Human immunoglobulin binary light chain transgene constructs and uses thereof

Abstract

Provided is a human immunoglobulin light chain transgene construct that encodes two different rearranged light chain VJ regions, arranged in such a way that one of the two alternative light chains is expressed from the construct upon recombination in B cells. Also provided is a transgenic animal containing the transgene. Thus, the binary light chain transgene allows the expression of two alternative fixed light chains in the animal. Also provided is a method for using the transgenic animal.

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 / 439,795, 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 December 13, 2023, is named ZL8016-WO-PCT_SL.xml and is 43,080 bytes in size. [Background technology]

[0003] Bispecific antibodies (bsAbs) combine two different antigen-binding sites in a single molecule. bsAbs may be advantageous over monospecific antibodies due to increased specificity in targeting and different mechanisms of action, potentially resulting in greater clinical efficacy (e.g., as reviewed in Sedykh et al. (2018) Drug Design Devel. Ther. 12:195-208; Labrijn et al. (2019) Nat. Rev. Drug Discov. 18:585-608). However, the production of bsAbs remains challenging due to the need for precise pairing between two different heavy and light chains and the associated manufacturability issues. One approach that has been attempted to reduce the complexity of bsAb production and manufacturing is the use of a common light chain (e.g., as shown schematically in Figure 1) combined with two different heavy chains. This approach takes advantage of the well-established fact that for many antibodies, the heavy chain is the primary driver of affinity and specificity. Thus, the use of a common light chain avoids the risk of inappropriate heavy / light chain pairing and limits production to only three peptide chains, while still retaining the desired antigen-binding specificity for the bsAb.

[0004] Approaches for preparing common light chain transgenes and the use of common light chains in bsAbs have been described (e.g., Merchant et al. (1998) Nat. 16:677-681; J. Biotech. Jackman et al. (2010) J. Biol. Chem. 285:20850-20859; DeNardis et al. (2017) J. Biol. Chem. 292:14706-14717; Sharkey et al. (2017) MABS 9:257-268; Van See Blarcom et al. (2018) MABS 10:256-268, WO 2011 / 097603, WO 2013 / 134263, WO 2015 / 153765, WO 2020 / 132557, WO 2020 / 205504).

[0005] Although some progress has been made, there is a need for additional approaches and compositions for designing, preparing and using common light chain transgenes, particularly for use in bispecific antibodies. Summary of the Invention

[0006] The present disclosure provides a human immunoglobulin light chain transgene construct, referred to herein as a binary fixed light chain construct, encoding two different rearranged VJ regions positioned in opposite orientations within the construct. The construct utilizes recombination signal sequences (RSSs) and RAG-mediated gene activation to silence the locus prior to recombination and result in functional expression of one VJ region after recombination. More specifically, upon RAG-mediated recombination in B cells of an animal (e.g., a mouse) carrying the transgene, one or the other of the two alternative VJ regions is expressed. Thus, a binary fixed light chain transgene results in stochastic selection between two different pre-rearranged light chain VJ regions. The resulting immunoglobulin repertoire in the transgenic animal contains both of these fixed light chains, thereby providing the animal with two different common light chain options. This is advantageous in increasing the likelihood of successful antibody production against an antigen of interest in the animal.

[0007] Thus, in one aspect, the present disclosure provides a transgene construct comprising: (a) a first immunoglobulin light chain variable cassette (VL1) and a second immunoglobulin light chain variable cassette (VL2), each of VL1 and VL2 comprising a promoter, a light chain V region, a light chain J region, and a splice donor site; (b) a stop cassette (SC) containing a splice acceptor site and a polyadenylation signal; (c) a first recombination signal sequence (RSS) 12-mer (RSS1), a second recombination signal sequence 12-mer (RSS2), and an RSS 23-mer (RSS3); The transgene construct comprises a 5' to 3' Includes VL1-RSS1-SC-RSS2-VL2-RSS3, VL2 refers to the transgene construct in the antisense orientation relative to VL1. When the transgene construct is carried by a B cell, before RAG-mediated recombination, VL1 and VL2 are inactive, and after RAG-mediated recombination, either VL1 or VL2 is active.

[0008] In embodiments, the light chain V region and light chain J region are human kappa sequences. Non-limiting examples of suitable V regions and J regions are disclosed herein. In one embodiment, VL1 or VL2 comprises a Vk 1-39 region. In one embodiment, VL1 or VL2 comprises a Jk JK2 region. In one embodiment, VL1 or VL2 comprises a Vk 1-39 region and a Jk JK2 region. In one embodiment, VL1 or VL2 comprises a Vk 4-1 region. In one embodiment, VL1 or VL2 comprises a Jk JK4 region. In one embodiment, VL1 or VL2 comprises a Vk 4-1 region and a Jk JK4 region. In one embodiment, VL1 comprises a Vk 1-39 region and VL2 comprises a Vk 4-1 region. In one embodiment, VL1 comprises the Vk 1-39 region and the Jk JK2 region, and VL2 comprises the Vk 4-1 region and the Jk JK4 region. In one embodiment, VL1 comprises the Vk 4-1 region and VL2 comprises the Vk 1-39 region. In one embodiment, VL1 comprises the Vk 4-1 region and the Jk JK4 region, and VL2 comprises the Vk 1-39 region and the Jk JK2 region.

[0009] In embodiments, the light chain V region and light chain J region are human lambda sequences. Non-limiting examples of suitable lambda V regions and J regions are disclosed herein. In one embodiment, VL1 or VL2 comprises a Vλ2-14 region. In one embodiment, VL1 or VL2 comprises a JλJL2 region. In one embodiment, VL1 or VL2 comprises a Vλ2-14 region and a JλJL2 region. In one embodiment, VL1 or VL2 comprises a Vλ1-40 region. In one embodiment, VL1 or VL2 comprises a JλJL1 region. In one embodiment, VL1 or VL2 comprises a Vλ1-40 region and a JλJL1 region. In one embodiment, VL1 comprises a Vλ2-14 region and VL2 comprises a Vλ1-40 region. In one embodiment, VL1 comprises a Vλ2-14 region and a JλJL2 region, and VL2 comprises a Vλ1-40 region and a JλJL1 region. In one embodiment, VL1 comprises a Vλ1-40 region and VL2 comprises a Vλ2-14 region. In one embodiment, VL1 comprises a Vλ1-40 region and a JλJL1 region, and VL2 comprises a Vλ2-14 region and a JλJL2 region.

[0010] In embodiments, the transgene construct further comprises a light chain constant region downstream of RSS3. In one embodiment, the light chain constant region is a human kappa constant region. In one embodiment, the transgene construct further comprises an enhancer downstream of RSS3 and upstream of the light chain constant region. In one embodiment, the enhancer comprises an intronic human kappa enhancer (hEKi).

