Human immunoglobulin binary light chain transgenic constructs and uses thereof

Abstract

A human immunoglobulin light chain transgenic construct is provided that encodes two different rearranged light chain V-J regions that are expressed from the construct such that one of the two alternating light chains is recombined in B cells. Transgenic animals comprising the transgene are also provided. Thus, a binary light chain transgene allows two alternating fixed light chains to be expressed in these animals. Methods of using these transgenic animals are also provided.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 439,795, filed on January 18, 2023, under 35 U.S.C. § 119(e), which is hereby incorporated by reference in its entirety for all purposes.

[0003] Sequence Listing

[0004] This application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated 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 of the Invention

[0005] Bispecific antibodies (bsAbs) combine two different antigen-binding sites in a single molecule. Due to increased target specificity and distinct mechanisms of action, bsAbs can be superior to monospecific antibodies, potentially leading to higher clinical efficacy (reviewed in, e.g., 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 correct pairing between two different heavy and light chains and associated manufacturability issues. One approach that has been attempted to reduce the complexity of bsAb production and manufacturing is to use a common light chain in combination with two different heavy chains (e.g., as Figure 1 schematically illustrated). This approach takes advantage of the established fact that for many antibodies, the major drivers of affinity and specificity are the heavy chains. Thus, using a common light chain avoids the risk of incorrect heavy chain / light chain pairing and limits manufacturing to only three peptide chains while still retaining the desired antigen-binding specificity of the bsAb.

[0006] Methods of making common light chain transgenes and using common light chains in bsAbs have been described (see, e.g., Merchant et al. (1998) Nat. Biotech. 16:677-681; 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 Blarcom et al. (2018) MABS 10:256-268; PCT Publication No. WO 2011 / 097603; PCT Publication No. WO 2013 / 134263; PCT Publication No. WO 2015 / 153765; PCT Publication No. WO 2020 / 132557; PCT Publication No. WO 2020 / 205504).

[0007] Although some progress has been made, additional methods and compositions are needed to design, make, and use common light chain transgenes, especially for use in bispecific antibodies. SUMMARY OF THE INVENTION

[0008] The present disclosure provides human immunoglobulin light chain transgene constructs that encode two different rearranged V-J regions positioned in opposite orientations in the construct, which transgene constructs are referred to herein as binary fixed light chain constructs. The constructs co-opt recombination signal sequences (RSSs) and RAG-mediated gene activation to silence the locus prior to recombination and to cause functional expression of one of the V-J regions 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 two alternative V-J regions is expressed. Thus, the binary fixed light chain transgene results in a random selection between two different pre-rearranged light chain V-J regions. The resulting immunoglobulin repertoire in the transgenic animal includes two fixed light chains, thereby providing two different common light chain selections in the animal. This facilitates increasing the likelihood of successfully generating antibodies against an antigen of interest in the animal.

[0009] Accordingly, in one aspect, the present disclosure relates to a transgene construct comprising:

[0010] (a) a first immunoglobulin light chain variable cassette (VL1) and a second immunoglobulin light chain variable cassette (VL2), wherein VL1 and VL2 each comprise a promoter, a light chain V region, a light chain J region, and a splice donor site;

[0011] (b) a termination cassette (SC) comprising a splice acceptor site and a polyadenylation signal; and

[0012] (c) The first recombination signal sequence (RSS) 12mer (RSS1), the second recombination signal sequence 12mer (RSS2), and the RSS 23mer (RSS3);

[0013] wherein the transgenic construct comprises, from 5' to 3',

[0014] VL1 - RSS1 - SC - RSS2 - VL2 - RSS3

[0015] and wherein VL2 is in an antisense orientation relative to VL1.

[0016] When the transgenic construct is carried by a B cell, VL1 and VL2 are inactive prior to RAG - mediated recombination and either VL1 or VL2 is active after RAG - mediated recombination.

[0017] In an embodiment, the light chain V and J regions are human κ sequences. Non - limiting examples of suitable V and J regions are disclosed herein. In one embodiment, VL1 or VL2 comprises the Vk 1 - 39 region. In one embodiment, VL1 or VL2 comprises the Jk JK2 region. In one embodiment, VL1 or VL2 comprises the Vk 1 - 39 region and the Jk JK2 region. In one embodiment, VL1 or VL2 comprises the Vk 4 - 1 region. In one embodiment, VL1 or VL2 comprises the Jk JK4 region. In one embodiment, VL1 or VL2 comprises the Vk 4 - 1 region and the Jk JK4 region. In one embodiment, VL1 comprises the Vk 1 - 39 region and VL2 comprises the 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.

