenging to generate antibodies in rodents by standard methods. These include targets with epitopes that are difficult to access or rarely combined with typical CDRs (e.g., targets with clefts or minimal surface accessibility), such as multi-pass membrane proteins (e.g., G protein coupled receptors (GPCRs) and ion channels), as well as enzyme active sites and allosteric epitopes. Such targets may also be difficult to stabilize as immunogen preparations, increasing their successful use as antigenic challenges.

[0006] Despite these obstacles, antibodies against intractable antigens, such as transmembrane receptors, have been described, including antibodies against the M2 ion channel of influenza A (Wei et al. (2011) PLoS One 6:e28309), the formyl peptide receptor 1 (FPR1) GPCR (Douthwaite et al. (2015) MABS 7:152-66), the voltage-gated potassium channel Kv1.3 (Wang et al. (2016) Proc. Natl. Acad. Sci. USA 113:11501-11506), and the 5-hydroxytryptamine 2B (5HT2B) GPCR (Ishchenko et al. (2017) Proc. Natl. Acad. Sci. USA 114:8223-8228). These antibodies have been observed to have or have been modified to have unusually long CDR H3 regions.

[0007] Some of these antibodies are produced in cattle or camels, which are known to have alternative immunoglobulin locus scaffold structures that can produce CDRH3 regions that are longer than those typical in rodents or humans (reviewed in de los Rios et al. (2015) Curr. Opin. Struct. Biol. 33:27-41; see also De Genst et al. (2006) Proc. Natl. Acad. Sci. USA 103:4586-4591; Wang et al. (2013) Cell 153:1379-1393; Sok et al. (2017) Nature 548:108-111). For example, the bovine Ig locus has limited combinatorial diversity potential because it has only 12 VH regions, but it can produce unusually long CDR H3 regions that can reach lengths exceeding 60 amino acids, whereas human CDR H3 regions are typically only 8-16 amino acids in length. The camelid Ig locus can produce heavy chain-only antibodies with dedicated variable domains (VHH) that have long CDR H3 regions.

[0008] Methods utilizing long CDR H3 regions for antibody production have been described, including transgenic chickens with long CDR H3 regions (U.S. Patent Publication 20210230253) and gene packaging libraries with long CDR H3 regions (U.S. Patent Publication 20200399785).

[0009] While some progress has been made, additional methods and compositions are needed for the design, preparation, and use of heavy chain long CDR3 transgenes, particularly for generating antibodies against intractable antigens. Summary of the Invention

[0010] The present disclosure provides human immunoglobulin heavy chain transgenic constructs encoding long CDR H3 regions. The extended CDR H3 regions are generated by DD fusion segments contained in the transgene, which comprise two D regions linked together. The long CDR H3 heavy chain transgenes of the present disclosure can be introduced into animal hosts to produce antibodies, particularly against difficult antigens, such as multi-pass transmembrane receptors (e.g., GPCRs and ion channels) that may be more readily bound by antibodies containing long CDR H3.

[0011] Thus, in one aspect, the present disclosure relates to a transgenic construct encoding an immunoglobulin heavy chain variable region, the transgenic construct comprising:

[0012] (a) a plurality of human unrearranged immunoglobulin heavy chain variable segments (VH), wherein each VH is 98 to 101 amino acids in length; the plurality of human unrearranged immunoglobulin heavy chain variable segments are operably linked to

[0013] (b) a plurality of human DD fusion segments; said plurality of human DD fusion segments being operably linked to

[0014] (c) Multiple human J segments.

[0015] In one embodiment, the plurality of human DD fusion segments comprises at least one naturally occurring human DD fusion pair. In one embodiment, the plurality of human DD fusion segments comprises at least one synthetic human DD fusion pair. In one embodiment, the plurality of human DD fusion segments comprises at least one synthetic human DD fusion pair and at least one naturally occurring human DD fusion pair.

[0016] In one embodiment, the transgenic construct encodes at least five, at least ten, at least fifteen, at least twenty, or at least twenty-five VH regions. In one embodiment, the transgenic construct encodes the following human VH regions in 5' to 3' direction: 3-73, 3-72, 2-70D, 1-69, 4-61, 5-51, 3-49, 3-43, 4-39, 4-32, 2-26, 1-24, 3-23, 3-15, 3-9, 1-8, 2-5, 7-4-1, 1-2, and 6-1.

[0017] In one embodiment, the transgenic construct encodes a synthetic 1.1 / 2-8D-D fusion or a synthetic 2-8 / 1-1D-D fusion. In one embodiment, the transgenic construct encodes a synthetic 1-1 / 2-8D-D fusion and a synthetic 2-8 / 1-1D-D fusion.

[0018] In one embodiment, the transgenic construct encodes at least one naturally occurring DD fusion pair selected from the group consisting of: 2-2 / 3-3; 5-12 / 4-17; 5-5 / 3-22; 6-6 / 3-10; 6-6 / 6-19; 5-12 / 5-5; 6-13 / 6-19; 2-2 / 6-13; 5-12 / 6-19; 6-13 / 2-21; 5-5 / 3-10; 2-15 / 3-22; 5-12 / 2-15; 2-15 / 5-24; 6 / 19-1 / 26; 2-15 / 5-5; 5-12 / 3-22; 2-15 / 4-17; 5-5 / 6-13; 6-19 / 3-22; 2-15 / 2-21 and 3-3 / 3-10.

[0019] In one embodiment, the transgenic construct encodes the naturally occurring DD fusion pairs 2-2 / 3-3; 5-12 / 4-17; 5-5 / 3-22; 6-6 / 3-10; 6-6 / 6-19; 5-12 / 5-5; 6-13 / 6-19; 2-2 / 6-13; 5-12 / 6-19; 6-13 / 2-21; 5-5 / 3-10; 2-15 / 3-22; 5-12 / 2-15; 2-15 / 5-24; 6 / 19-1 / 26; 2-15 / 5-5; 5-12 / 3-22; 2-15 / 4-17; 5-5 / 6-13; 6-19 / 3-22; 2-15 / 2-21 and 3-3 / 3-10.