[0011] In one embodiment, the VL1 or VL2 of the transgene construct comprises a CDR3 comprising the sequence set forth in SEQ ID NO: 1. In one embodiment, the VL1 or VL2 of the transgene construct comprises a CDR3 comprising the sequence set forth in SEQ ID NO: 2. In one embodiment, the VL1 or VL2 of the transgene construct comprises the sequence set forth in SEQ ID NO: 3. In one embodiment, the VL1 or VL2 of the transgene construct comprises the sequence set forth in SEQ ID NO: 4. In one embodiment, the transgene construct comprises the sequence set forth in SEQ ID NO: 5.

[0012] In one embodiment, the VL1 or VL2 of the transgene construct comprises a CDR3 comprising the sequence set forth in SEQ ID NO: 6. In one embodiment, the VL1 or VL2 of the transgene construct comprises a CDR3 comprising the sequence set forth in SEQ ID NO: 7.

[0013] 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 heavy chain, such that the mouse expresses an antibody comprising a heavy chain paired with a light chain comprising either a VL1 or VL2 light chain V region.

[0014] 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 whether the antibody uses a VL1 or VL2 light chain V region. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram of the bispecific antibody approach, in which two different heavy chains and one fixed light chain are used to create two different binding specificities.

[0016] [Figure 2] Schematic diagram of binary light chain transgene constructs showing an inactive state before RAG activation and alternating activation of VJK1 or VJK2 after RAG activation. PR = promoter; SD = splice donor; SA = splice acceptor; pA ​​= polyA / stop cassette; hEKi = intronic human kappa enhancer; SA / pA = cassette derived from the human IG lambda C2 locus and containing two consensus pA signals.

[0017] [Figure 3] FIG. 1 is a schematic diagram showing how the binary fixed light chain model increases the hit frequency compared to the single fixed light chain model.

[0018] [Figure 4] FIG. 1 is a schematic diagram showing the architectural elements of a representative binary locked light chain construct of the present disclosure.

[0019] [Figure 5] FIG. 1 is a schematic diagram showing the structure of a representative binary locked light chain construct of the present disclosure.

[0020] [Figure 6]Schematic diagram showing insertion of a representative binary fixed light chain construct into the mouse kappa locus by recombination. LP = landing pad; FLC = fixed (AKA binary) light chain; neoR and puroR refer to neomycin and puromycin resistance cassettes, respectively.

[0021] [Figure 7] FIG. 1 is a schematic diagram of a plasmid map of a representative transgene construct of the present disclosure.

[0022] [Figure 8] 1 shows the results of PCR amplicon and DNA sequencing analysis of recombinant transgene alleles from transgenic mice.

[0023] [Figure 9] 1 shows the results of RT-PCR analysis of splenocyte RNA from transgenic mice, demonstrating expression of correctly spliced ​​transgene mRNA.

[0024] [Figure 10A] Figure 10 shows the results of ELISA assays for transgenic mice. Figure 10A is a schematic diagram of the antibody format to be detected, carrying fixed human κLC encoded by the transgene. Figure 10B shows IgK levels in naive (non-immunized) mice. Figure 10C shows the ELISA results for mice immunized with a COVID-19 spike protein antigen preparation. [Figure 10B] Figure 10 shows the results of ELISA assays for transgenic mice. Figure 10A is a schematic diagram of the antibody format to be detected, carrying fixed human κLC encoded by the transgene. Figure 10B shows IgK levels in naive (non-immunized) mice. Figure 10C shows the ELISA results for mice immunized with a COVID-19 spike protein antigen preparation. [Figure 10C]Figure 10 shows the results of ELISA assays for transgenic mice. Figure 10A is a schematic diagram of the antibody format to be detected, carrying fixed human κLC encoded by the transgene. Figure 10B shows IgK levels in naive (non-immunized) mice. Figure 10C shows the ELISA results for mice immunized with a COVID-19 spike protein antigen preparation.

[0025] [Figure 11] Schematic diagram of the binary lambda-locked light chain (λFLC) transgene construct, showing the inactive state of both of its light chain variable regions before RAG-mediated recombination and the activation of either λFLC1 or λFLC2 after RAG-mediated recombination. DETAILED DESCRIPTION OF THE INVENTION

[0026] The binary locked light chain transgene construct of the present disclosure encodes two distinct VJ regions in opposite orientations, as shown schematically in Figure 2. The construct utilizes a recombination signal sequence (RSS) and the endogenous Recombination Activation Gene (RAG) gene activation system to silence expression of both VJ regions prior to recombination and to generate functional expression of one or the other VJ region after recombination. This design helps ensure that pre-rearranged human light chains are inactive prior to B cell recombination and ensures that normal B cell development is not hindered by having a transcriptionally active, functional light chain too early in development. Because typical B cell differentiation proceeds by rearranging heavy chains before light chains (e.g., Yancopoulos and Alt (1986) Annu Rev Immunol. 4:339-68), every attempt was made to preserve this natural biology inherent in B cell development. The overall idea behind this approach is therefore to provide the transgenic animal with one of two alternative choices regarding the human light chain it expresses, as shown schematically in Figure 3. Thus, for antigens with specific HC, LC pairs are eliminated or are not antigen-reactive, and alternative choices are available, increasing the chances of obtaining the antibody of interest.

[0027] Various aspects of the present disclosure are described in further detail below. Unless otherwise defined, all technical, labeling, 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.

[0028] I. Structure design The design and construction of the binary locked light chain transgene is described in detail in Example 1. As shown in Figures 2, 4, and 5, the construct contains two VJ region cassettes in opposite orientations (i.e., antisense) with a STOP cassette between them. The construct also contains an intervening RSS sequence to facilitate recombination.

[0029] Thus, in one embodiment, the transgene construct comprises: (a) a first immunoglobulin light chain variable cassette (VL1) and a second immunoglobulin light chain variable cassette (VL2), each of VL1 and VL2 comprising a promoter, a light chain V region, a light chain J region, and a splice donor site; (b) a termination cassette (SC) containing a splice acceptor site and a polyadenylation signal; (c) a first recombination signal sequence (RSS) 12-mer (RSS1), a second recombination signal sequence 12-mer (RSS2), and an RSS 23-mer (RSS3); The transgene construct comprises a 5' to 3' Includes VL1-RSS1-SC-RSS2-VL2-RSS3, VL2 is in antisense (ie, reverse or opposite) orientation to VL1.

[0030] In this transgene configuration, neither VL cassette is capable of producing a functional light chain transcript prior to RAG-mediated recombination because the artificial splice / pA stop signal prevents proper splicing and expression of the upstream VL1 and the downstream VL2 is in the incorrect antisense orientation. Furthermore, the RSSs are out of their normal context in that they do not link coding sequences (the resulting non-homologous end joining occurs within and should not be affected by the VL and kappa constant region introns).

[0031] As further illustrated in Figures 2, 4, and 5, after RAG-mediated recombination, either: (i) VL1 becomes active through excision of the SA / pA stop cassette and VL2 cassette and operably linking the VL1 promoter and coding sequence with the downstream enhancer and constant region sequences, or (ii) VL2 becomes active through inversion of the VL2 cassette, resulting in operably linking the VL2 promoter and coding sequence with the downstream enhancer and constant region sequences.