[0018] In an embodiment, the light chain V region and J region are human lambda sequences. Non-limiting examples of suitable lambda V and J regions are disclosed herein. In one embodiment, VL1 or VL2 comprises the V lambda 2-14 region. In one embodiment, VL1 or VL2 comprises the J lambda JL2 region. In one embodiment, VL1 or VL2 comprises the V lambda 2-14 region and the J lambda JL2 region. In one embodiment, VL1 or VL2 comprises the V lambda 1-40 region. In one embodiment, VL1 or VL2 comprises the J lambda JL1 region. In one embodiment, VL1 or VL2 comprises the V lambda 1-40 region and the J lambda JL1 region. In one embodiment, VL1 comprises the V lambda 2-14 region and VL2 comprises the V lambda 1-40 region. In one embodiment, VL1 comprises the V lambda 2-14 region and the J lambda JL2 region, and VL2 comprises the V lambda 1-40 region and the J lambda JL1 region. In one embodiment, VL1 comprises the V lambda 1-40 region and VL2 comprises the V lambda 2-14 region. In one embodiment, VL1 comprises the V lambda 1-40 region and the J lambda JL1 region, and VL2 comprises the V lambda 2-14 region and the J lambda JL2 region.

[0019] In an embodiment, the transgenic 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 an embodiment, the transgenic construct further comprises an enhancer downstream of RSS3 and upstream of the light chain constant region. In one embodiment, the enhancer comprises the intronic human kappa enhancer (hEKi).

[0020] In one embodiment, VL1 or VL2 of the transgenic construct comprises a CDR3 containing the sequence shown in SEQ ID NO: 1. In one embodiment, VL1 or VL2 of the transgenic construct comprises a CDR3 containing the sequence shown in SEQ ID NO: 2. In one embodiment, VL1 or VL2 of the transgenic construct comprises the sequence shown in SEQ ID NO: 3. In one embodiment, VL1 or VL2 of the transgenic construct comprises the sequence shown in SEQ ID NO: 4. In one embodiment, the transgenic construct comprises the sequence shown in SEQ ID NO: 5.

[0021] In one embodiment, VL1 or VL2 of the transgenic construct comprises a CDR3 containing the sequence shown in SEQ ID NO: 6. In one embodiment, VL1 or VL2 of the transgenic construct comprises a CDR3 containing the sequence shown in SEQ ID NO: 7.

[0022] In another aspect, the present disclosure relates to a transgenic animal comprising the transgenic construct of the present disclosure. In one embodiment, the transgenic animal is a mouse. In one embodiment, the transgenic mouse further comprises a transgenic construct encoding an immunoglobulin heavy chain such that the mouse expresses an antibody comprising a heavy chain paired with a light chain comprising a light chain V region of VL1 or VL2.

[0023] In another aspect, the present 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 of the present disclosure (e.g., a mouse) such that an antibody that binds to the antigen of interest is produced. In one embodiment, the method further comprises isolating the antibody of interest from the animal and determining whether the antibody uses a light chain V region of VL1 or VL2. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a schematic diagram of a method for generating bispecific antibodies using two different heavy chains and one fixed light chain to generate two different binding specificities.

[0025] Figure 2 is a schematic diagram of a binary light chain transgenic construct showing the inactive state before RAG activation and the alternative activation of VJK1 or VJK2 after RAG activation. PR>=promoter; SD=splice donor; SA=splice acceptor; pA=polyA / termination cassette; hEKi=intronic human κ enhancer; SA / pA=cassette derived from the human IGλ C2 locus and containing 2 consensus pA signals.

[0026] Figure 3 is a schematic diagram illustrating the degree to which the binary fixed light chain model increases the hit frequency compared to the single fixed light chain model.

[0027] Figure 4 is a schematic diagram illustrating the construction elements of a representative binary fixed light chain construct of the present disclosure.

[0028] Figure 5 is a schematic diagram illustrating the structure of a representative binary fixed light chain construct of the present disclosure.

[0029] Figure 6 is a schematic diagram illustrating the insertion of a representative binary fixed light chain construct into the mouse κ locus via recombination. LP=landing pad; FLC=fixed (AKA binary) light chain; neo R and puro R refer to the neomycin and puromycin resistance cassettes, respectively.