[0020] In one embodiment, the transgenic construct further encodes at least one native D segment.

[0021] In one embodiment, the transgenic construct encodes the following D segments and DD fusion pairs in the 5′ to 3′ direction: 1-1 / 2-8; 2-2 / 3-3; 3-3; 5-12 / 4-17; 5-5 / 3-22; 6-6 / 3-10; 6-6 / 6-19; 2-8; 5-12 / 5-5; 6-13 / 6-19; 2-2 / 6-13; 5-12 / 6-19; 6 -13 / 2-21; 5-5 / 3-10; 2-15 / 3-22; 3-16; 5-12 / 2-15; 2-15 / 5-24; 6 / 19-1 / 26; 2-15 / 5-5; 5-12 / 3-22; 2-15 / 4-17; 5-5 / 6-13; 6-19 / 3-22; 2-15 / 2-21; 3-3 / 3-10 and 2-8 / 1-1.

[0022] In one embodiment, the transgenic construct encodes the J1-J6 segment.

[0023] In one embodiment, the transgenic construct further encodes constant regions (eg, mouse or human Ig constant regions) downstream (3') of the multiple J segments.

[0024] In one embodiment, the transgenic construct further comprises lox sites to facilitate cre / lox-mediated RMCE (recombinase-mediated cassette exchange). In one embodiment, the transgenic construct further comprises a guide recombination sequence (GRS) to facilitate CRISPR / CAS-mediated recombination.

[0025] In one embodiment, the transgenic construct comprises the sequence shown in SEQ ID NO:1.

[0026] In one embodiment, the transgenic construct is carried on a bacterial artificial chromosome (BAC).

[0027] In another aspect, the present disclosure relates to a transgenic animal comprising a transgenic construct of the present disclosure. In one embodiment, the transgenic animal is a mouse. In one embodiment, the transgenic mouse further comprises a transgenic construct encoding an immunoglobulin light chain, such that the mouse expresses antibodies comprising a light chain paired with a heavy chain comprising a long CDR3 region.

[0028] In another aspect, the present disclosure relates to a method for producing an antibody against an antigen of interest, the method comprising administering the antigen of interest to a transgenic animal (e.g., a mouse) of the present disclosure, such that an antibody that binds to the antigen of interest is produced. In one embodiment, the method further comprises isolating the antibody of interest from the animal and determining its heavy chain CDR3 sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1A-Figure 1C An overview of the sequences of the indicated native D, synthetic DD fusions, and naturally occurring DD fusions in their "sense" orientation is provided. SEQ ID NOs: 2-109. The polynucleotide sequence of the D segment is in reading frame 1; due to the binding mechanism of antibodies, reading frame 2 or 3 may be utilized. The amino acid sequence includes all three possible frames. Yellow highlighted text = in-frame stop codon; green = native; pink = synthetic DD fusion; orange = native DD fusion.

[0030] Figure 2A- Figure 2B An alignment of the open reading frames of the indicated D segments and DD fusions is provided; this does not take into account additional sequence that may result from variations in the joining region or inversions of the D or DD segments. Figure 2A. SEQ ID NOs: 110-156. Figure 2B .SEQ ID NO: 157-228.

[0031] Figure 3 is a summary of the transgenic positions of the indicated D segments and DD fusions; these positions correspond to their naturally occurring positions within the human IGH locus.

[0032] Figure 4 is a schematic diagram illustrating representative examples of long CDR3 heavy chain constructs of the present disclosure.

[0033] Figure 5 Schematic diagram of a representative vector construct for the long CDR3 heavy chain transgene of the present disclosure.

[0034] Figure 6 Schematic diagram of the knock-in protocol for site-specific delivery of a long CDR3 heavy chain transgene donor into the mouse Ig heavy chain locus. DETAILED DESCRIPTION

[0035] The long CDR3 heavy chain transgenic constructs of the present disclosure encode a combination of a longer-than-average VH region and a DD fusion, such as Figure 4 Various aspects of the present disclosure are described in further detail below.

[0036] Unless otherwise defined, all technical terms, symbols and other scientific terms or technical terms used herein are intended to have the meanings commonly understood by those skilled in the art to which the present disclosure belongs. In some cases, for the sake of clarity and / or ease of reference, terms with commonly understood meanings are defined herein, and such definitions contained herein should not be construed as being substantially different from the general understanding of the art. The techniques and procedures described or referred to herein are generally well understood and commonly used by those skilled in the art using conventional methodologies, such as the widely used molecular cloning methodologies described in the following references: Sambrook et al., Molecular Cloning: A Laboratory Manual 4th Edition (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, unless otherwise indicated, procedures involving the use of commercially available kits and reagents are generally performed according to the protocols and / or parameters defined by the manufacturer.

[0037] I. Construct Design

[0038] The design and construction of the long CDR3 human heavy chain transgene are described in detail in Example 1. Figure 4 As exemplified in , the construct encodes a combination of a longer-than-average VH region and a DD fusion. Thus, in one aspect, the present disclosure relates to a transgenic construct encoding an immunoglobulin heavy chain variable region, the transgenic construct comprising:

[0039] (a) a plurality of human unrearranged immunoglobulin heavy chain variable segments (VH), wherein each VH in its unrearranged form is 98 to 101 amino acids in length; the plurality of human unrearranged immunoglobulin heavy chain variable segments operably linked to

[0040] (b) a plurality of human DD fusion segments; said plurality of human DD fusion segments being operably linked to

[0041] (c) Multiple human J segments.

