Human immunoglobulin heavy chain long CDR3 transgene constructs and uses thereof

Abstract

A human immunoglobulin heavy chain transgene construct is provided that encodes a long CDR3 region. The heavy chain transgene comprises multiple VH regions that are longer than average and operably linked to multiple DD fusion segments. Transgenic animals containing the transgene are also provided. Methods for using the transgenic animals are also provided.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 439,797, filed January 18, 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML file format, which is incorporated herein by reference in its entirety. The XML copy created on January 12, 2024, is named ZL8017-WO-PCT_SL.xml and is 128,639 bytes in size. [Background technology]

[0003] Immunotherapy has revolutionized the treatment of a wide variety of diseases, including cancer and autoimmune disorders. While therapeutic antibodies have been successfully produced against numerous antigens, certain types of targets remain that have proven challenging for antibody generation using standard methods in rodents. These include targets with epitopes that are inaccessible or poorly bound by typical CDRs (e.g., targets with clefts or minimal surface access), 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 can also be difficult to stabilize as immunogen preparations, adding to the challenges of their successful use as antigens.

[0004] Despite these obstacles, antibodies against difficult-to-treat antigens such as transmembrane receptors have been described, including those against the influenza A M2 ion channel (Wei et al. (2011) PLoS One 6:e28309), formyl peptide receptor 1 (FPR1) GPCR (Douthwaite et al. (2015) MABS 7:152-66), voltage-gated potassium channel Kv1.3 (Wang et al. (2016) Proc. Natl. Acad. Sci. USA 113:11501-11506), and 5-hydroxytryptamine 2B (5HT2B) GPCR (Ishchenko et al. (2017) Proc. Natl. Acad. Sci. USA 114:8223-8228). These antibodies were observed to have, or were modified to have, unusually long CDR H3 regions.

[0005] Some of these antibodies are produced in cattle or camels, which are known to have alternative immunoglobulin locus scaffold structures that may result in longer CDR H3 regions than are 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 only 12 VH regions, limiting the potential for combinatorial diversity, but can generate unusually long CDR H3 regions that can reach lengths of over 60 amino acids, whereas human CDR H3 regions are typically only 8-16 amino acids long. Camelid Ig loci can give rise to heavy chain-only antibodies with dedicated variable domains (VHHs) with long CDR H3 regions.

[0006] Approaches utilizing long CDR H3 regions for antibody generation have been described, including transgenic chickens with long CDR H3 regions (U.S. Patent Application Publication No. 20210230253) and libraries of genetic packages with long CDR H3 regions (U.S. Patent Application Publication No. 20200399785).

[0007] Although some progress has been made, further approaches and compositions are needed for the design, preparation and use of heavy chain-length CDR3 transgenes, particularly for use in generating antibodies against intractable antigens. Summary of the Invention

[0008] The present disclosure provides human immunoglobulin heavy chain transgene constructs encoding long CDR H3 regions. The extended CDR H3 regions result from the inclusion in the transgene of a D-D fusion segment comprising two D regions joined together. The long CDR H3 heavy chain transgenes of the present disclosure can be introduced into an animal host to produce antibodies, particularly antibodies against difficult-to-treat antigens such as multi-pass transmembrane receptors (e.g., GPCRs and ion channels) that can be more readily bound by long CDR H3-containing antibodies.

[0009] Thus, in one aspect, the present disclosure provides a transgene construct encoding an immunoglobulin heavy chain variable region, comprising: (a) a plurality of human unrearranged immunoglobulin heavy chain variable segments (VHs), each VH being 98 to 101 amino acids in length; (b) a plurality of human DD fusion segments operably linked thereto; (c) a transgene construct comprising a plurality of human J segments.

[0010] 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.

[0011] In one embodiment, the transgene construct encodes at least 5, at least 10, at least 15, at least 20, or at least 25 VH regions. In one embodiment, the transgene construct encodes human VH regions in the 5' to 3' direction as follows: 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.

[0012] In one embodiment, the transgene construct encodes a synthetic 1-1 / 2-8 DD fusion or a synthetic 2-8 / 1-1 DD fusion. In one embodiment, the transgene construct encodes a synthetic 1-1 / 2-8 DD fusion and a synthetic 2-8 / 1-1 DD fusion.