[0030] Figure 7 is a schematic diagram of the plasmid map of a representative transgenic construct of the present disclosure.

[0031] Figure 8 Shows the results of PCR amplicons and DNA sequencing analysis of recombinant transgenic alleles from transgenic mice.

[0032] Figure 9 Shows the results of RT-PCR analysis of splenocyte RNA from transgenic mice, showing the expression of correctly spliced transgenic mRNA.

[0033] Figures 10A - 10C Shows the results of ELISA assays of transgenic mice. Figure 10A Is a schematic illustration of the antibody format to be detected, which carries a fixed human κLC encoded by the transgene. Figure 10B Shows the IgK levels in naïve (unimmunized) mice. Figure 10C Shows the ELISA results of mice immunized with a COVID-19 spike protein antigen preparation.

[0034] Figure 11 Is a schematic diagram of a binary λ fixed light chain (λFLC) transgenic construct, showing the inactive state of two light chain variable regions before RAG-mediated recombination and the activation of λFLC1 or λFLC2 after RAG-mediated recombination. Detailed Description

[0035] The binary fixed light chain transgenic constructs of the present disclosure encode two different V-J regions in opposite orientations, as Figure 2 schematically illustrated. These constructs utilize the recombination signal sequence (RSS) and the endogenous recombination activating gene (RAG) gene activation system to silence the expression of the two V-J regions before recombination and to cause the functional expression of one or the other V-J region after recombination. This design ensures that the pre-rearranged human light chain is inactive before B cell recombination to help ensure that normal B cell development is not disrupted by having a transcriptionally active and functional light chain too early in development. Since typical B cell differentiation proceeds by rearranging the heavy chain before the light chain (see, for example, Yancopoulos and Alt (1986) Annu Rev Immunol. 4:339-68), all attempts are made to preserve this natural biology inherent in B cell development. Thus, the overall intent behind this method is to give the transgenic animal one of two alternate choices with respect to the human light chain that it will express, as Figure 3 schematically illustrated. Thus, for an antigen for which a particular HC:LC pair is excluded or non-reactive, the other alternative is available, thereby increasing the likelihood of obtaining an antibody of interest.

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

[0037] I. Construct Design

[0038] The design and construction of the binary fixed light chain transgene are described in detail in Example 1. As Figure 2 , Figure 4 and Figure 5 illustrate, the construct contains two V-J region cassettes that are in opposite orientations (i.e., antisense) to each other, with a termination cassette therebetween. The construct also contains intervening RSS sequences for facilitating recombination.

[0039] Thus, in one embodiment, the transgene construct comprises:

[0040] (a) a first immunoglobulin light chain variable cassette (VL1) and a second immunoglobulin light chain variable cassette (VL2), wherein VL1 and VL2 each comprise a promoter, a light chain V region, a light chain J region, and a splice donor site;

[0041] (b) a termination cassette (SC) comprising a splice acceptor site and a polyadenylation signal; and

[0042] (c) a first recombination signal sequence (RSS) 12mer (RSS1), a second recombination signal sequence 12mer (RSS2), and an RSS23mer (RSS3);

[0043] wherein the transgene construct comprises

[0044] VL1-RSS1-SC-RSS2-VL2-RSS3

[0045] And wherein VL2 is in an antisense (i.e., reverse or opposite) orientation relative to VL1.

[0046] Due to this structure of the transgene, neither VL cassette can generate a functional light chain transcript prior to RAG-mediated recombination because the artificial splice / pA termination signal prevents proper splicing and expression of upstream VL1, and downstream VL2 is in an incorrect antisense orientation. Additionally, the RSSs are removed from their normal context because they are not linked to coding sequences (resulting in non-homologous end joining occurring within the VL and κ constant region introns and should have no effect).

[0047] As Figure 2 、 Figure 4 and Figure 5 Further illustrated, after RAG-mediated recombination: (i) VL1 becomes active by excision of the SA / pA termination cassette and the VL2 cassette and the operable linkage of the VL1 promoter and coding sequence to the downstream enhancer and constant region sequences, or (ii) VL2 becomes active by inversion of the VL2 cassette, resulting in the operable linkage of the VL2 promoter and coding sequence to the downstream enhancer and constant region sequences.

[0048] Thus, when the transgene construct of the present disclosure is carried by a B cell, VL1 and VL2 are inactive prior to RAG-mediated recombination and VL1 or VL2 is active after RAG-mediated recombination.