[0042] Non-limiting examples of human VH regions with lengths of 98 to 101 amino acids include the following regions: 6-1, 2-5, 2-26, 2-70, 2-70D, 3-15, 3-49, 3-72, 3-73, 3-9, 3-23, 3-43, 4-30-2, 4-30-4, 4-31, 4-32, 4-39, 4-61, 5-51, 7-4-1, 1-2, 1-3, 1-8, 1-18, 1-24, 1-38-4, 1-45, 1-46, 1-58, and 1-69. In one embodiment, the transgenic construct encodes at least five, at least ten, at least fifteen, at least twenty, or at least twenty-five VH regions, e.g., selected from the above list.

[0043] In one embodiment, the transgenic construct encodes the following human VH regions in the 5' to 3' direction: 3-73, 3-72, 2-70D, 1-69, 4-61, 5-51, 3-49, 3-43, 4-39, 4-32, 2-26, 1-24, 3-23, 3-15, 3-9, 1-8, 2-5, 7-4-1, 1-2, and 6-1.

[0044] In one embodiment, the plurality of human DD fusion segments comprises at least one naturally occurring human DD fusion pair. In one embodiment, the plurality of human DD fusion segments comprises at least one synthetic human DD fusion pair. In one embodiment, the plurality of human DD fusion segments comprises at least one synthetic human DD fusion pair and at least one naturally occurring human DD fusion pair. Non-limiting examples of naturally occurring and synthetic human DD fusion pairs are shown in Figures 1, 2, and Figure 3 (described in further detail in Example 1). Naturally occurring DD fusion pairs have also been described in the art, such as in Larimore et al. (2012) J Immunol. 189: 3221-3230; Briney et al. (2012) Immunol. 137: 56-64; Yu and Guan (2014) Front. Immunol. 5: 250; Safonova and Pevzner (2019) Front. Immunol. 10: 987; and Safonova and Pevzner (2020) Genome Res. 30: 1547-1558, the entire contents of each of which are expressly incorporated by reference.

[0045] In one embodiment, the transgenic construct encodes a synthetic 1-1 / 2-8D-D fusion or a synthetic 2-8 / 1-1D-D fusion. In one embodiment, the transgenic construct encodes a synthetic 1-1 / 2-8D-D fusion and a synthetic 2-8 / 1-1D-D fusion.

[0046] In one embodiment, the transgenic construct encodes at least one, at least two, at least three, at least four, or at least five, at least six, at least seven, at least eight, at least nine, at least ten or more naturally occurring DD fusion pairs selected from the group consisting of: 2-2 / 3-3; 5-12 / 4-17; 5-5 / 3-22; 6-6 / 3-10; 6-6 / 6-19; 5-12 / 5-5; 6-13 / 6-19; 2-2 / 6-13; 5-12 / 6-19; 6-13 / 2-21; 5-5 / 3-10; 2-15 / 3-22; 5-12 / 2-15; 2-15 / 5-24; 6 / 19-1 / 26; 2-15 / 5-5; 5-12 / 3-22; 2-15 / 4-17; 5-5 / 6-13; 6-19 / 3-22; 2-15 / 2-21 and 3-3 / 3-10.

[0047] In one embodiment, the transgenic construct encodes the naturally occurring DD fusion pairs 2-2 / 3-3; 5-12 / 4-17; 5-5 / 3-22; 6-6 / 3-10; 6-6 / 6-19; 5-12 / 5-5; 6-13 / 6-19; 2-2 / 6-13; 5-12 / 6-19; 6-13 / 2-21; 5-5 / 3-10; 2-15 / 3-22; 5-12 / 2-15; 2-15 / 5-24; 6 / 19-1 / 26; 2-15 / 5-5; 5-12 / 3-22; 2-15 / 4-17; 5-5 / 6-13; 6-19 / 3-22; 2-15 / 2-21 and 3-3 / 3-10.

[0048] In one embodiment, the transgenic construct further encodes at least one human native D segment (i.e., a D segment that is not a DD fusion). In an embodiment, the transgenic construct further encodes at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more natural D segments. In one embodiment, the natural D segments are longer than average, e.g., encoding a translated sequence of ≥9 a.a., ≥10 a.a., ≥11 a.a., or 9-12 amino acids in length. Non-limiting examples of human natural D segments of 9-12 amino acids in length include segments 2-2, 2-8, 2-15, 2-21, 3-3, 3-9, 3-10, 3-16, and 3-22. In one embodiment, the transgenic construct comprises natural human D segments 3-3, 2-8, and 3-16.

[0049] In one embodiment, the transgenic construct encodes the following D segments and DD fusion pairs in the 5′ to 3′ direction: 1-1 / 2-8; 2-2 / 3-3; 3-3; 5-12 / 4-17; 5-5 / 3-22; 6-6 / 3-10; 6-6 / 6-19; 2-8; 5-12 / 5-5; 6-13 / 6-19; 2-2 / 6-13; 5-12 / 6-19; 6 -13 / 2-21; 5-5 / 3-10; 2-15 / 3-22; 3-16; 5-12 / 2-15; 2-15 / 5-24; 6 / 19-1 / 26; 2-15 / 5-5; 5-12 / 3-22; 2-15 / 4-17; 5-5 / 6-13; 6-19 / 3-22; 2-15 / 2-21; 3-3 / 3-10 and 2-8 / 1-1.

[0050] In one embodiment, the transgenic construct encodes the human J1-J6 segment.

[0051] In one embodiment, the transgenic construct further encodes constant regions (eg, mouse or human Ig constant regions) downstream (3') of the multiple J segments.

[0052] In one embodiment, the transgenic construct further comprises lox sites to facilitate cre / lox-mediated RMCE (recombinase-mediated cassette exchange). In one embodiment, the transgenic construct further comprises a guide recombination sequence (GRS) to facilitate CRISPR / CAS-mediated recombination.

[0053] The nucleotide sequence of the transgenic construct can be further optimized for the intended purpose. For example, the construct can be altered to codon optimize (e.g., to increase expression of the coded region). Additionally or alternatively, the construct can be altered to avoid excessive somatic hypermutation (SHM), for example, by analyzing the hypermutation regions in the CDR1, CDR2, and / or CDR3 of the heavy chain variable region and eliminating sequences that may enhance SHM. Methods for codon optimization and SHM reduction are well established in the art.