[0032] Thus, when the transgene construct of the present disclosure is carried by a B cell, before RAG-mediated recombination, VL1 and VL2 are inactive, and after RAG-mediated recombination, either VL1 or VL2 is active.

[0033] In one embodiment, the light chain V and J regions used in the VL1 and VL2 cassettes are kappa regions (e.g., human kappa regions). Non-limiting examples of human κ light chain constructs are described in detail in Example 1. In another embodiment, the light chain V and J regions used in the VL1 and VL2 cassettes are lambda regions (e.g., human lambda regions). Non-limiting examples of human λ light chain constructs are described in detail in Example 2. In another embodiment, one of the VL1 / VL2 cassettes uses a kappa region and the other VL1 / VL2 cassette uses a lambda region. For example, the human κVL1 or VL2 cassette described in Example 1 can be combined with the human λVL1 or VL2 cassette described in Example 2 to create a binary locked light chain construct comprising a κ cassette and a λ cassette.

[0034] The selection of light chain V and J regions to be used in VL1 and VL2 can be based on one or more of several possible criteria. For example, if a particular V / J region is known to exhibit a tendency to bind to an antigen of interest, such region can be selected for use with that intended antigen. Alternatively, for use with a wide range of antigens, V regions can be selected based on (i) frequency of expression in the normal human population, (ii) ability to pair with diverse human heavy chain families, if known, and / or (iii) ability to pair with known J domains. Information regarding V region usage and preferred VK / VH pairings is available in the art and can be used in construct design. For example, a VK region can be selected based on preferred pairing with a VH region. Non-limiting examples of such preferred pairings are described in DeKosky et al. (2015) Nat. Med. 21:86-91, the entire contents of which are expressly incorporated herein by reference.

[0035] In one embodiment, the V region used in VL1 and VL2 is Vk region 1-5, 1-6, 1-8, 1D-8, 1-9, 1-12, 1D-12, 1-13, 1D-13, 1-16, 1D-16, 1-17, 1D-17, 1-27, 1-33, 1D-33, 1-37, 1D-37, 1-39, 1D-39, 1D-42, 1D-43, and a human Vk region independently selected from the group consisting of 1-NL1, 2-24, 2-28, 2D-28, 2-29, 2D-29, 2-30, 2D-30, 2-40, 2D-40, 3-7, 3D-7, 3-11, 3D-11, 3-15, 3D-15, 3-20, 3D-20, 4-1, 5-2, 6-21, 6D-21, and 6D-41.

[0036] In one embodiment, the V regions used in VL1 and VL2 are human Vk regions independently selected from the group consisting of Vk regions 1-5, 1-39, 3-11, 3-15, 3-20, 3-28, and 4-1.

[0037] In one embodiment, the V regions used in VL1 and VL2 are human Vk regions independently selected from the group consisting of Vk regions 1-39, 3-20, and 4-1. In one embodiment, VL1 or VL2 comprises a Vk 1-39 region. In one embodiment, VL1 or VL2 comprises a Vk 4-1 region. In one embodiment, VL1 comprises a Vk 1-39 region and VL2 comprises a Vk 4-1 region. In one embodiment, VL1 comprises a Vk 4-1 region and VL2 comprises a Vk 1-39 region.

[0038] In one embodiment, the J region used in VL1 and VL2 is a human Jk region (e.g., when a Vk region is used). In one embodiment, the J region used in VL1 and VL2 is a human Jλ region (e.g., when a Vλ region is used). In one embodiment, the J region used is a human Jk region selected from the group consisting of Jk, JK1, JK2, JK3, JK4, and JK5. Information available in the art regarding commonly observed IGKV-IGKJ pairings can be used in designing the VL1 and VL2 regions. For example, a Jk region can be selected based on preferred pairings with a Vk region. Non-limiting examples of such preferred pairings are described in Collins et al. (2008) Immunogenetics 60:669-676, the entire contents of which are expressly incorporated herein by reference. In one embodiment, VL1 or VL2 comprises a Jk JK2 region. In one embodiment, VL1 or VL2 comprises a Vk 1-39 region and a Jk JK2 region. In one embodiment, VL1 or VL2 comprises a Jk JK4 region. In one embodiment, VL1 or VL2 comprises a Vk 4-1 region and a Jk JK4 region. In one embodiment, VL1 comprises a Vk 1-39 region and a Jk JK2 region, and VL2 comprises a Vk 4-1 region and a Jk JK4 region. In one embodiment, VL1 comprises a Vk 4-1 region and a Jk JK4 region, and VL2 comprises a Vk 1-39 region and a Jk JK2 region.

[0039] In one embodiment, the V regions used in VL1 and VL2 are human Vλ regions independently selected from the group consisting of, for example, Vλ regions 2-14, 3-19, 3-21, 3-1, 1-51, and 1-40.

[0040] In one embodiment, the V regions used in VL1 and VL2 are human Vλ regions independently selected from the group consisting of Vλ regions 2-14, 3-19, and 1-40. In one embodiment, VL1 or VL2 comprises a Vλ2-14 region. In one embodiment, VL1 or VL2 comprises a Vλ1-40 region. In one embodiment, VL1 comprises a Vλ2-14 region and VL2 comprises a Vλ1-40 region. In one embodiment, VL1 comprises a Vλ1-40 region and VL2 comprises a Vλ2-14 region.

[0041] In one embodiment, the J regions used in VL1 and VL2 are human Jλ regions (e.g., when Vλ regions are used). In one embodiment, the J regions used are human JλJL1 or JL2. In one embodiment, VL1 or VL2 comprises a JλJL2 region. In one embodiment, VL1 or VL2 comprises a Vλ2-14 region and a JλJL2 region. In one embodiment, VL1 or VL2 comprises a JλJL1 region. In one embodiment, VL1 or VL2 comprises a Vλ1-40 region and a JλJL1 region. In one embodiment, VL1 comprises a Vλ2-14 region and a JλJL2 region, and VL2 comprises a Vλ1-40 region and a JλJL1 region. In one embodiment, VL1 comprises a Vλ1-40 region and a JλJL1 region, and VL2 comprises a Vλ2-14 region and a JλJL2 region.

[0042] Each VL1 and VL2 cassette also comprises a promoter operably linked to the VJ coding sequence to drive expression of the VJ coding region upon activation of the cassette. Suitable promoters are well established in the art and include endogenous mouse or human immunoglobulin promoters as well as heterologous promoters. In one embodiment, the VL1 and VL2 cassettes use the endogenous promoter of the V region incorporated into the cassette.

[0043] Each VL1 and VL2 cassette also contains an operably linked splice donor site, while the intervening stop cassette (SC) contains an operably linked splice acceptor site. Standard splice donor and acceptor site sequences well established in the art are used. The SC also contains polyadenylation signal(s), for which standard sequences are used and well established in the art. In one embodiment, the stop cassette is derived from the human Ig lambda C2 locus and contains two consensus polyadenylation signals.