[0049] In one embodiment, the light chain V and J regions used in the VL1 and VL2 cassettes are κ regions (e.g., human κ 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 λ regions (e.g., human λ 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 κ region and the other of the VL1 / VL2 cassettes uses a λ region. For example, a human κ VL1 or VL2 cassette as described in Example 1 can be combined with a human λ VL1 or VL2 cassette as described in Example 2 to produce a binary fixed light chain construct comprising a κ cassette and a λ cassette.

[0050] The selection of the 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 show a tendency to bind to an antigen of interest, such a region can be selected for use with that intended antigen. Alternatively, for use with a wide range of antigens, the V region can be selected based on: (i) the expression frequency in the normal population; (ii) the ability to pair with multiple human heavy chain families, if known; and / or (iii) the ability to pair with a known J domain. Information on the frequency of V region usage and preferred VK / VH pairings is available in the art and can be used in the design of constructs. For example, the VK region can be selected based on its preferred pairing with the VH region. Non-limiting examples of such preferred pairings are described in DeKosky et al. (2015) Nat. Med. 21:86-91, the entire content of which is expressly incorporated herein by reference.

[0051] In one embodiment, the V regions used in VL1 and VL2 are independently human Vk regions selected from the group consisting of: Vk regions 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, 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.

[0052] In one embodiment, the V regions used in VL1 and VL2 are independently human Vk regions selected from the group consisting of: Vk regions 1-5, 1-39, 3-11, 3-15, 3-20, 3-28 and 4-1.

[0053] In one embodiment, the V regions used in VL1 and VL2 are independently human Vk regions selected from the group consisting of: Vk regions 1-39, 3-20 and 4-1. In one embodiment, VL1 or VL2 contains the Vk 1-39 region. In one embodiment, VL1 or VL2 contains the Vk 4-1 region. In one embodiment, VL1 contains the Vk 1-39 region and VL2 contains the Vk 4-1 region. In one embodiment, VL1 contains the Vk 4-1 region and VL2 contains the Vk 1-39 region.

[0054] In one embodiment, the J regions used in VL1 and VL2 are human Jk regions (e.g., when Vk regions are used). 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 region used is a human Jk region selected from Jk, JK1, JK2, JK3, JK4, and JK5. Information available in the art regarding commonly observed IGKV-IGKJ pairings can be used in the design of the VL1 and VL2 regions. For example, the Jk region can be selected based on its preferred pairing with the Vk region. Non-limiting examples of such preferred pairings are described in Collins et al. (2008) Immunogenetics 60:669-676, the entire content of which is expressly incorporated herein by reference. In one embodiment, VL1 or VL2 comprises the Jk JK2 region. In one embodiment, VL1 or VL2 comprises the Vk 1-39 region and the Jk JK2 region. In one embodiment, VL1 or VL2 comprises the Jk JK4 region. In one embodiment, VL1 or VL2 comprises the Vk 4-1 region and the Jk JK4 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 the Jk JK4 region, and VL2 comprises the Vk 1-39 region and the Jk JK2 region.

[0055] In one embodiment, the V regions used in VL1 and VL2 are human Vλ regions, such as independently selected from the group consisting of: Vλ region 2-14, 3-19, 3-21, 3-1, 1-51, and 1-40.

[0056] In one embodiment, the V regions used in VL1 and VL2 are human Vλ regions independently selected from the group consisting of: Vλ region 2-14, 3-19, and 1-40. In one embodiment, VL1 or VL2 comprises the Vλ 2-14 region. In one embodiment, VL1 or VL2 comprises the Vλ 1-40 region. In one embodiment, VL1 comprises the Vλ 2-14 region, and VL2 comprises the Vλ 1-40 region. In one embodiment, VL1 comprises the Vλ 1-40 region, and VL2 comprises the Vλ 2-14 region.

[0057] 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 region used is human Jλ JL1 or JL2. In one embodiment, VL1 or VL2 comprises the Jλ JL2 region. In one embodiment, VL1 or VL2 comprises the Vλ 2-14 region and the Jλ JL2 region. In one embodiment, VL1 or VL2 comprises the Jλ JL1 region. In one embodiment, VL1 or VL2 comprises the Vλ 1-40 region and the Jλ JL1 region. In one embodiment, VL1 comprises the Vλ 2-14 region and the Jλ JL2 region, and VL2 comprises the Vλ 1-40 region and the Jλ JL1 region. In one embodiment, VL1 comprises the Vλ 1-40 region and the Jλ JL1 region, and VL2 comprises the Vλ 2-14 region and the Jλ JL2 region.