[0054] Transgenic construct can also comprise the sequence that allows transgenic targeting to be inserted into specific gene seat (for example, endogenous mouse heavy chain gene seat).Knock-in technology with targeted transgenic replacement endogenous gene seat is confirmed in the art.In a preferred embodiment, transgenic construct comprises the recombination sequence (guiding recombination sequence or GRS) that allows transgenic to be knocked into endogenous mouse heavy chain gene seat.

[0055] In one embodiment, the long CDR3 heavy chain construct comprises the nucleotide sequence shown in SEQ ID NO:1.

[0056] II. Construct Preparation

[0057] The transgenic constructs disclosed herein can be prepared using standard recombinant DNA techniques. Cloning vectors containing polylinkers can be used as starting vectors for inserting the DNA fragment of interest. Suitable cloning vectors are well established in the art. In addition, plasmids or other vectors (e.g., YACs) carrying human unrearranged light chain immunoglobulin sequences have been described in the art (see, e.g., U.S. Patents 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay; and U.S. Patents 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963, all to Kucherlapati et al.) and can be used as a source of heavy chain V, D, and J region sequences. Alternatively, the desired sequences can be synthesized by standard methods. The appropriate DNA fragments are then operatively linked into the cloning vector by ligation, and the vector is subsequently characterized (eg, by restriction fragment analysis or sequencing, etc.) to ensure correct arrangement of the fragments.

[0058] In one embodiment, the transgenic construct is carried on a bacterial artificial chromosome (BAC).BAC technology for carrying Ig transgenes is well established in the art.

[0059] Non-limiting examples of long CDR3 heavy chain vectors of the present disclosure are Figure 5 Schematically illustrated in FIG.

[0060] In order to prepare the transgenic construct for microinjection or other transgenic technology, the transgenic construct can be separated from the carrier carried by cleaving with appropriate restriction enzymes to release the transgenic construct fragment. Standard techniques can be used to separate the fragments, such as by pulsed field gel electrophoresis on an agarose gel, followed by separation of the fragments from the agarose gel, such as by [β]-agarase digestion or by electroelution. For example, agarose gel slices containing transgenic construct fragments can be cut from the gel, and standard methods can be used to digest the agarose with [β]-agarase (e.g., from Takara). Alternatively, the preparation of the transgenic for knocking in purpose can be carried out by standard BAC or plasmid purification techniques, and the closed circular form is separated for direct transfection or is introduced into recipient mouse cells or embryos.

[0061] III. Preparation of transgenic animals

[0062] Another aspect of the present disclosure relates to a transgenic non-human host animal (that is, transgenic construct is integrated into the genome of host animal) comprising a transgenic construct of the present disclosure, so that animal expression comprises the immune repertoire of the antibody using the heavy chain comprising long CDR3 district. Standard methods known in the art for introducing exogenous nucleic acid into the genome of non-human animal are used to prepare transgenic non-human host animal of the present disclosure. In a preferred embodiment, knock-in technology is used to insert transgenic construct into the genome of host animal, to replace all or part of endogenous heavy chain locus (for example, endogenous mouse heavy chain locus) with transgenic (for example, human heavy chain transgenic). Alternatively, transgenic construct can be introduced into the genome of host animal by, for example, prokaryotic microinjection for random genome insertion or transfection into mouse embryonic stem (mES) cells.

[0063] For knock-in methods, typically loxP flanking sites are included in the construct so that these sites promote recombination between the loxP flanking sites in the host and the loxP flanking sites in the transgenic donor when the Cre recombinase is expressed. Recombination is performed in embryonic stem cells (e.g., mouse embryonic stem cells), and then the embryonic stem cells with the modification of interest are implanted in living blastocysts, which are then grown into mature chimeric animals (e.g., mice), some of which have the original blastocyst cell genetic information, while other cells have the modification introduced into the embryonic stem cell. The subsequent offspring of the chimeric animal will then have the gene knocked in. Knock-in technology is summarized in, for example, Manis (2007) New Engl. J. Med. 357: 2426-2429.

[0064] As an alternative knock-in method, constructs can include flanking guide recombination sequences (GRS) to promote CRISPR / CAS-mediated recombination. These are 500bp-1500bp sequences, which flank transgenic inserts and have specific homology with the endogenous mouse sequence adjacent to a specific CRISPR / CAS cleavage site in the mouse genome. The identical CRISPR / CAS cleavage site is attached to the end of the GRS flanking sequence to allow CRISPR / CAS-mediated digestion to cut the endogenous mouse genome and circular BAC transgenic donor simultaneously. In this way, the cut end of the mouse CRISPR / CAS site can be used for homologous recombination-mediated repair via similar cutting and linearized transgenic donor inserts, thereby causing site-specific knock-in.

[0065] In a preferred embodiment, knock-in technology is used to insert transgenic construct into the genome of mouse to replace all or part of endogenous heavy chain locus.In a preferred embodiment, transgenic construct is the people's heavy chain construct that is inserted into endogenous mouse heavy chain locus by homologous recombination, thus lacks mouse VH, DH and JH and at least some parts of CH sequence.In another embodiment, the heavy chain transgenic that lacks constant region is inserted into endogenous heavy chain locus, so that at least some parts of VH, DH and JH sequence are missing, but CH sequence remains intact and is operably connected to the functional heavy chain variable region of transgenic, thus produces chimeric antibody (it can be reverse engineered to be fully human) in mouse.