[0044] To promote RAG-mediated recombination, a recombination signal sequence (RSS) is included in the construct. Two operably linked RSS 12mer sequences (RSS1 and RSS2) are located upstream and downstream (in opposite orientation) of the SC, respectively. An operably linked RSS 23mer sequence (RSS3) is located downstream of VL2. Standard RSS 12mer and 23mer sequences well established in the art are used. In one embodiment, the two RSS 12mer sequences are the sequences of human Vκ1-39. * 01 allele and are used in two opposite orientations. In one embodiment, the RSS 23mer is derived from the human IGKJ1 * It is derived from the 01 allele.

[0045] In embodiments, the transgene construct further comprises a light chain constant region downstream of RSS3. In one embodiment, the light chain constant region is a human Ig kappa constant region. In one embodiment, the light chain constant region is a human Ig lambda constant region. In one embodiment, the light chain constant region is a mouse kappa constant region. In one embodiment, the light chain constant region is a mouse lambda constant region.

[0046] In one embodiment, the transgene construct further comprises an enhancer downstream of RSS3 and upstream of the light chain constant region. Suitable enhancers are well established in the art and include endogenous mouse or human immunoglobulin enhancers, as well as heterologous enhancers. In one embodiment, the enhancer comprises an intronic human kappa enhancer (hEKi).

[0047] The transgene construct may further include immunoglobulin locus genomic sequences upstream (5') of the VL1 and VL2 cassettes to act as a type of genetic "buffer" and to include minor or sensitive regulatory elements from the Ig locus. For example, in one embodiment, the VL1 cassette uses the Vκ1-39 variable region, and approximately 9.7 kb of 5' genomic sequence from Vκ1-39 is integrated upstream of the VL1 cassette. Similarly, in one embodiment, the VL2 cassette uses the Vκ4-1 variable region, and approximately 1.6 kb of 5' genomic sequence from Vκ4-1 is integrated upstream of the VL2 cassette.

[0048] The transgene construct may further comprise immunoglobulin locus genomic sequence upstream (5') of the constant region coding sequence. For example, in one embodiment, the construct incorporates the human Ig kappa constant region and contains approximately 2.8 kb of genomic DNA 5' to the Ck region coding sequence, but this region remains intact regardless of whether the VL1 or VL2 cassette is functionally activated.

[0049] 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). Approaches for codon optimization are well established in the art.

[0050] The transgene construct may further comprise sequences that allow for targeted insertion of the transgene into a specific locus, for example, the endogenous mouse light 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 recombination sequences that allow the transgene to be knocked into the endogenous mouse kappa locus, thereby deleting all of the endogenous mouse Vk, Jk, and Ck sequences. In one embodiment, the construct comprises an upstream loxP site and a downstream lox2272 site. Insertion of a transgene into the genome of a host via recombination is further described in Section III.

[0051] In one embodiment, VL1 or VL2 encodes the commonly observed CDR3 sequence of VK 1-39 / JK2, the amino acid sequence of which CDR3 is set forth in SEQ ID NO: 1. In one embodiment, VL1 or VL2 encodes the commonly observed CDR3 sequence of VK 4-1 / JK4, the amino acid sequence of which CDR3 is set forth in SEQ ID NO: 2. In one embodiment, VL1 or VL2 encodes the VK 1-39 / JK2 amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, VL1 or VL2 encodes the VK 4-1 / JK4 amino acid sequence set forth in SEQ ID NO: 4.

[0052] In one embodiment, the binary locked light chain construct comprises the nucleotide sequence shown in SEQ ID NO:5.

[0053] In one embodiment, VL1 or VL2 encodes the CDR3 set forth in SEQ ID NO: 6, which represents the consensus Vλ2-14 / JL2 CDR3 sequence. In one embodiment, VL1 or VL2 encodes the CDR3 set forth in SEQ ID NO: 7, which represents the consensus Vλ1-40 / JL1 CDR3 sequence.

[0054] 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). Nos. 5,874,299, and 5,770,429 (all to Lonberg and Kay), and 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 V-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.

[0055] A non-limiting example of a binary locked light chain vector of the present disclosure is shown schematically in FIG.

[0056] 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, for example, [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, the transgene can be prepared as an intact supercoiled plasmid by using a sequence-specific recombinase to effect recombination of the transgene insert with a compatible recombinase site already present in the mouse Igκ locus.

[0057] 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 a fixed light chain comprising the VJ region of a VL1 cassette or the VJ region of a VL2 cassette. 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 or into cells of a non-human animal (e.g., embryonic stem cells). In a preferred embodiment, the transgene construct is inserted into the genome of the host animal or host cell using knock-in technology to replace all or part of an endogenous light chain locus (e.g., an endogenous κ chain locus) with the transgene (e.g., a κ chain transgene).

[0058] For the knock-in approach, loxP-flanked sites are typically included in the construct, allowing site-specific recombination between a donor transgene and a host genome that has been modified with similar loxP sites to facilitate site- and orientation-specific recombination upon expression of Cre recombinase (so-called recombinase-mediated cassette exchange, or RMCE). Recombination is performed in embryonic stem cells (e.g., mouse embryonic stem cells). 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 and Turan et al. (2011) J. Mol. Biol. 407:193-221.

[0059] In a preferred embodiment, the transgene construct is inserted into the genome of a mouse whose endogenous light chain locus has been modified to delete all Vk, Jk, and Ck sequences, while simultaneously introducing loxP sites compatible with the donor transgene. In a preferred embodiment, the transgene construct is a κ light chain construct inserted by homologous recombination into an endogenous mouse κ locus modified to delete all mouse Vk, Jk, and Ck sequences. In a preferred embodiment, the endogenous κ locus is first engineered to delete Vk through CK (a deletion of approximately 3.5 million bp), while leaving a region containing compatible loxP and lox2272 sites (referred to herein as the "landing pad" site). The binary LC transgene construct is then introduced into the landing pad site of ES cells along with cre recombinase (referred to as recombinase-mediated cassette exchange, or RMCE), as shown schematically in FIG. 6.

[0060] In another embodiment, a kappa light chain transgene lacking the constant region is inserted into the endogenous kappa locus such that the Vk and Jk sequences are deleted but the Ck sequences remain intact and are operably linked to a functional variable region of the transgene, thereby producing a chimeric antibody in mice (which can be back-engineered to be fully human).

[0061] The standard method for preparing transgenic non-human animals, particularly transgenic mice, is to perform genetic modifications, such as knock-ins, in embryonic stem (ES) cells. These modified stem cells can then be used to generate chimeric mice via microinjection of preimplantation-stage mouse embryos, which are then bred to transmit the gene of interest or knock-in allele. At this point, the knock-in allele can be bred or cross-bred for lineage expansion and further study. Southern blot analysis, PCR, or other such techniques for analyzing genomic DNA can be used to detect the presence of unique nucleic acid fragments present in transgenic animals or ES cells but not in non-transgenic animals or ES cells. Selective breeding of transgenic offspring allows for the achievement of homozygosity for the transgene.

[0062] 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.

[0063] 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.