[0058] Each VL1 cassette and VL2 cassette also contains a promoter operably linked to the V-J coding sequence to drive the expression of the V-J coding region upon activation of the cassette. Suitable promoters are well-recognized in the art and include endogenous murine or human immunoglobulin promoters, as well as heterologous promoters. In one embodiment, the VL1 cassette and VL2 cassette use the endogenous promoter of the V region incorporated into the cassette.

[0059] Each VL1 cassette and VL2 cassette also contains an operably linked splice donor site, while the intermediate termination cassette (SC) contains an operably linked splice acceptor site. Standard splice donor and acceptor site sequences well-recognized in the art are used. SC also contains a polyadenylation signal, the standard sequence of which is used and well-recognized in the art. In one embodiment, the termination cassette is derived from the human Igλ C2 locus and contains two consensus polyadenylation signals.

[0060] Recombination signal sequences (RSSs) are included in the construct to facilitate RAG-mediated recombination. Two operably linked RSS 12mer sequences (RSS1 and RSS2) are located upstream and downstream (in opposite orientations) of SC, respectively. An operably linked RSS 23mer sequence (RSS3) is located downstream of VL2. Standard RSS 12mer and 23mer sequences well-recognized in the art are used. In one embodiment, the two RSS 12mer sequences are derived from the human VK 1-39*01 allele and are used in two opposite orientations. In one embodiment, the RSS 23mer is from the human IGKJ1*01 allele.

[0061] In an embodiment, the transgenic construct further comprises a light chain constant region downstream of RSS3. In one embodiment, the light chain constant region is a human Igκ constant region. In one embodiment, the light chain constant region is a human Igλ constant region. In one embodiment, the light chain constant region is a murine κ constant region. In one embodiment, the light chain constant region is a murine λ constant region.

[0062] In one embodiment, the transgenic construct further comprises an enhancer downstream of RSS3 and upstream of the light chain constant region. Suitable enhancers are well-recognized in the art and include endogenous murine or human immunoglobulin enhancers, as well as heterologous enhancers. In one embodiment, the enhancer comprises the intronic human κ enhancer (hEKi).

[0063] The transgenic construct may also include genomic sequences of the immunoglobulin locus upstream (5') of the VL1 and VL2 cassettes to act as various genetic "buffers" and to include minor or subtle regulatory elements from the Ig locus. For example, in one embodiment, the VL1 cassette uses the VK 1-39 variable region, and approximately 9.7 kb of the 5' genomic sequence from VK 1-39 is incorporated upstream of the VL1 cassette. Similarly, in one embodiment, the VL2 cassette uses the VK 4-1 variable region, and approximately 1.6 kb of the 5' genomic sequence from VK 4-1 is incorporated upstream of the VL2 cassette.

[0064] The transgenic construct may also include genomic sequences of the immunoglobulin locus upstream (5') of the constant region coding sequence. For example, in one embodiment, the construct incorporates the human Igκ constant region and includes approximately 2.8 kb of genomic DNA 5' of the Cκ region coding sequence, which remains intact regardless of whether the VL1 or VL2 cassette is functionally activated.

[0065] The nucleotide sequence of the transgenic construct can be further optimized for the intended purpose. For example, the construct can be altered for codon optimization (e.g., to increase the expression of the encoded region). Methods of codon optimization are well-recognized in the art.

[0066] The transgenic construct may also contain sequences that allow for targeted insertion of the transgene into a specific locus (e.g., the endogenous murine light chain locus). Knock-in techniques for replacing an endogenous locus with a targeted transgene are well-recognized in the art. In a preferred embodiment, the transgenic construct contains recombination sequences that allow for knock-in of the transgene into the endogenous murine κ locus, thereby deleting all endogenous murine Vk, Jk, and Cκ sequences. In one embodiment, the construct contains an upstream loxP site and a downstream lox2272 site. Insertion of the transgene into the host genome via recombination is further described in Section III.

[0067] In one embodiment, VL1 or VL2 encodes the CDR3 sequence of Vk1-39 / JK2 that is commonly observed, and the amino acid sequence of this CDR3 is shown in SEQ ID NO: 1. In one embodiment, VL1 or VL2 encodes the CDR3 sequence of Vk4-1 / JK4 that is commonly observed, and the amino acid sequence of this CDR3 is shown in SEQ ID NO: 2. In one embodiment, VL1 or VL2 encodes the Vk1-39 / JK2 amino acid sequence shown in SEQ ID NO: 3. In one embodiment, VL1 or VL2 encodes the Vk4-1 / JK4 amino acid sequence shown in SEQ ID NO: 4.