[0066] Another method for producing transgenic non-human animals, particularly transgenic mice, is pronuclear microinjection. This technique is well established in the art (see, for example, Wagner, TE et al. (1981) Proc. Natl. Acad. Sci. USA 78: 6376-6380; Wagner and Hoppe, U.S. Pat. No. 4,873,191). Generally speaking, the method involves introducing exogenous genetic material into the pronucleus of a mammalian fertilized egg (e.g., a mouse fertilized egg) by microinjection to obtain a genetically transformed fertilized egg, which is then transplanted into a pseudopregnant female. The embryos are then allowed to develop to term, and the genomes of the resulting offspring are analyzed for the presence of the transgenic material.

[0067] Southern blot analysis, PCR or other techniques for analyzing genomic DNA are used to detect the presence of unique nucleic acid fragments that will not be present in non-transgenic animals but will be present in transgenic animals. Selective breeding of transgenic offspring allows homozygosity for the transgene to be achieved.

[0068] If the long CDR3 heavy chain transgene is randomly inserted into the genome, it is preferred to also disable the endogenous heavy chain locus to obtain a limited heavy chain selection repertoire in animals (e.g., mice). The endogenous heavy chain locus can be engineered to be inoperable by standard knockout techniques, such as by deleting all or part of the endogenous heavy chain V, D, J, and C regions, rendering them non-functional.

[0069] Although preferred embodiments of the present disclosure include transgenic mice, the present invention encompasses other non-human host animals, including but not limited to rats, rabbits, pigs, goats, sheep, cattle, and chickens. Techniques for producing transgenic animals of each of these species have been described in the art. For example, the preparation of transgenic rats is described in Tesson, L. et al. (2005) Transgenic Res. 14: 531-546, including techniques such as DNA microinjection, lentiviral vector-mediated DNA transfer to early embryos, and sperm-mediated transgenesis. Transgenic methods in rats are also described in Mullin, LJ et al. (2002) Methods MoI. Biol. 180: 255-270. The preparation of transgenic rabbits is described, for example, in Fan, J. et al. (1999) Pathol. Int. 49: 583-594; Fan, J. and Watanabe, T. (2000) J. Atheroscler. Thromb. 7: 26-32; Bosze, Z. et al. (2003) Transgenic Res. 12: 541-553. The preparation of transgenic pigs is described, for example, in Zhou, C.Y. et al. (2002) Xenotransplantation 9: 183-190; Vodicka, P. et al. (2005) Ann. NY Acad. Sci. 1049: 161-171.

[0070] Alternative transgenic technologies for pronuclear microinjection in pigs include adenovirus-mediated DNA introduction into porcine sperm (see, e.g., Farre, L et al. (1999) Mol. Reprod. Dev. 53: 149-158) and linker-based sperm-mediated gene transfer (Chang, K. et al. (2002) BMC Biotechnol. 2: 5). The preparation of transgenic goats is described, e.g., in Ebert, KM et al. (1991) Biotechnology (NY) 9: 835-838; Baldassarre, H. et al. (2004) Reprod. Fertil. Dev. 16: 465-470. Somatic cell nuclear transfer in goats is described, e.g., in Behboodi, E. et al. (2004) Transgenic Res. 11: 215-224. The preparation of transgenic sheep is described, for example, in Ward, KA and Brown, BW (1998) Reprod. Fertil. Dev. 10: 659-665. The preparation of transgenic cattle is described, for example, in Donovan, DM et al. (2005) Transgenic Res. 14: 563-567. Gene transfection of donor cells for bovine embryo nuclear transfer is described, for example, in Lee SL et al. (2005) Mol. Reprod. Dev. 72: 191-200. The preparation of transgenic domestic farm animals is also described in Niemann, H. et al. (2005) Rev. Sci. Tech. 24: 285-298. The preparation of transgenic chickens is described, for example, in Pain, B. et al. (1999) Cells Tissues Organs 165:212-219; Lillico, SG et al. (2005) Drug Discov. Today 10:191-196; and Ishii, Y. et al. (2004) Dev. Dyn. 229:630-642.

[0071] Animals (e.g., mice) of the present disclosure carrying long CDR3 heavy chain transgenic constructs can be hybridized with animals (e.g., mice) carrying immunoglobulin light chain transgenics to produce animals (e.g., mice) expressing antibodies comprising light chains paired with heavy chains comprising long CDR3 regions. Immunoglobulin light chain transgenic animals (e.g., mice) are well established in the art.

[0072] IV. Use of Transgenic Animals

[0073] Transgenic animals disclosed herein can be used to produce antibodies for multiple antigens of interest.For the animal that only carries long CDR3 heavy chain transgenic and endogenous light chain gene seat, this animal will produce chimeric light chain / heavy chain antibody, if desired, these chimeric light chain / heavy chain antibodies can be reverse engineered so that long CDR3 heavy chain is paired with the light chain of same species. Alternatively, for the animal (for example, mouse) that carries long CDR3 heavy chain Ig transgenic (for example, people) and light chain Ig transgenic (for example, people), complete heterologous antibodies (for example, fully human antibodies) can be prepared in host transgenic animals. For the animal that carries chimeric Ig transgenic gene seat (for example, the people variable region that is attached to mouse constant region of heavy chain and / or light chain), the combination of humanized gene seat can be with other humanized gene seats or alternatively with its wild-type mouse counterpart functional pairing.

[0074] Therefore, in yet another aspect, the present disclosure relates to a method for producing an antibody against an antigen of interest, the method comprising administering an antigen of interest to a transgenic animal of the present disclosure. In one embodiment, the animal is a transgenic mouse, and the antigen is administered to the mouse so as to produce an antibody bound to the antigen of interest in the mouse. In one embodiment, the animal is a transgenic mouse carrying a human Ig long CDR3 heavy chain transgene and a human Ig light chain transgene, and the antigen is administered to the mouse so as to produce a human or human-mouse chimeric antibody bound to the antigen of interest in the mouse. In one embodiment, the antigen is a GPCR or an ion channel protein. In one embodiment, the method may also comprise separating the antibody of interest from a host animal (e.g., a mouse) and determining the heavy chain CDR3 sequence of the antibody.