[0013] In one embodiment, the transgene 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.

[0014] In one embodiment, the transgene 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.

[0015] In one embodiment, the transgene construct further encodes at least one naturally occurring D segment.

[0016] In one embodiment, the transgene constructs are any of the following: 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 encode D segments and DD fusion pairs in the 5' to 3' direction.

[0017] In one embodiment, the transgene construct encodes the J1-J6 segments.

[0018] In one embodiment, the transgene construct further encodes a constant region downstream (3') of multiple J segments (eg, a murine or human Ig constant region).

[0019] In one embodiment, the transgene construct further comprises lox sites to facilitate cre / lox-mediated RMCE (Recombinase-Mediated Cassette Exchange). In one embodiment, the transgene construct further comprises a Guide Recombination Sequence (GRS) to facilitate CRIS PR / CAS-mediated recombination.

[0020] In one embodiment, the transgene construct comprises the sequence shown in SEQ ID NO:1.

[0021] In one embodiment, the transgene construct is carried on a bacterial artificial chromosome (BAC).

[0022] In another aspect, the present disclosure relates to a transgenic animal comprising a transgene construct of the present disclosure. In one embodiment, the transgenic animal is a mouse. In one embodiment, the transgenic mouse further comprises a transgene construct encoding an immunoglobulin light chain, such that the mouse expresses an antibody comprising a light chain paired with a heavy chain comprising a long CDR3 region.

[0023] In another aspect, the disclosure relates to a method of generating 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 disclosure, such that an antibody that binds to the antigen of interest is generated. In one embodiment, the method further comprises isolating the antibody of interest from the animal and determining its heavy chain CDR3 sequence. [Brief explanation of the drawings]

[0024] [Figure 1A]A summary of the sequences of the indicated native D, synthetic DD fusions, and naturally occurring DD fusions is provided in their "sense" orientation. SEQ ID NO: 2 to SEQ ID NO: 109. The polynucleotide sequence of the D segment is in reading frame 1. Due to the antibody binding mechanism, reading frame 2 or reading frame 3 can 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 = natural DD fusion. [Figure 1B] A summary of the sequences of the indicated native D, synthetic DD fusions, and naturally occurring DD fusions is provided in their "sense" orientation. SEQ ID NO: 2 to SEQ ID NO: 109. The polynucleotide sequence of the D segment is in reading frame 1. Due to the antibody binding mechanism, reading frame 2 or reading frame 3 can 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 = natural DD fusion. [Figure 1C] A summary of the sequences of the indicated native D, synthetic DD fusions, and naturally occurring DD fusions is provided in their "sense" orientation. SEQ ID NO: 2 to SEQ ID NO: 109. The polynucleotide sequence of the D segment is in reading frame 1. Due to the antibody binding mechanism, reading frame 2 or reading frame 3 can 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 = natural DD fusion.

[0025] [Figure 2A] Alignments of the open reading frames of the indicated D segments and DD fusions are provided. This does not account for additional sequence that may result from junction variations or inversions of the D or DD segments. Figure 2A: SEQ ID NO: 110 to SEQ ID NO: 156. Figure 2B: SEQ ID NO: 157 to SEQ ID NO: 228. [Figure 2B]Alignments of the open reading frames of the indicated D segments and DD fusions are provided. This does not account for additional sequence that may result from junction variations or inversions of the D or DD segments. Figure 2A: SEQ ID NO: 110 to SEQ ID NO: 156. Figure 2B: SEQ ID NO: 157 to SEQ ID NO: 228.

[0026] [Figure 3] 1 is a summary of the transgenic locations of the indicated D segments and DD fusions, which correspond to their naturally occurring locations within the human IGH locus.

[0027] [Figure 4] FIG. 1 is a schematic diagram showing representative examples of long CDR3 heavy chain constructs of the present disclosure.

[0028] [Figure 5] FIG. 1 is a schematic diagram of a representative vector construct for a long CDR3 heavy chain transgene of the present disclosure.

[0029] [Figure 6] FIG. 1 is a schematic diagram of the knock-in scheme used to site-specifically deliver a long CDR3 heavy chain transgene into the mouse Ig heavy chain locus. DETAILED DESCRIPTION OF THE INVENTION

[0030] The long CDR3 heavy chain transgene constructs of the present disclosure encode a combination of a longer than average VH region and a DD fusion, as shown schematically in Figure 4. Various aspects of the present disclosure are described in further detail below.