[0068] In one embodiment, the binary fixed light chain construct comprises the nucleotide sequence shown in SEQ ID NO: 5.

[0069] In one embodiment, VL1 or VL2 encodes a CDR3 as shown in SEQ ID NO: 6, which represents the consensus Vλ2-14 / JL2 CDR3 sequence. In one embodiment, VL1 or VL2 encodes a CDR3 as shown in SEQ ID NO: 7, which represents the consensus Vλ 1-40 / JL1 CDR3 sequence.

[0070] II. Construct Preparation

[0071] The transgenic constructs of the present disclosure can be prepared using standard recombinant DNA techniques. A cloning vector containing a multiple cloning site can be used as the starting vector for inserting DNA fragments of interest. Suitable cloning vectors are well established in the art. In addition, plasmids or other vectors (e.g., YAC) carrying human non-rearranged light chain immunoglobulin sequences have been described in the art (see, for example, U.S. Patent 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; 5,874,299; 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.), and can be used as a source of V and J region sequences. Alternatively, the desired sequences can be synthesized by standard methods. The appropriate DNA fragments are then operably linked into the cloning vector by ligation, and the vector is subsequently characterized (e.g., by restriction fragment analysis or sequencing, etc.) to ensure the correct arrangement of the fragments.

[0072] Non-limiting examples of the binary fixed light chain vectors of the present disclosure are illustrated schematically in Figure 7 as shown.

[0073] To prepare a transgenic construct for microinjection or other transgenic techniques, the transgenic construct can be isolated from the vector it is carried on by cleavage with an appropriate restriction enzyme to release the transgenic construct fragment. Fragments can be isolated using standard techniques, such as by pulsed field gel electrophoresis on an agarose gel, followed by isolation of the fragment from the agarose gel, such as by [β]-agarase digestion or by electroelution. For example, an agarose gel slice containing the transgenic construct fragment can be excised from the gel and the agarose can be digested with [β]-agarase (e.g., from Takara) using standard methodologies. Alternatively, the transgene can be prepared as a complete supercoiled plasmid and sequence-specific recombinases can be utilized to effect recombination of the transgene insert with a compatible recombinase site already present in the murine Igκ locus.

[0074] III. Preparation of Transgenic Animals

[0075] Another aspect of the present disclosure relates to a transgenic non-human host animal comprising a transgenic construct of the present disclosure (i.e., the transgenic construct is integrated into the genome of the host animal) such that the animal expresses an immunoglobulin repertoire of antibodies comprising a fixed light chain using a V-J region comprising a VL1 cassette or a V-J region comprising 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 non-human animal cell (e.g., embryonic stem cells). In a preferred embodiment, the transgenic construct is inserted into the genome of the host animal or host cell using knock-in technology to replace all or a portion of an endogenous light chain locus (e.g., an endogenous κ chain locus) with the transgene (e.g., a κ chain transgene).

[0076] For knock-in methods, loxP flanking sites are typically included in the construct such that these sites enable site-specific recombination between the donor transgene and the host genome, which has been modified with similar loxP sites to facilitate site- and orientation-specific recombination upon Cre recombinase expression (so-called recombinase-mediated cassette exchange or RMCE). Recombination is performed in embryonic stem cells (e.g., mouse embryonic stem cells), and then the embryonic stem cells with the modification of interest are implanted into a live blastocyst, which then grows into a mature chimeric animal (e.g., a mouse), some of whose cells have the genetic information of the original blastocyst cells while other cells have the modification introduced into the embryonic stem cells. Subsequently, the subsequent offspring of the chimeric animal will have the gene knock-in. The knock-in technique is outlined, for example, in Manis (2007) New Engl. J. Med. 357:2426-2429 and Turan et al. (2011) J. Mol. Biol. 407:193–221.

[0077] In a preferred embodiment, the transgenic 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 introducing loxP sites compatible with the donor transgene. In a preferred embodiment, the transgenic construct is a κ light chain construct that is inserted into the modified endogenous mouse κ locus by homologous recombination, in which all mouse Vk, Jk, and Ck sequences have been deleted. In a preferred embodiment, the endogenous κ locus is first engineered to delete VK to CK (a deletion of approximately 3,500,000 bp) while leaving a region containing compatible loxP and lox2272 sites, herein referred to as the "landing pad" site. Subsequently, the binary LC transgenic construct is introduced into the landing pad site in ES cells with cre recombinase (referred to as recombinase-mediated cassette exchange, or RMCE), as Figure 6 schematically illustrated.