[0075] Transgenic animals can be immunized with an antigen of interest by standard methods known in the art, and the antibodies produced in the animals can also be isolated and characterized by standard validation methods. Polyclonal antibodies can be isolated directly from host animals and monoclonal antibodies can be prepared by standard methods (such as hybridoma technology). Procedures for preparing monoclonal antibodies using hybridomas are well established in the art (see, for example, U.S. Patent No. 4,977,081, PCT Publication No. WO 97 / 16537, and European Patent No. 491057B1, the disclosures of which are incorporated herein by reference). Alternatively, the production of monoclonal antibodies in vitro from cloned cDNA molecules is also well established in the art (see, for example, Andris-Widhopf et al. (2000) J. Immunol. Methods 242: 159; and Burton (1995) Immunotechnology 1: 87, the disclosures of which are incorporated herein by reference). B cell clones from immunized transgenic animals can be isolated, and cDNA encoding the antibody can be isolated and cloned into expression vectors by standard molecular biology techniques. Further recombinant engineering of cloned Ig cDNAs is also possible and established in the art.

[0076] V. Definitions

[0077] As used herein, the term "DD fusion segment" is intended to refer to the direct connection of two different immunoglobulin heavy chain D region nucleic acid sequences. For example, a DD fusion segment of a 5-12D segment and a 4-17D segment is referred to herein as a 5-12+4-17D-D fusion segment (or simply a "DD fusion"). A "naturally occurring DD fusion pair" refers to a DD fusion of two constituent D segments observed in nature resulting from a V(DD)J recombination event. Such naturally occurring DD fusion pairs have been described in the art, such as in Larimore et al. (2012) J. Immunol. 189: 3221-3230; Briney et al. (2012) Immunol. 137: 56-64; Yu and Guan (2014) Front. Immunol. 5: 250; Safonova and Pevzner (2019) Front. Immunol. 10: 987; and Safonova and Pevzner (2020) Genome Res. 30: 1547-1558. A "synthetic DD fusion pair" refers to a DD fusion resulting from a V(DD)J recombination event that has not been observed in nature. Regardless of whether the DD fusion pair is "naturally occurring" (i.e., has been observed in nature) or "synthetic" (i.e., not observed in nature), the DD fusion segments used in the transgenes of the present disclosure can be engineered ex vivo by genetically joining the sequences of the two D segments by standard methods.

[0078] As used herein, "long CDR3," "long H CDR3," "long CDR H3," or "long HCDR3" refers to a heavy chain CDR3 region that is longer than the typical or average length of a human heavy chain CDR3 region, for example, typically longer than 8-16 amino acids in length. After VDJ recombination, the transgenes of the present disclosure produce heavy chain variable regions with CDR3s of various lengths, and when a significant portion (e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% or more) of the HCDR3 produced is longer than the typical or average length of the HCDR3, the transgene is considered to be a transgene encoding a long CDR3.

[0079] As used herein, the term "operably linked" is intended to describe a configuration of a nucleic acid sequence that is in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, it is operably linked to that sequence. With respect to the connection of two protein coding regions, operably linked means that the linked nucleic acid sequences are contiguous and in reading frame. With respect to splice donor / acceptor and RSS sequences, operably linked means that the sequences are able to fulfill their functional purpose.

[0080] As used herein, the term "transgene" refers to a gene that is introduced as an exogenous gene into a site within the host genome (eg, the mouse light chain Ig locus).

[0081] As used herein, the term "transgenic construct" refers to a nucleic acid preparation suitable for introduction into the genome of a host animal.

[0082] As used herein, the term "transgenic mouse" refers to a mouse comprising cells having a transgene as defined herein. The transgene can be present in all or some cells of the mouse.

[0083] As used herein, the term "unrearranged" with reference to an immunoglobulin V segment refers to an immunoglobulin V segment in its germline configuration, in which the V segment is not recombined to be directly adjacent to a D segment or a J segment.

[0084] The present invention is further illustrated by the following examples, which should not be construed as further limiting.The contents of the figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0085] Example

[0086] Example 1: Preparation of human long CDR3 heavy chain constructs

[0087] In this example, the preparation of human immunoglobulin heavy chain transgenic constructs is described, wherein the CDR3 domains of the constructs are on average larger (longer) than the CDR3 domains found in normal naive human B cell heavy chain CDR3s. The basic principle of this approach is that certain antigens may have functionally important epitopes that are difficult to access or rarely found in normal length heavy chain CDR3 domain antibodies. By using a set of CDR3 domains that are longer (larger) than the average, the likelihood of the antibody interacting with such epitopes is increased.

[0088] Conceptual Framework

[0089] It has been reported in the art that long CDR3s have the potential to be difficult membrane targets. In particular, GPCRs, ion channels, and other membrane targets with clefts or limited surface accessibility may benefit from long CDR3 mAbs to better access functional sites (see, e.g., Douthwaite et al. (2015) MAbs 7: 152-166; Corti et al. (2013) Annu. Rev. Immunol. 31: 705-742; Wei et al. (2011) PLoS One 6: e28309; Wang et al. (2016) Proc. Natl. Acad. Sci. USA 113: 11501-11506; Ishchenko et al. (2017) Proc. Natl. Acad. Sci. USA 114: 8223-8228). Camelid and bovine antibodies with long CDR3 domains have been used for this purpose (see, e.g., Wang et al. (2013) Cell 153: 1379-1393; de los Rios et al. (2015) Curr. Opin. Struct. Biol. 33: 27-41; Sok et al. (2017) Nature 548: 108-111).

[0090] Construct design and transgene preparation

[0091] Using the mechanism of CDR3 elongation, transgenes were designed that bias expression toward longer domains. First, the longest VH and D segments were identified, and transgenes were designed to selectively utilize some longer VH segments and longer D segments. Additionally, DD fusions were included that utilized some shorter D segments in order to retain some naturally occurring D amino acid sequence while also retaining a longer CDR3 length.