[0064] Animals (e.g., mice) of the present disclosure carrying a binary light chain transgene construct can be cross-bred with animals (e.g., mice) carrying an immunoglobulin heavy chain transgene, thereby producing animals (e.g., mice) that express antibodies comprising a heavy chain paired with a light chain comprising either a VL1 or VL2 light chain V region. Immunoglobulin heavy chain transgenic animals (e.g., mice) are well established in the art.

[0065] In one embodiment, a transgenic animal (e.g., a mouse) of the present disclosure is heterozygous for the binary light chain construct, and the other endogenous mouse light chain allele has been inactivated such that after recombination of the transgene, either VL1 or VL2 is expressed in all light chains of the transgenic cell. In another embodiment, a transgenic animal (e.g., a mouse) of the present disclosure is homozygous for the binary light chain construct, in which case both alleles of the binary LC construct can be expressed in the animal (e.g., a mouse) such that both VL1 and VL2 are utilized in the light chain repertoire.

[0066] 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 binary light chain transgene and an endogenous heavy chain locus, the animals produce chimeric light chain / heavy chain antibodies that can be reverse engineered to pair light and heavy chains of the same species, if desired. Alternatively, for animals (e.g., mice) carrying both a binary light chain Ig transgene (e.g., human) and a heavy chain Ig transgene (e.g., human, or human-mouse chimera), partial or fully xenogenic antibodies (e.g., fully human antibodies) can be prepared in the host transgenic animal.

[0067] 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 binary light chain transgene and a human Ig heavy chain transgene, and the antigen is administered to the mouse such that a human antibody that binds to the antigen of interest is generated in the mouse. In one embodiment, the method may further include isolating the antibody of interest from the host animal (e.g., mouse) and determining whether the antibody uses a VL1 or VL2 light chain V region.

[0068] 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 using standard methods such as hybridoma technology or single B-cell cloning. Procedures for producing monoclonal antibodies using hybridomas are well established in the art (see, e.g., U.S. Pat. No. 4,977,081, International Publication No. PCT 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). Techniques to aid in the identification or recovery of single B cells for cloning or cDNA capture are well established (for review, see, e.g., Pedrioli and Oxenius 2021) Trends Immunol. 42:1143-1158). B cell clones from immunized transgenic animals can be isolated, and cDNA encoding the 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.

[0069] In one embodiment, a fixed light chain encoded by a transgene construct of the present disclosure is identified as binding to a target of interest, and the fixed light chain is then incorporated into a bispecific antibody (bsAb) that binds to the target of interest. An exemplary bsAb approach incorporating the use of a fixed light chain with two different heavy chains is shown schematically in Figure 1.

[0070] V. Definition As used herein, a "common light chain" or "common immunoglobulin light chain" or "single light chain" refers to a light chain variable region that can pair with multiple heavy chain variable regions to produce antibodies that bind to different antigens. For example, both arms of a bispecific antibody may utilize the same light chain (i.e., the "common" light chain) and different heavy chains (which primarily determine the binding specificity of the arms).

[0071] 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.

[0072] As used herein, "promoter" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some embodiments, this sequence may be the core promoter sequence. In some embodiments, this sequence may also include an enhancer sequence and other regulatory element(s) required for expression of the gene product.

[0073] As used herein, the term "rearranged" with respect to an immunoglobulin V segment refers to a configuration in which the V segment is positioned immediately adjacent to a J segment so as to encode essentially a complete VL domain. Rearranged variable region loci can be identified by comparison with germline DNA.

[0074] 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 light chain Ig locus).

[0075] As used herein, the term "transgene construct" refers to a nucleic acid preparation suitable for introduction into the genome of a host animal.

[0076] 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.

[0077] 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]

[0078] Example 1: Preparation of a human kappa binary consensus light chain construct This example describes the preparation of transgene constructs for expressing one of two alternative "fixed" human κ light chains. The fixed nature of the light chain means that the Vκ and J segments to be expressed are already recombined in a manner that prevents the normal stochastic Vκ-J recombination that occurs naturally during early B cell development.

[0079] Conceptual Framework The intention behind the binary common light chain concept is to provide transgenic mice with one of two alternative choices for the human κLC they express. By doing so, this helps enable a common light chain (CLC) approach that can be used in bispecific antibody (bsAb) platforms. One of the more successful bsAb approaches utilizes a CLC shared between two unrelated heavy chains that, together with the CLC, encode two different antigen specificities while utilizing the same LC, as shown schematically in Figure 1. In this bispecific antibody format, Ab1 and Ab2 are derived from transgenic mice with normal, unrearranged human or mouse VH repertoires but with fixed light chains known to be HC promiscuous. This format is known to exhibit low immunogenicity, expression levels are similar to those of conventional antibodies, and bispecific antibody products have normal Fc-receptor interactions. Furthermore, this format does not suffer from some of the issues with HCs that plague other, more complex bispecific antibody schemes. The combination of LC pairings and the manipulations required to enforce proper pairing or selectively purify properly paired species (for a review of such schemes, see, e.g., Kontermann and Brinkmann (2015) Drug Discovery Today 20(7)).

[0080] An advantageous feature of the binary common light chain constructs described herein is that two different CLCs are provided for the mouse to "choose from," so that if a particular HC:LC pair is eliminated or not antigen-reactive, another option is available. The choice of κ light chain for selecting a fixed or common allele is typically based on (i) frequency of expression in the normal human population, (ii) ability to pair with diverse human HC families (if known), and (iii) ability to pair with known J domains.

[0081] Binary light chain construct design A representative schematic of a binary light chain transgene construct is shown in Figure 2. The binary transgene displays a recombination signal sequence (RSS) and a recombination activating gene (RAG) gene activation system with a pre-silenced locus, allowing selection for functional expression after recombination, resulting in stochastic selection between two different pre-rearranged kappa light chains. Neither cassette is active prior to RAG action. The artificial splice / pA termination signal prevents properly oriented upstream sequences from driving functional light chain expression. The RSSs are outside their normal context in that they do not bind coding sequences. Rather, they reside within non-coding introns so as not to interfere with coding sequences or RNA splicing behavior (thus, non-homologous end joining in this context should have no effect).

[0082] Following RAG-mediated recombination, either (i) V1-J1 becomes active through excision of the SA / pA termination cassette and V2-J2 cassette and operably linking the V1-J1 promoter and coding sequence with the downstream enhancer and constant region sequences, or (ii) V2-J2 becomes active through inversion of the V2-J2 cassette, resulting in operably linking the V2-J2 promoter and coding sequence with the downstream enhancer and constant region sequences.

[0083] As shown schematically in Figure 3, the use of a binary fixed light chain approach, which affords the ability to express two possible light chains, should result in an increased hit frequency, as having two light chain choices increases the odds of successful pairing with any given heavy chain and provides more binding solutions for the two antigens of interest. Ultimately, the mouse "decides" on the best light chain match.