[0078] In another embodiment, a κ light chain transgene lacking a constant region is inserted into the endogenous κ locus such that the Vk and Jk sequences are deleted, but the Ck sequence remains intact and is operably linked to the functional variable region of the transgene, thereby producing a chimeric antibody in the mouse (which can be reverse-engineered into a fully human one).

[0079] Standard methods for producing transgenic non-human animals, particularly transgenic mice, involve genetic modification in embryonic stem (ES) cells, such as knock-ins. Such modified stem cells can then be used to generate chimeric mice by implanting them into preimplantation stage mouse embryos via microinjection, and the chimeras are in turn bred to transmit the gene or knock-in allele of interest. At this point, the knock-in allele can be inbred or outbred for line expansion and further study. Southern blot analysis, PCR, or other techniques for analyzing genomic DNA are used to detect the presence of a unique nucleic acid fragment that will not be present in non-transgenic animals or ES cells but will be present in transgenic animals or ES cells. Selective breeding of transgenic offspring allows for the achievement of homozygosity of the transgene.

[0080] Although the preferred embodiments of the present disclosure include transgenic mice, the invention encompasses other non-human host animals, including but not limited to rats, rabbits, pigs, goats, sheep, cattle, and chickens. Techniques for producing transgenic animals of each of these species have been described in the art. For example, the preparation of transgenic rats is described in Tesson, L. et al. (2005) Transgenic Res. 14:531-546, including techniques such as DNA microinjection, lentiviral vector-mediated DNA transfer into early embryos, and sperm-mediated transgenesis. Transgenic methods in rats are also described in Mullin, L. J. et al. (2002) Methods Mol. Biol. 180:255-270. The preparation of transgenic rabbits is described in, for example, Fan, J. et al. (1999) Pathol. Int. 49:583-594; Fan, J. and Watanabe, T. (2000) J. Atheroscler. Thromb. 7:26-32; Bosze, Z. et al. (2003) Transgenic Res.. 12:541-553. The preparation of transgenic pigs is described in, for example, Zhou, CY. et al. (2002) Xenotransplantation 9:183-190; Vodicka, P. et al. (2005) Ann. N. Y. Acad. Sci. 1049:161-171.

[0081] Alternative transgenic techniques for pronuclear microinjection in pigs include adenovirus-mediated DNA introduction into pig sperm (see, e.g., Farre, L. et al. (1999) Mol. Reprod. Dev. 53:149-158) and adapter-based sperm-mediated gene transfer (Chang, K. et al. (2002) BMC Biotechnol. 2:5). The preparation of transgenic goats is described, e.g., in Ebert, K.M. et al. (1991) Biotechnology (NY) 9:835-838; Baldassarre, H. et al. (2004) Reprod. Fertil. Dev. 16:465-470. Somatic cell nuclear transfer in goats is described, e.g., in Behboodi, E. et al. (2004) Transgenic Res. 11:215-224. The preparation of transgenic sheep is described, e.g., in Ward, K.A. and Brown, B.W. (1998) Reprod. Fertil. Dev. 10:659-665. The preparation of transgenic cattle is described, e.g., in Donovan, D.M. et al. (2005) Transgenic Res. 14:563-567. Gene transfection of donor cells for bovine embryo nuclear transfer is described, e.g., in Lee S. L. et al. (2005) Mol. Reprod. Dev. 72:191-200. The preparation of transgenic domestic farm animals is also reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298. The preparation of transgenic chickens is described, e.g., in Pain, B. et al. (1999) Cells Tissues Organs 165:212-219; Lillico, S.G. et al. (2005) Drug Discov. Today 10:191-196; and Ishii, Y. et al. (2004) Dev. Dyn. 229:630-642.

[0082] An animal (e.g., a mouse) of the present disclosure carrying a binary light chain transgenic construct can be crossed with an animal (e.g., a mouse) carrying an immunoglobulin heavy chain transgenic construct, thereby producing an animal (e.g., a mouse) that expresses an antibody having a heavy chain that pairs with a light chain comprising a light chain V region comprising VL1 or VL2. Immunoglobulin heavy chain transgenic animals (e.g., mice) are well-recognized in the art.