[0092] The selection of VH segments was based on length and uniqueness. VH segments with a length of 98-101 amino acids were selected for possible inclusion in the construct.

[0093] Natural long D segments of wild-type configuration were also selected for possible inclusion in the constructs, examples of which include: 2-02, 2-08, 2-15, 2-21, 3-03, 3-09, 3-10, 3-16, and 3-22.

[0094] Regarding DD fusions, the natural formation of DD fusions during recombination can be explained by the presence of so-called "cryptic nonamers", which are nonamer sequences that are beyond their normal background but are still able to support recombination (usually part of the RSS motif that can undergo VDJ recombination) (Safonova and Pevzner (2020) Genome Res. 30: 1547-1558). Some D segments have a higher probability of non-classical nonamers in the spacing that will allow recombination, examples of which include: 2-02, 2-15, 3-03, 3-09, 3-10, 3-16, 3-22, 6-06, 6-19, and 6-25. In addition, based on experience, it seems that the occurrence of DD fusions generally follows their germline order (5'D fused to 3'D), making 3'D segments less common to fuse to 5' segments.

[0095] The sequences of exemplary native D segments, synthetic DD fusions, and naturally occurring DD fusions are shown in FIG1 . In total, the 27 different D or DD components shown in FIG1 comprise 47 different ORFs, which are aligned in FIG2 , where F1, F2, and F3 correspond to different reading frames. These illustrate the overall structure and sequence of elements that can be included in a transgene. However, it should also be noted that recombinant D segments can be found in reverse orientation as part of VDJ recombination sequences (e.g., see Meek et al., (1989) J Exp Med. 170: 39-57). Therefore, for the purposes of this disclosure, all D and DD segments included herein are intended to be encompassed in their forward orientation as well as their reverse orientation.

[0096] The strategy for placing the various D and DD fusions into the constructs was (in order of preference): (i) if possible, place the DD fusion at a location within the D domain where one of the DD pairs is normally located; (ii) if this is not possible in its own genomic locus, try to match the expression of the naturally occurring D segment there to the expression level of one of the D segments present in the DD fusion; and (iii) find a remaining slot where possible. Figure 3 The endogenous D position is shown in the transgenic construct. Figure 3 The last two columns of the are the endogenous D segment expression levels (as reported in the IMGT database; https: / / www.imgt.org / genefrequency / query) were compared with the expression levels of the DD fusion pairs reported in Safonova and Pevzner (2019) Front. Immunol. 10: 987. While there is some correlation between the two, the primary goal of placing the DD fusion cassette into its “natural” location means that the rank order of the two parameters cannot match.

[0097] Schematic diagram of a representative long CDR3 construct is shown in Figure 4 and a representative nucleotide sequence of the construct is shown in SEQ ID NO: 1.

[0098] The construct comprises the following native VH segments in 5' to 3' order: 3-73, 3-72, 2-70D, 1-69, 4-61, 5-51, 3-49, 3-43, 4-39, 4-32, 2-26, 1-24, 3-23, 3-15, 3-9, 1-8, 2-5, 7-4-1, 1-2, and 6-1. Each VH segment is 98-101 amino acids in length and uses the native RSS and octamer sites.

[0099] The construct also contains three native DH sequences (3-3, 2-8, and 3-16), two novel synthetic DD fusions (1-1 / 2-8 and 2-8 / 1-1), and 22 naturally occurring DD fusion pairs (2-2 / 3-3; 5-12 / 4-17; 5-5 / 3-22; 6-6 / 3-10; 6-6 / 6-19; 5-12 / 5-5; 6-13 / 6-19; 2-2 / The long D region is 9-20 amino acids in length and retains as much of its native position as possible, with the RSS site remaining intact. The 5′ to 3′ order of the D segments and DD fusions in the constructs is as follows: 1-1 / 2-8; 2-2 / 3-3; 3-3; 5-12 / 4-17; 5-5 / 3-22; 6-6 / 3-10; 6-6 / 6-19; 2-8; 5-12 / 5-5; 6-13 / 6-19; 2-2 / 6-13; 5-12 / 6-19; 6-13 / 2-2 1; 5-5 / 3-10; 2-15 / 3-22; 3-16; 5-12 / 2-15; 2-15 / 5-24; 6 / 19-1 / 26; 2-15 / 5-5; 5-12 / 3-22; 2-15 / 4-17; 5-5 / 6-13; 6-19 / 3-22; 2-15 / 2-21; 3-3 / 3-10 and 2-8 / 1-1.

[0100] The DD fusion segments inserted into the constructs used the DNA sequences shown in Figure 1. These sequences were inserted into and replaced the native D segment coding sequence, the amino acid sequence of which is shown in Figure 2. For this replacement, the coding sequence was placed directly between the flanking RSS sites on either side of the CDS of the segment. The RSS sequence was not changed.

[0101] LoxP flanks the insert at the 5' end. An ADAM / IGHD GRS1 site is also present at the 5' end, and a J-Mu GRS site is present at the 3' end to facilitate recombination between the donor transgene and the mouse host genome. GRS donor-delivered knock-ins have demonstrated success to the point where this is the preferred mechanism for site-specific donor transgene delivery. The PGK-Puro cassette is flanked by FRT sites, allowing for removal of the cassette following GRS donor gene delivery.

[0102] from Figure 4The segment from the end of the "long DH" to the J segment (J1-J6) shown is of germline configuration and is unchanged relative to the human GenBank reference sequence, except for the final DD fusion (2-8 / 1-1), which places this segment within the J sequence in the region where the endogenous 7-27D allele normally occurs.