[0031] Unless otherwise defined, all technical terms, designations, and other scientific terms used herein are intended to have the meaning generally understood by those of ordinary skill in the art to which this disclosure pertains. In some cases, terms having generally understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a deviation from that understood in the art. The techniques and procedures described or referenced herein are generally well understood by those of ordinary skill in the art and are commonly employed using conventional methodologies, such as the widely used molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and / or parameters unless otherwise noted.

[0032] I. Structure design The design and construction of a long CDR3 human heavy chain transgene is described in detail in Example 1. As shown in Figure 4, the construct encodes a combination of a longer than average VH region and a DD fusion. Thus, in one aspect, the disclosure provides a transgene construct encoding an immunoglobulin heavy chain variable region, comprising: (a) a plurality of human unrearranged immunoglobulin heavy chain variable segments (VH), each VH in its unrearranged form being 98 to 101 amino acids in length, operably linked thereto; (b) a plurality of human DD fusion segments operably linked thereto; (c) a transgene construct comprising a plurality of human J segments.

[0033] Non-limiting examples of human VH regions between 98 and 101 amino acids in length 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 transgene construct encodes at least 5, at least 10, at least 15, at least 20, or at least 25 VH regions, e.g., selected from the foregoing list.

[0034] In one embodiment, the transgene construct encodes a human VH region in the 5' to 3' direction as follows: 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.

[0035] 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 3 (described in further detail in Example 1). Naturally occurring DD fusion pairs are also described 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 specifically incorporated by reference.

[0036] In one embodiment, the transgene construct encodes a synthetic 1-1 / 2-8 DD fusion or a synthetic 2-8 / 1-1 DD fusion. In one embodiment, the transgene construct encodes a synthetic 1-1 / 2-8 DD fusion and a synthetic 2-8 / 1-1 DD fusion.

[0037] In one embodiment, the transgene constructs are 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 15 / 2-21, 2-15 / 3-22, 2-15 / 4-17, 5-5 / 6-13, 6-19 / 3-22, 2-15 / 2-21, and 3-3 / 3-10.

[0038] In one embodiment, the transgene 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.

[0039] In one embodiment, the transgene construct further encodes at least one human native D segment (i.e., a D segment that is not a DD fusion). In one embodiment, the transgene 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 native D segments. In one embodiment, the native D segment(s) are longer than average, e.g., encode a translated sequence of ≥ 9 aa, ≥ 10 aa, ≥ 11 aa, or are 9-12 amino acids in length. Non-limiting examples of human native D segments that are 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 transgene construct includes native human D segments 3-3, 2-8, and 3-16.

[0040] In one embodiment, the transgene constructs are any of the following: 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 / 3-2 Encoding D segments and DD fusion pairs in the 5' to 3' direction: 2, 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.

[0041] In one embodiment, the transgene construct encodes human J1-J6 segments.

[0042] In one embodiment, the transgene construct further encodes a constant region downstream (3') of multiple J segments (eg, a murine or human Ig constant region).

[0043] In one embodiment, the transgene construct further comprises lox sites to facilitate cre / lox-mediated RMCE (recombinase-mediated cassette exchange). In one embodiment, the transgene construct further comprises guide recombination sequences (GRS) to facilitate CRIS PR / CAS-mediated recombination.

[0044] The nucleotide sequence of the transgene construct can be further optimized for the intended purpose. For example, the construct can be altered for codon optimization (e.g., to increase expression of the coding region). Additionally, or alternatively, the construct can be modified to avoid excessive somatic hypermutation (SHM), for example, by analyzing hypermutable regions in CDR1, CDR2, and / or CDR3 of the heavy chain variable region and eliminating sequence(s) that may enhance SHM. Approaches for codon optimization and SHM reduction are well established in the art.

[0045] The transgene construct may further comprise a sequence that allows for targeted insertion of the transgene into a specific locus, for example, the endogenous mouse heavy chain locus. Knock-in techniques for replacing endogenous loci with targeted transgenes are well established in the art. In a preferred embodiment, the transgene construct comprises a recombination sequence (guide recombination sequence, or GRS) that allows the transgene to be knocked into the endogenous mouse heavy chain locus.