[0084] Light chain V region selection The frequency of VK region expression in the human population was used as a starting point for light chain V region selection. The IMGT® database provides information on VK frequency, leading to the determination that VK 1-39 and 3-20 were most frequently used, but 4-1, 3-11, 3-15, 3-28, and 1-5 were also frequently observed. VH / VK pairings were also analyzed based on the data disclosed in DeKosky et al. (2015) Nat. Med. 21:86-91. This analysis revealed that the combination of VK 1-39 and 4-1 should allow for diverse VH pairings across most VH families.

[0085] Further BLAST analysis was performed to identify commonly used LCDR3s from VK 1-39+JK2 and VK 4-1+JK4. For VK 1-39+JK2, a common LCDR3 was identified as having the sequence CQQSYSTPYTF (SEQ ID NO: 1). For VK 4-1+JK4, a common LCDR3 was identified as having the sequence CQQYYSTPLTF (SEQ ID NO: 2). These most common LCDR3s were then used to prepare optimized alleles for VK 1-39+JK2 and VK 4-1+JK4, with the amino acid sequences shown in SEQ ID NOs: 3 and 4, respectively.

[0086] Further design of the light chain sequence may include altering somatic hypermutation (SHM) sites and optimizing codons for expression. When balancing considerations for sequence modification, optimization for better expression was chosen over altering SHM sites. Transgene construction and mouse preparation

[0087] The construction of the binary fixed light chain transgene construct is shown diagrammatically in Figure 4. The RSS(12) element (in two opposite orientations) binds the huVK 1-39 * The RSS(23) element is derived from the huIGKJ1 allele. *The IGKC fragment is derived from the 01 allele. The cassette incorporating splice acceptor (SA) and polyadenylation (pA) sequences is derived from the human IGKC lambda C2 locus and contains two consensus pA signals. Approximately 9.7 kb of genomic 5' sequence from VK 1-39 is included at the 5' end, providing a genetic "buffer" of sorts, but also containing potential minor or subtle regulatory elements from this region. The VK 4-1 element has a smaller upstream regulatory element (1.6 kb), but due to the mechanism of binary switching, this should always be "protected" by the 1-39 5' segment. Additionally, approximately 2.8 kb of information 5' of the human IGKC coding region is included in this construct, which remains intact regardless of which VK segment is functionally recombined. The start of this region is defined by the location of the RSS (23) site. Additionally, there is approximately 560 bp of information 3' of the human IGKC segment, which means that it aligns with the coordinates of the CRIS PR-edited mouse genomic IGKC locus landing pad.

[0088] The binary locked light chain transgene is further illustrated schematically in Figure 5, which shows how the orientation of the 1-39 cassette is opposite to that of the 4-1 cassette (i.e., the orientation of the 4-1 cassette is antisense to that of the 1-39 cassette). The splice acceptor / 3'UTR / polyA downstream of 1-39 should prevent any splicing or translation of a functional kappa light chain containing the kappa C exon. Furthermore, the 4-1 cassette in the antisense orientation is also blocked for functional splicing. The action of RAG proteins on the construct during B cell development results in RSS recombination, stochastically activating either the 1-39 or 4-1 cassette. The nucleotide sequence of a representative binary locked light chain transgene (as shown in Figure 5) is set forth in SEQ ID NO:5.

[0089] As shown schematically in Figure 6, the binary-locked light chain transgene construct was knocked into the mouse kappa locus (where all mouse Vk, Jk, and kC information had previously been deleted) using standard techniques known in the art and via cre / lox-mediated RMCE (recombinase-mediated cassette exchange). The resulting mice expressed either the fully human 1-39 / J2 / kC or 4-1 / J4 / kC light chains. Twenty-three ES clones were successfully knocked in, and four were sequence-verified by targeted locus amplification (TLA) technology. Mice containing the binary-locked light chain transgene can be bred to homozygosity. Mice can be cross-bred with fully diverse heavy chain mice (e.g., humanized or human HC transgenic mice).

[0090] The final plasmid-based transgene construct, including the appropriate vector sequences, is shown schematically in FIG.

[0091] Functional validation of binary locked light chain mice can include analysis of genomic DNA to show that recombination occurs in B cells to generate two different light chain alleles, RNA analysis to show that in-frame spliced ​​transcripts can be generated in mouse B cells, and examination of kappa light chain protein levels. Titers and immune responsiveness to test antigens can also be tested and compared to wild-type mice. Characterization of transgenic mice

[0092] To demonstrate that recombination occurs in B cells to generate the RSS-recombined fixed light chain allele, genomic DNA was prepared from either ear or spleen biopsies, under the assumption that a higher frequency of B cells (and therefore recombination) would be found in spleen biopsies compared to ear tissue. Standard PCR was performed using gene-specific primers (see directional arrows above the schematic diagram of the transgene), as seen in Figure 8. PCR amplicons of the appropriate size for the two recombined alleles were found only in the spleen sample (as expected, see arrows above the agarose gel photograph) and not in the ear DNA sample, where few or no B cells were found. Furthermore, the amplicon indicated by the arrow was gel purified and subjected to DNA sequencing according to the sequence chromatogram inset. The results indicate that the junction sequences resulting from RSS-mediated recombination are heterogeneous. This is the expected outcome for such junctions, which ultimately resolve via the process of non-homologous end-joining (NHEJ).

[0093] To demonstrate that such rearranged fixed light chain loci can properly express spliced ​​mRNA transcripts, we performed RT-PCR on spleen RNA samples from transgenic common light chain mice. As seen in Figure 9, sequence analysis of individual cDNA clones, when compared to computer-generated virtual reference sequences, indicates that in-frame transcripts corresponding to two fixed light chain alleles, the fully human 1-39 / J2 / kC light chain or the fully human 4-1 / J4 / kC light chain, can be recovered. While many of these sequences correspond 100% to the reference sequences, others may vary due to occasional somatic hypermutation events.

[0094] To establish that recombinant and expressed fixed light chain alleles can contribute to normal immune responses, transgenic mice were immunized with COVID-19 spike protein, and serum titers were determined by ELISA. Prior to immunization, mice were bled to determine baseline levels of human κ light chain expression. Three different mouse genotypes were analyzed. The first mouse was a compound heterozygote consisting of one fixed light chain transgene allele and one null allele of mouse κLC (so-called KLaP, lacking all Vk and Jk sequences), the second mouse was a compound heterozygote consisting of one wild-type mouse κ light chain allele and one null allele of mouse κLC (KLaP), and the third mouse was a normal wild-type mouse. As can be seen in Figures 10A–10C, naive mice before immunization were indistinguishable in terms of serum Igκ levels using a non-species-specific anti-κ detection reagent. After immunization, strong titers against the COVID spike protein were detected via a sandwich ELISA using a first-stage capture of the COVID spike, followed by a second-stage detection of all kappa light chains (non-species specific). Serum titer midpoint dilution determinations (EC50) showed that mice containing the fixed light chain exhibited strong titers, differing only approximately 2-3-fold from wild-type responses. Hybridoma fusion of immunized mice followed by selection of antigen-specific clones yielded over 200 antigen-specific hits. Sequencing of a subset of 48 randomly selected clones and subsequent light chain components revealed that 45 of these were specific for the 1-39JK2 allele and 3 for the 4-1JK4 allele. From the total group of 48 clones, 13 distinct murine VH heavy chain partners were identified. Together, these results demonstrate that the binary transgene system is fully functional and can serve as a source of common light chains to facilitate the construction of bispecific antibodies and the development of therapeutics.