[0103] like Figure 4 and Figure 5 As exemplified in , a long CDR3 bacterial artificial chromosome (BAC) transgene donor was used for CRISPR / Cas-mediated one-step deletion / delivery of a transgene via a GRS sequence. Figure 6 Provides a schematic illustration of the GRS knock-in strategy and the resulting knock-in alleles when a BAC donor is used in conjunction with mouse Ig-specific CRISPR / Cas reagents in mouse ES cells capable of mediating homologous recombination events. Mice containing a long CDR3 heavy chain transgene can be bred to homozygosity. Mice can be crossed with light chain transgenic mice to generate HC / LC transgenic mice expressing a repertoire of long CDR3 transgenes.

[0104] Sequence Listing Overview

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Claims

1. A transgenic construct encoding an immunoglobulin heavy chain variable region, the transgenic construct comprising: (a) a plurality of human unrearranged immunoglobulin heavy chain variable segments (VH), wherein each VH is 98 to 101 amino acids in length; the plurality of human unrearranged immunoglobulin heavy chain variable segments are operably linked to (b) a plurality of human DD fusion segments; said plurality of human DD fusion segments being operably linked to (c) Multiple human J segments. 2 . The transgenic construct of claim 1 , wherein the plurality of human DD fusion segments comprises at least one naturally occurring human DD fusion pair.

3. The transgenic construct of claim 1, wherein the plurality of human DD fusion segments comprises at least one synthetic human DD fusion pair. 4 . The transgenic construct of claim 1 , wherein the plurality of human DD fusion segments comprises at least one synthetic human DD fusion pair and at least one naturally occurring human DD fusion pair.

5. The transgenic construct of claim 1, encoding at least twenty VH regions.

6. The transgenic construct of claim 1 , encoding the following human VH regions in a 5′ to 3′ direction: 3-73, 3-72, 2-70D, 1-69, 4-61, 5-51, 3-49, 3-43, 4-39, 4-32, 2-26, 1-24, 3-23, 3-15, 3-9, 1-8, 2-5, 7-4-1, 1-2, and 6-1.

7. The transgenic construct of claim 1, encoding a synthetic 1-1 / 2-8D-D fusion or a synthetic 2-8 / 1-1D-D fusion.

8. The transgenic construct of claim 1, encoding a synthetic 1-1 / 2-8D-D fusion and a synthetic 2-8 / 1-1D-D fusion.

9. The transgenic construct of claim 1 , encoding at least one naturally occurring DD fusion pair selected from the group consisting of: 2-2 / 3-3; 5-12 / 4-17; 5-5 / 3-22; 6-6 / 3-10; 6-6 / 6-19; 5-12 / 5-5; 6-13 / 6-19; 2-2 / 6-13; 5-12 / 6-19; 6-13 / 2-21; 5-5 / 3-10; 2-15 / 3-22; 5-12 / 2-15; 2-15 / 5-24; 6 / 19-1 / 26; 2-15 / 5-5; 5-12 / 3-22; 2-15 / 4-17; 5-5 / 6-13; 6-19 / 3-22; 2-15 / 2-21 and 3-3 / 3-10.

10. The transgenic construct of claim 1 , encoding the naturally occurring DD fusion pairs 2-2 / 3-3; 5-12 / 4-17; 5-5 / 3-22; 6-6 / 3-10; 6-6 / 6-19; 5-12 / 5-5; 6-13 / 6-19; 2-2 / 6-13; 5-12 / 6-19; 6-13 / 2-21; 5-5 / 3-10; 2-15 / 3-22; 5-12 / 2-15; 2-15 / 5-24; 6 / 19-1 / 26; 2-15 / 5-5; 5-12 / 3-22; 2-15 / 4-17; 5-5 / 6-13; 6-19 / 3-22; 2-15 / 2-21; and 3-3 / 3-10.

11. The transgenic construct of claim 1 , further encoding at least one native D segment.

12. The transgenic construct of claim 11, encoding the following D segments and DD fusions in the 5' to 3' direction: 1-1 / 2-8; 2-2 / 3-3; 3-3; 5-12 / 4-17; 5-5 / 3-22; 6-6 / 3-10; 6-6 / 6-19; 2-8; 5-12 / 5-5; 6-13 / 6-19; 2-2 / 6-13; 5-12 / 6-19; 6-13 / 2-21; 5-5 / 3-10; 2-15 / 3-22; 3-16; 5-12 / 2-15; 2-15 / 5-24; 6 / 19-1 / 26; 2-15 / 5-5; 5-12 / 3-22; 2-15 / 4-17; 5-5 / 6-13; 6-19 / 3-22; 2-15 / 2-21; 3-3 / 3-10 and 2-8 / 1-1.

13. The transgenic construct according to any one of claims 1 to 12, encoding the J1-J6 segment.

14. The transgenic construct of any one of claims 1 to 13, further encoding an immunoglobulin (Ig) constant region downstream of the plurality of J segments.

15. The transgenic construct of claim 14, wherein the Ig constant region is a mouse Ig constant region.

16. The transgenic construct of claim 14, wherein the Ig constant region is a human Ig constant region.

17. The transgenic construct according to any one of claims 1 to 16, further comprising lox sites to facilitate cre / lox-mediated RMCE (recombinase-mediated cassette exchange).

18. The transgenic construct of any one of claims 1 to 16, further comprising a guide recombination sequence (GRS) to promote CRISPR / CAS-mediated recombination.

19. The transgenic construct of claim 1, comprising the sequence shown in SEQ ID NO:

1.

20. The transgenic construct according to any one of claims 1 to 19, which is carried on a bacterial artificial chromosome (BAC).

21. A transgenic mouse comprising the transgenic construct of any one of claims 1 to 20.

22. The transgenic mouse of claim 21, further comprising a transgenic construct encoding an immunoglobulin light chain such that the mouse expresses antibodies comprising a light chain and a heavy chain.

23. A method for producing an antibody against an antigen of interest, the method comprising administering the antigen of interest to the transgenic mouse according to claim 22, so that an antibody that binds to the antigen of interest is produced.

24. The method of claim 23, further comprising isolating the antibody of interest from the mouse and determining its heavy chain CDR3 sequence.

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