[0046] In one embodiment, the long CDR3 heavy chain construct comprises the nucleotide sequence shown in SEQ ID NO:1.

[0047] II. Preparation of Constructs The transgene constructs of the present disclosure can be prepared using standard recombinant DNA techniques. Cloning vectors containing polylinkers are useful as starting vectors for the insertion of a DNA fragment of interest. Suitable cloning vectors are well established in the art. Additionally, plasmids or other vectors (e.g., YACs) carrying human unrearranged light chain immunoglobulin sequences have been described in the art (e.g., U.S. Pat. Nos. 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, (See U.S. Patent Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963 (all to Kucherlapati et al.)), can be used as a source of heavy chain V-region, D-region, and J-region sequences. Alternatively, the desired sequences can be synthesized by standard methods. The appropriate DNA fragments are then operably joined into a cloning vector via ligation, followed by characterization of the vector (e.g., by restriction fragment analysis or sequencing) to ensure proper placement of the fragments.

[0048] In one embodiment, the transgene construct is carried on a bacterial artificial chromosome (BAC). BAC technology for carrying Ig transgenes is well established in the art.

[0049] A non-limiting example of a long CDR3 heavy chain vector of the present disclosure is shown schematically in FIG.

[0050] To prepare a transgene construct for microinjection or other transgenesis techniques, the transgene construct can be isolated from the vector in which it is carried by digestion with an appropriate restriction enzyme to release the transgene construct fragment. The fragment can be isolated using standard techniques, such as digestion on an agarose gel, followed by isolation of the fragment from the agarose gel by [β]-agarase pulsed-field gel electrophoresis or electroelution. For example, an agarose gel slice containing the transgene construct fragment can be excised from the gel, and the agarose can be digested with [β]-agarase (e.g., from Takara) using standard methodology. Alternatively, preparation of the transgene for knock-in purposes can be performed by standard BAC or plasmid purification techniques, followed by isolation of the closed circular form for direct transfection or introduction into recipient mouse cells or embryos.

[0051] III. Generation of transgenic animals Another aspect of the present disclosure relates to a transgenic non-human host animal (i.e., the transgene construct is integrated into the genome of the host animal) comprising a transgene construct of the present disclosure, such that the animal expresses an immune repertoire comprising antibodies that use heavy chains comprising a long CDR3 region. The transgenic non-human host animals of the present disclosure are prepared using standard methods known in the art for introducing exogenous nucleic acid into the genome of a non-human animal. In a preferred embodiment, the transgene construct is inserted into the genome of the host animal using knock-in technology to replace all or part of the endogenous heavy chain locus (e.g., an endogenous mouse heavy chain locus) with a transgene (e.g., a human heavy chain transgene). Alternatively, the transgene construct can be introduced into the genome of the host animal by, for example, pronuclear microinjection for random genomic insertion or transfection into mouse embryonic stem (mES) cells.

[0052] For the knock-in approach, loxP-flanked sites are typically included in the construct, whereby upon expression of Cre recombinase, these sites promote recombination between the host loxP-flanked sites and the loxP-flanked sites in the transgene donor. Recombination is carried out in embryonic stem cells (e.g., mouse embryonic stem cells), and the embryonic stem cells with the desired modification are then implanted into viable blastocysts, which then develop into mature chimeric animals (e.g., mice) in which some cells carry the genetic information of the original blastocyst cells and other cells carry the modification introduced into the embryonic stem cells. Subsequent offspring of the chimeric animals carry the gene knock-in. Knock-in technology is summarized, for example, in Manis (2007) New Engl. J. Med. 357:2426-2429.

[0053] As an alternative knock-in approach, constructs can include flanking guide recombination sequences (GRSs) to facilitate CRISPR / CAS-mediated recombination. These are 500-1500 bp sequences flanking the transgene insert and have specific homology to endogenous mouse sequences adjacent to specific CRISPR / CAS cleavage sites in the mouse genome. Adding the same CRISPR / CAS cleavage sites to the ends of the GRS-flanking sequences allows CRISPR / CAS-mediated digestion to simultaneously cleave the endogenous mouse genome as well as the circular BAC transgene donor. In this way, the cleaved ends of the mouse CRISPR / CAS sites are available for homologous recombination-mediated repair via a similarly cleaved and linearized transgene donor insert, resulting in site-specific knock-in.