[0095] Example 2: Preparation of a human lambda binary common light chain construct This example describes the preparation of a transgene construct for expressing one of two alternative "fixed" human λ light chains. Similar work was done to design and generate a fixed human λ light chain transgene, following the approach described in Example 1 for generating fixed light chain mice for human κ light chains. This process involved several steps. (i) Determining VL usage in humans, with the idea that highly expressed alleles are well tolerated and favor pairing with a broad array of potential heavy chain variable domains; (ii) Combining VL usage data with JL usage data to define specific VL-JL junction species that can be used to generate transgenes for knock-in; (iii) creating in silico versions of fixed or pre-recombined human lambda alleles (cassettes) in the context of the human genome sequence that surrounds them; and (iv) Construction of the cassette into a functional transgene that can be used for gene targeting (i.e., also containing constant regions, enhancers if necessary, and engineered sites to allow site-specific gene delivery or knock-in). Selection of λ light chain V region

[0096] The frequency of lambda V-region expression in the human population was used as a starting point for selecting lambda light chain V-regions. The IMGT® database provides information on Vλ frequency, leading to the determination that Vλ2-14 and 3-19 were most frequently used, but 3-21, 3-1, and 1-51 were also frequently observed. The reported IGVL frequencies in human SARS-2 patients (J. Exp. Med. (2022) Vol. 219, No. 9e20220367) were also examined. Furthermore, Vλ and Jλ frequencies in human naive repertoires were also analyzed based on the data disclosed in DeKosky et al. (2015) Nat. Med. 21:86-91. Analysis of the data reported in DeKosky et al. led to the identification of three top Vλ-Jλ pairs for each of the three patients studied. The dataset was sorted by Vλ segment frequency and subjected to pivot analysis. Based on this analysis, two candidate VL-JL pairs were identified: VL2-14 / JL2 and VL1-40 / JL2. However, using J2 segments in both VL-JL pairs in the binary construct would generate nearly identical CDR3 domains from each. One feature of the binary fixed light chain approach is that the transgene provides a "choice" between two different λ light chains; therefore, sequence divergence is an important consideration, so a second VL-JL selection was performed based on divergence parameters. For this reason, a non-J2 segment (e.g., a JL1 segment) could be used in one of the two VL-JL pairs in the final construct. Thus, the VL-JL pair selected for use in the binary fixed λ light chain transgene was VL2-14 / JL2 + VL1-40 / JL1.

[0097] Selection of VL2-14 / JL2 DNA Sequences: Because there are many possible junctions between the VL-JL segments, it was useful to rank the different junction pairs to select specific VL2-14 / JL2 DNA sequences for use in transgene constructs. Based on the DeKosky dataset, the identified CDR3 regions were analyzed because they contain the VL-JL junctions. From the thousands of VL2-14 / J2 LCDR3 sequences returned from each of the three donors, the most frequently recovered LCDR3s were identified and aligned using the commercially available Geneious Prime software to generate a consensus sequence. Furthermore, the CDR3 sequences were translated to derive amino acid sequences, and alignments were performed to determine the most common amino acid sequence of the CDR3. LCDR3 sequence analysis led to the following consensus sequence (the sequence extends all the way to the end of the J segment): CSSYTSSSTLVVFGGGTKLTVL (SEQ ID NO: 6). This sequence was subjected to pBLAST, which confirmed that it was commonly found in the NCBI database. The full-length variable domain of VL2-14 / JL2 was also subjected to pBLAST, which revealed that the full-length variable domain sequence also returned a BLAST hit. Overall, sequence analysis of the VL2-14 / J2 combination confirmed that it is found in GenBank, is expressed, and is likely to be active.

[0098] Selection of VL2-1-40 / JL1 DNA sequence: Similar work was performed on the 1-40 / J1 pair as for the 2-14 / J2 pair. Nucleotide sequences from the DeKosky dataset were aligned to determine a "consensus" LCDR3 from the VL1-40 / J1 transcript. The nucleotide sequences were also translated, and an alignment of the translated sequences was prepared. LCDR3 sequence analysis yielded the following consensus sequence (extending all the way to the end of the J segment): CQSYDSSLSGYVFGTGTKVTVLG (SEQ ID NO: 7). The LCDR3 sequence, as well as the sequence assembled into the complete variable domain, were subjected to pBLAST, which confirmed that the sequences were readily found in the NCBI database. Overall, similar to the VL2-14 / J2 analysis, sequence analysis of the VL1-40 / J1 combination indicated that they were well tolerated and likely commonly found in the human lambda light chain repertoire.

[0099] Construction of a fixed light chain transgene Regarding the overall strategy, the "binary" approach of fixed light chain mice was validated with the human κ light chain, as described in Example 1 and shown schematically in Figure 2. Building on the success of the kappa-fixed light chain model, the lambda-fixed light chain model was created by substituting the coding sequence identified in the λ light chain V region selection described above for the kappa coding sequence. This resulted in a lambda binary-fixed light chain construct containing two lambda variable region cassettes (λFLC1 and λFLC2) arranged in opposite orientations, both of which are inactive in the germline, with one of the two cassettes becoming active upon RAG recombination, as shown schematically in Figure 11. As in the κ construct described in Example 1, the green and yellow triangles represent recombination signal sequences (RSSs), which are normally acted upon during recombination to link a given light chain V segment to a given light chain J segment. In that context, RSS sites are used to join coding sequences together. In this transgene context, they are placed in non-coding regions but are still intended to perform the same recombination / ligation function.

[0100] The two lambda-locked light chain cassettes are designed to "pre-rearrange" the Vλ and Jλ segments, so that there is no CDR3 variability or junctional diversity in that region. Furthermore, given the success of the traditional κ approach, much of the original κ light chain non-coding transgene sequence is used to create the λ transgene. Furthermore, the lambda transgene can be delivered site-specifically to the mouse κ light chain locus, for example, by recombinase-mediated cassette exchange (RMCE), as described herein (e.g., Example 1 and Figure 6). Alternatively, CRISPR-based recombination methods can be used to deliver the lambda transgene to an endogenous light chain locus, such as the kappa locus.

[0101] Mice containing the lambda binary locked light chain transgene can be bred to homozygosity. Mice can be cross-bred with fully heterogeneous heavy chain mice (e.g., humanized or human HC transgenic mice).

[0102] Functional validation of binary locked light chain mice can include analysis of genomic DNA to show that recombination occurs in B cells to generate two different light chain alleles, RNA analysis to show that in-frame spliced ​​transcripts can be generated in mouse B cells, and examination of lambda light chain protein levels. Titers and immune responsiveness to test antigens can also be tested and compared to wild-type mice.