[0054] In a preferred embodiment, a transgene construct is inserted into the genome of a mouse using knock-in technology to replace all or part of the endogenous heavy chain locus. In a preferred embodiment, the transgene construct is a human heavy chain construct that is inserted into the endogenous mouse heavy chain locus by homologous recombination, thereby deleting the mouse VH, DH, and at least a portion of the JH and CH sequences. In another embodiment, a heavy chain transgene lacking a constant region is inserted into the endogenous heavy chain locus, deleting at least a portion of the VH, DH, and JH sequences, but leaving the CH sequence intact, operably linked to a functional heavy chain variable region in the transgene, thereby allowing the mouse to produce chimeric antibodies (which can be reverse engineered to be fully human).

[0055] Another method for producing transgenic non-human animals, particularly transgenic mice, is the method of pronuclear microinjection. This technique is well established in the art (see, e.g., Wagner, TE et al. (1981) Proc. Natl. Acad. Sci. USA 78:6376-6380; U.S. Pat. No. 4,873,191 by Wagner and Hoppe). Generally, this method involves introducing exogenous genetic material into the pronucleus of a mammalian zygote (e.g., a mouse zygote) by microinjection to obtain a genetically transformed zygote, followed by implantation of the genetically transformed zygote into a pseudopregnant female animal. The embryo is then allowed to develop to term, and the genome of the resulting offspring is analyzed for the presence of the transgenic material.

[0056] Southern blot analysis, PCR, or other such techniques for analyzing genomic DNA are used to detect the presence of unique nucleic acid fragments present in the transgenic animals but not in the non-transgenic animals. Selective breeding of transgenic offspring makes it possible to achieve homozygosity for the transgene.

[0057] When the long CDR3 heavy chain transgene is randomly inserted into the genome, it is also preferable to disable the endogenous heavy chain locus to limit the repertoire of heavy chain options in the animal (e.g., mouse). The endogenous heavy chain locus can be engineered to be inactive by standard knockout techniques, for example, by deleting all or part of the endogenous heavy chain V, D, J, and C regions so that they are non-functional.

[0058] 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, cows, and chickens. Techniques for generating transgenic animals of each of these species are described in the art. For example, the preparation of transgenic rats is described in Tesson, L. et al. (2005) Transgenic Res. 14:531-546 and includes techniques such as DNA microinjection, lentiviral vector-mediated DNA transfer into early embryos, and sperm-mediated gene transfer. Methods for transgenesis in rats are also described in Mullin, L. et al. (2002) Methods Mol. 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. Theroscler. 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. N.Y. Acad. Sci. 1049:161-171.

[0059] Alternative transgenesis techniques to pronuclear microinjection in pigs include adenovirus-mediated transfer of DNA into pig sperm (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 production of transgenic goats has been described, for example, 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 has been described, for example, in Behabodi, E. et al. (2004) Transgenic Res. 11:215-224. The preparation of transgenic sheep is described, for example, in Ward, K. A. and Brown, B. W. (1998) Reprod. Fertil. Dev. 10:659-665. The preparation of transgenic cattle is described, for example, in Donovan, D. M. et al. (2005) Transgenic Res. 14:563-567. Gene transfection of donor cells for nuclear transfer of bovine embryos is described, for example, in Lee, S. L. et al. (2005) Mol. Reprod. Dev. 72:191-200. The preparation of transgenic livestock is also reviewed 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, Lilico, SG et al. (2005) Drug Discov. Today 10:191-196, and Ishii, Y. et al. (2004) Dev. Dyn. 229:630-642.

[0060] Animals (e.g., mice) of the present disclosure carrying a long CDR3 heavy chain transgene construct can be cross-bred with animals (e.g., mice) carrying an immunoglobulin light chain transgene, thereby producing animals (e.g., mice) that express antibodies comprising a light chain paired with a heavy chain that includes a long CDR3 region. Immunoglobulin light chain transgenic animals (e.g., mice) are well established in the art.

[0061] IV. Use of Transgenic Animals The transgenic animals of the present disclosure are useful for generating antibodies against a wide variety of antigens of interest. For animals carrying only a long CDR3 heavy chain transgene and an endogenous light chain locus, the animal produces chimeric light / heavy chain antibodies that can be reverse engineered to pair a long CDR3 heavy chain with a light chain of the same species, if desired. Alternatively, for animals (e.g., mice) carrying both a long CDR3 heavy chain Ig transgene (e.g., human) and a light chain Ig transgene (e.g., human), complete xenogenous antibodies (e.g., fully human antibodies) can be prepared in the host transgenic animal. For animals carrying chimeric Ig transgenes (e.g., human variable regions added to mouse constant regions of the heavy and / or light chain), combinations of humanized loci can be functionally paired with other humanized loci or with their wild-type mouse counterparts.

[0062] Thus, in another aspect, the present disclosure relates to a method for generating an antibody against an antigen of interest, the method comprising administering the 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 such that an antibody that binds to the antigen of interest is generated in the mouse. In one embodiment, the animal is a transgenic mouse carrying both a human Ig long CDR3 heavy chain transgene and a human Ig light chain transgene, and the antigen is administered to the mouse such that a human or human-mouse chimeric antibody that binds to the antigen of interest is generated in the mouse. In one embodiment, the antigen is a GPCR or ion channel protein. In one embodiment, the method may further comprise isolating the antibody of interest from the host animal (e.g., mouse) and determining the heavy chain CDR3 sequence of the antibody.

[0063] The transgenic animals can be immunized with the antigen(s) of interest using standard methodologies known in the art, and antibodies produced in the animals can also be isolated and characterized using standard, established methods. Polyclonal antibodies can be isolated directly from the host animal, and monoclonal antibodies can be prepared by standard methods such as hybridoma technology. Procedures for producing monoclonal antibodies using hybridomas are well established in the art (see, e.g., U.S. Pat. No. 4,977,081, PCT International Publication No. WO 97 / 16537, and European Patent No. 491057(B1), the disclosures of which are incorporated herein by reference). Alternatively, in vitro production of monoclonal antibodies from cloned cDNA molecules is also established in the art (see, e.g., 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 antibodies can be isolated and cloned into expression vectors by standard molecular biology techniques. Further recombinant manipulation of cloned Ig cDNAs is also possible and well established in the art.

[0064] V. Definition As used herein, the term "DD fusion segment" is intended to refer to the direct linkage 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 that has been observed in nature, whose two constituent D segments result from a V(DD)J recombination event. Such naturally occurring DD fusion pairs have been described in the art, for example, 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 that has not been observed in nature, resulting from a V(DD)J recombination event. Regardless of whether the DD fusion pair is "naturally occurring" (i.e., observed in nature) or "synthetic" (i.e., not observed in nature), the DD fusion segment(s) used in the transgenes of the present disclosure can be engineered ex vivo by genetically linking the sequences of the two D segments by standard methods.

[0065] As used herein, the terms "long CDR3," "long H CDR3," "long CDR H3," or "long HCDR3" refer to a heavy chain CDR3 region that is longer than the typical or average length of a human heavy chain CDR3 region, e.g., typically longer than 8 to 16 amino acids in length. A transgene of the present disclosure generates heavy chain variable regions having CDR3s of various lengths after VDJ recombination, and is considered to be a transgene encoding a long CDR3 when a significant portion of the generated HCDR3s, e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% or more, is longer than the typical or average length of HCDR3s.

[0066] As used herein, the term "operatively linked" is intended to describe the configuration of a nucleic acid sequence that is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to the linking of two protein-coding regions, operably linked means that the nucleic acid sequences being linked are contiguous and in reading frame. With respect to splice donor / acceptor and RSS sequences, operably linked means that the sequences are capable of achieving their functional purpose.

[0067] As used herein, the term "transgene" refers to a gene that is introduced as an exogenous source into a site within the host genome (e.g., the mouse heavy chain Ig locus).

[0068] As used herein, the term "transgene construct" refers to a nucleic acid preparation suitable for introduction into the genome of a host animal.

[0069] As used herein, the term "transgenic mouse" refers to a mouse containing cells that carry a transgene, as defined herein. The transgene can be present in all or some of the cells of the mouse.