[0103] Sequence Listing Summary [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

[0104]

Claims

1. 1. A transgene construct comprising: (a) a first immunoglobulin light chain variable cassette (VL1) and a second immunoglobulin light chain variable cassette (VL2), each of VL1 and VL2 comprising a promoter, a light chain V region, a light chain J region, and a splice donor site; (b) a termination cassette (SC) containing a splice acceptor site and a polyadenylation signal; (c) a first recombination signal sequence (RSS) 12-mer (RSS1), a second recombination signal sequence 12-mer (RSS2), and an RSS 23-mer (RSS3); wherein the transgene construct comprises a 5' to 3' VL1-RSS1-SC-RSS2-VL2-RSS3, A transgene construct in which VL2 is in the antisense orientation relative to VL1.

2. The transgene construct of claim 1, wherein in B cells harboring the transgene construct, VL1 and VL2 are inactive before RAG-mediated recombination, and either VL1 or VL2 is active after RAG-mediated recombination.

3. 2. The transgene construct of claim 1, wherein the light chain V region and the light chain J region are human κ sequences.

4. 4. The transgene construct of claim 3, wherein VL1 or VL2 comprises the Vκ 1-39 region.

5. 4. The transgene construct of claim 3, wherein VL1 or VL2 comprises a Jκ JK2 region.

6. 4. The transgene construct of claim 3, wherein VL1 or VL2 comprises a Vκ 1-39 region and a Jκ JK2 region.

7. 4. The transgene construct of claim 3, wherein VL1 or VL2 comprises a Vκ4-1 region.

8. 4. The transgene construct of claim 3, wherein VL1 or VL2 comprises a Jκ JK4 region.

9. 4. The transgene construct of claim 3, wherein VL1 or VL2 comprises a Vκ4-1 region and a JκJK4 region.

10. 4. The transgene construct of claim 3, wherein VL1 comprises the Vκ 1-39 region and VL2 comprises the Vκ 4-1 region.

11. 4. The transgene construct of claim 3, wherein VL1 comprises a Vκ 1-39 region and a Jκ JK2 region, and VL2 comprises a Vκ 4-1 region and a Jκ JK4 region.

12. 4. The transgene construct of claim 3, wherein VL1 comprises a Vκ 4-1 region and VL2 comprises a Vκ 1-39 region.

13. 4. The transgene construct of claim 3, wherein VL1 comprises a Vκ 4-1 region and a Jκ JK4 region, and VL2 comprises a Vκ 1-39 region and a Jκ JK2 region.

14. The transgene construct of any one of claims 1 to 13, further comprising a light chain constant region downstream of RSS3.

15. 15. The transgene construct of claim 14, wherein the light chain constant region is a human kappa constant region.

16. 16. The transgene construct of claim 14 or 15, further comprising an enhancer downstream of RSS3 and upstream of the light chain constant region.

17. 17. The transgene construct of claim 16, wherein the enhancer comprises an intronic human kappa enhancer (mEKi).

18. 18. The transgene construct of any one of claims 1 to 17, wherein VL1 or VL2 comprises a CDR3 comprising the sequence shown in SEQ ID NO:

1.

19. 18. The transgene construct of any one of claims 1 to 17, wherein VL1 or VL2 comprises a CDR3 comprising the sequence shown in SEQ ID NO:

2.

20. 18. The transgene construct of any one of claims 1 to 17, wherein VL1 or VL2 comprises the sequence shown in SEQ ID NO:

3.

21. 18. The transgene construct of any one of claims 1 to 17, wherein VL1 or VL2 comprises the sequence shown in SEQ ID NO:

4.

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

5.

23. A transgenic animal comprising a transgene construct according to any one of claims 1 to 22.

24. The transgenic animal of claim 23, which is a mouse.

25. 25. The transgenic mouse of claim 24, further comprising a transgene construct encoding an immunoglobulin heavy chain such that the mouse expresses an antibody comprising a heavy chain paired with a light chain comprising the light chain V region of either VL1 or VL2.

26. 26. A method for producing antibodies against an antigen of interest, said method comprising administering said antigen of interest to the transgenic mouse of claim 25 so as to produce antibodies that bind to said antigen of interest.

27. 27. The method of claim 26, further comprising isolating an antibody of interest from the mouse and determining whether the antibody uses the light chain V region of VL1 or VL2.

28. 2. The transgene construct of claim 1, wherein the light chain V region and the light chain J region are human lambda sequences.

29. 29. The transgene construct of claim 28, wherein VL1 or VL2 comprises the Vλ2-14 region.

30. 29. The transgene construct of claim 28, wherein VL1 or VL2 comprises a JλJL2 region.

31. 29. The transgene construct of claim 28, wherein VL1 or VL2 comprises a Vλ2-14 region and a JλJL2 region.

32. 29. The transgene construct of claim 28, wherein VL1 or VL2 comprises a Vλ1-40 region.

33. 29. The transgene construct of claim 28, wherein VL1 or VL2 comprises a JλJL1 region.

34. 29. The transgene construct of claim 28, wherein VL1 or VL2 comprises a Vλ1-40 region and a JλJL1 region.

35. 29. The transgene construct of claim 28, wherein VL1 comprises the Vλ2-14 region and VL2 comprises the Vλ1-40 region.

36. 29. The transgene construct of claim 28, wherein VL1 comprises a Vλ2-14 region and a JλJL2 region, and VL2 comprises a Vλ1-40 region and a JλJL1 region.

37. 29. The transgene construct of claim 28, wherein VL1 comprises the Vλ1-40 region and VL2 comprises the Vλ2-14 region.

38. 29. The transgene construct of claim 28, wherein VL1 comprises a Vλ1-40 region and a JλJL1 region, and VL2 comprises a Vλ2-14 region and a JλJL2 region.

39. The transgene construct of any one of claims 28 to 38, further comprising a light chain constant region downstream of RSS3.

40. 40. The transgene construct of any one of claims 1 to 39, wherein the light chain constant region is a human kappa constant region or a human lambda constant region.

41. 41. The transgene construct of any one of claims 28 to 40, wherein VL1 or VL2 comprises a CDR3 comprising the sequence shown in SEQ ID NO:

6.

42. 41. The transgene construct of any one of claims 28 to 40, wherein VL1 or VL2 comprises a CDR3 comprising the sequence shown in SEQ ID NO:

7.

43. A transgenic animal comprising a transgene construct according to any one of claims 28 to 42.

44. 44. The transgenic animal of claim 43, which is a mouse.

45. 45. The transgenic mouse of claim 44, further comprising a transgene construct encoding an immunoglobulin heavy chain such that the mouse expresses an antibody comprising a heavy chain paired with a light chain comprising the light chain V region of either VL1 or VL2.

46. 46. ​​A method for producing antibodies against an antigen of interest, said method comprising administering said antigen of interest to the transgenic mouse of claim 45 so as to produce antibodies that bind to said antigen of interest.

47. 47. The method of claim 46, further comprising isolating an antibody of interest from the mouse and determining whether the antibody uses the light chain V region of VL1 or VL2.

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