[0070] As used herein, the term "unrearranged" with respect to an immunoglobulin V segment refers to an immunoglobulin V segment in its germline configuration, where the V segment has not recombined so that it is immediately adjacent to a D or J segment.

[0071] 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. [Example]

[0072] Example 1: Preparation of human long CDR3 heavy chain constructs This example describes the preparation of a human immunoglobulin heavy chain transgene construct, in which the CDR3 domains of the constructs are, on average, larger (longer) than those found in normal, naive human B-cell heavy chain CDR3s. The rationale for this approach is that certain antigens may have functionally important epitopes that are rare or unavailable among normal long heavy chain CDR3 domain antibodies. The likelihood that the antibody will interact with such epitopes is increased by the use of a set of CDR3 domains that are longer (larger) than average.

[0073] Conceptual Framework It has been reported in the art that long CDR3s can be difficult membrane targets. In particular, GPCRs, ion channels, and other membrane targets with limited cleft or surface accessibility can benefit from long CDR3 mAbs to better access functional sites (see, for example, 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). Camel and bovine antibodies with long CDR3 domains have been utilized 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).

[0074] Construct design and transgene preparation Using a mechanism for CDR3 elongation, transgenes were designed to bias expression toward longer domains. First, the longest VH and D segments were identified, and transgenes were designed to selectively use some of the longer VH and D segments. Additionally, DD fusions were included that use some of the shorter D segments to retain some of the naturally occurring D amino acid sequence while also maintaining a longer CDR3.

[0075] The selection of VH segments was based on length and specificity: VH segments between 98 and 101 amino acids in length were selected for potential inclusion in the constructs.

[0076] Naturally occurring long D segments used in the wild-type configuration were also selected for possible inclusion in the construct, examples of which include 2-02, 2-08, 2-15, 2-21, 3-03, 3-09, 3-10, 3-16, and 3-22.

[0077] Regarding DD fusions, the natural formation of DD fusions during recombination can be explained by the existence of so-called "cryptic nonamers," which are nonamer sequences (usually part of the RSS motif that enables VDJ recombination) that deviate from their normal context but can still support recombination (Safonova and Pevzner (2020) Genome Res. 30:1547-1558). Some D segments have a higher probability of noncanonical nonamers in intervals that 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. Empirically, the occurrence of DD fusions largely follows their germline order (5'D fused to 3'D), and as a result, 3'D segments appear to be less commonly fused to 5' segments.

[0078] Sequences of exemplary native D segments, synthetic DD fusions, and naturally occurring DD fusions are shown in Figure 1. In total, the 27 different D or DD components shown in Figure 1 contain 47 different ORFs, which are aligned in Figure 2, where F1, F2, and F3 correspond to different reading frames. These illustrate the overall structure and sequence of elements that may be included in a transgene. However, it should also be noted that recombined D segments, as part of a VDJ recombination sequence, can be found in reverse orientation (see, e.g., Meek et al. (1989) J Exp Med. 170:39-57). Thus, for purposes of this disclosure, all D and DD segments included herein are intended to be encompassed in both their forward and reverse orientations.

[0079] The strategy for placing the various D and DD fusions in the constructs was as follows (in order of priority): (i) if possible, place the DD fusion at a location where one of the DD pairs normally resides within the D domain; (ii) if its own locus is not possible, attempt 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 the remaining slots, if possible. D segments and DD fusions (and their associated nucleotide and amino acid sequences) were genomically placed in the transgene constructs at the endogenous D locations shown in Figure 3. The last two columns of Figure 3 compare the endogenous D segment expression rank (IMGT database, https: / / www.imgt.org / genefrequency / query) with the expression levels of the DD fusion pairs as reported in Safonova and Pevzner (2019) Front. Immunol. 10:987. Although there is some correlation between the two, the primary objective of placing the DD fusion cassettes in their "native" locations means that the rank order cannot be consistent for both parameters.

[0080] A schematic diagram of a representative long CDR3 construct is shown in FIG. 4, and a representative nucleotide sequence of the construct is shown in SEQ ID NO:1.

[0081] This construct contains, in 5' to 3' order, the following naturally occurring VH segments: 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 to 101 amino acids in length and uses the naturally occurring RSS and octamer site.

[0082] This construct also contains three natural DH sequences (3-3, 2-8, and 3-16), two new 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, These include 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. The long D regions are 9–20 amino acids in length, and they retain their native positions whenever possible, with the RSS sites 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.

[0083] The DD fusion segments inserted into the constructs used the DNA sequences shown in Figure 1. These sequences were incorporated into and substituted for the native D segment coding sequence, the amino acid sequence of which is shown in Figure 2. To accomplish this substitution, the coding sequence was placed directly between the adjacent RSS sites on either side of the CDS of the segment. No changes were made to the RSS sequences.

[0084] LoxP flanks the insert at the 5' end. Additionally, an ADAM / IGHD GRS1 site at the 5' end and a J-Mu GRS site at the 3' end are present to facilitate recombination of the donor transgene and mouse host genome. Successful GRS donor delivery for knock-in has been validated to the point that this is the preferred mechanism for site-specific donor transgene delivery. The PGK-Puro cassette is flanked by FRT sites, which allow for removal of the cassette after GRS donor gene delivery.

[0085] The segments from the end of the "Long DH" to the J segments (J1-J6) as shown in Figure 4 are in the germline configuration and are unaltered with respect to the human GenBank reference sequence, except for the final DD fusion (2-8 / 1-1), which is located in the region of the J sequence where the endogenous 7-27D allele normally resides.

[0086] As shown in Figures 4 and 5, a long CDR3 bacterial artificial chromosome (BAC) transgene donor is used for CRISPR / Cas-mediated one-step deletion / delivery of the transgene via the GRS sequence. Figure 6 provides a graphical representation of the GRS knock-in strategy and the resulting knock-in alleles when the BAC donor is used in concert with mouse Ig-specific CRISPR / CAS reagents in mouse ES cells capable of mediating homologous recombination events. Mice containing the long CDR3 heavy chain transgene can be bred to homozygosity. Mice can be cross-bred with light chain transgenic mice to generate HC / LC transgenic mice expressing the long CDR3 transgene repertoire.

[0087] Sequence Listing Overview [Table 1-1] [Table 1-2] [Table 1-3] Table 1-4 Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 Table 1-22 Table 1-23 Table 1-24 Table 1-25 Table 1-26 Table 1-27 Table 1-28 Table 1-29 Table 1-30 Table 1-31 Table 1-32 Table 1-33 Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 2-6

[0088]

Claims

1. 1. A transgene construct encoding an immunoglobulin heavy chain variable region, comprising: (a) a plurality of human unrearranged immunoglobulin heavy chain variable segments (VH), each VH being 98 to 101 amino acids in length; and (b) a plurality of human DD fusion segments operably linked thereto; (c) a transgene construct comprising a plurality of human J segments.

2. 2. The transgene construct of claim 1, wherein said plurality of human DD fusion segments comprises at least one naturally occurring human DD fusion pair.

3. 2. The transgene construct of claim 1, wherein said plurality of human DD fusion segments comprises at least one synthetic human DD fusion pair.

4. 2. The transgene 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. 2. The transgene construct of claim 1, encoding at least 20 VH regions.

6. 2. The transgene construct of claim 1, encoding a human VH region in the 5' to 3' direction as follows: 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. 2. The transgene construct of claim 1, which encodes a synthetic 1-1 / 2-8 DD fusion or a synthetic 2-8 / 1-1 DD fusion.

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

9. 2. The transgene construct of claim 1, encoding at least one naturally occurring D-D 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. 2. The transgene construct of claim 1, encoding the naturally occurring D-D 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 transgene construct of claim 1 , further encoding at least one native D segment.

12. The following: 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 12. The transgene construct of claim 11, encoding a D segment and a D-D fusion in the 5' to 3' direction as follows: -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. A transgene construct according to any one of claims 1 to 12, encoding J1 to J6 segments.

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

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

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

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

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

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

1.

20. 20. The transgene construct of any one of claims 1 to 19 carried on a bacterial artificial chromosome (BAC).

21. A transgenic mouse comprising a transgene construct according to any one of claims 1 to 20.

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

23. 23. A method for producing antibodies against an antigen of interest, said method comprising administering said antigen of interest to the transgenic mouse of claim 22 so as to produce antibodies that bind to said antigen of interest.

24. 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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