[0083] In one embodiment, the transgenic animals of the present disclosure (e.g., mice) are heterozygous for the dual light chain construct and other endogenous mouse light chain alleles are inactivated such that after transgenic recombination, VL1 or VL2 is expressed in all of the light chains of the transgenic cells. In another embodiment, the transgenic animals of the present disclosure (e.g., mice) are homozygous for the dual light chain construct, in which case both alleles of the dual LC construct can be expressed in the animal (e.g., mouse) such that both VL1 and VL2 are used in the light chain repertoire.

[0084] IV. Use of Transgenic Animals

[0085] The transgenic animals of the present disclosure can be used to generate antibodies against a variety of antigens of interest. For an animal that only carries the dual light chain transgene and the endogenous heavy chain locus, the animal will produce chimeric light chain / heavy chain antibodies, which, if desired, can be reverse engineered to pair the light chain with a heavy chain of the same species. Alternatively, for an animal (e.g., mouse) that carries a dual light chain Ig transgene (e.g., human) and a heavy chain Ig transgene (e.g., human or human-mouse chimera), a partially or fully heterologous antibody (e.g., a fully human antibody) can be prepared in the host transgenic animal.

[0086] Thus, in another aspect, the present 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 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 produced in the mouse. In one embodiment, the animal is a transgenic mouse that carries both a human Ig dual 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 produced in the mouse. In one embodiment, the method may further comprise isolating the antibody of interest from the host animal (e.g., mouse) and determining whether the light chain V region of the antibody uses VL1 or VL2.

[0087] Transgenic animals can be immunized with an antigen of interest by standard methodologies known in the art, and the antibodies produced in the animals can also be isolated and characterized by standard validation methods. Polyclonal antibodies can be isolated directly from the host animal and monoclonal antibodies can be prepared by standard methods such as hybridoma technology or single B cell cloning. The procedures for preparing monoclonal antibodies using hybridomas are well established in the art (see, for example, U.S. Patent No. 4,977,081, PCT Publication WO97 / 16537, and European Patent No. 491057B1, the disclosures of which are incorporated herein by reference). Alternatively, the in vitro production of monoclonal antibodies from cloned cDNA molecules is also well established in the art (see, for example, Andris-Widhopf et al. (2000) J. Immunol. Methods 242:159; and Burton (1995) Immunotechnology 1:87, the disclosures of which are incorporated herein by reference). Techniques for facilitating the identification or recovery of single B cells for cloning or cDNA capture are also well established (reviewed, for example, in Pedrioli and Oxenius (2021) Trends Immunol. 42:1143-1158). B cell clones can be isolated from immunized transgenic animals, and the cDNA encoding the antibodies can be isolated and cloned into an expression vector by standard molecular biology techniques. Further recombinant engineering of the cloned Ig cDNA is also possible and well established in the art.

[0088] In one embodiment, a fixed light chain encoded by the transgenic 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. In Figure 1 is schematically illustrated an exemplary method of incorporating a fixed light chain with two different heavy chains into a bsAb.

[0089] V. Definitions

[0090] As used herein, "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, the two arms of a bispecific antibody can utilize the same light chain (i.e., the "common" light chain) and different heavy chains (the heavy chains largely determine the binding specificity of the arm).

[0091] As used herein, the term "operably linked" is intended to describe the configuration of a nucleic acid sequence that is in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, it is operably linked to that sequence. With respect to the joining of two protein-coding regions, operably linked means that the joined nucleic acid sequences are contiguous and in frame. For splice donor / acceptor and RSS sequences, operably linked means that the sequences are capable of achieving their functional purpose.

[0092] As used herein, a "promoter" refers to a nucleic acid sequence required for the expression of a gene product that is operably linked to the promoter / regulator sequence. In some embodiments, the sequence can be a core promoter sequence. In some embodiments, the sequence can also include enhancer sequences and other regulatory elements required for the expression of the gene product.

[0093] As used herein, the term "rearrangement" with respect to an immunoglobulin V segment refers to a configuration in which the V segment is positioned adjacent to the J segment such that it substantially encodes a complete VL domain. Rearranged variable locus genes can be identified by comparison to germline DNA.

[0094] As used herein, the term "transgene" refers to a gene that is introduced as an exogenous site (e.g., the murine light chain Ig locus) into the genome of a host.

[0095] As used herein, the term "transgene construct" refers to a nucleic acid preparation that is suitable for introduction into the genome of a host animal.

[0096] As used herein, the term "transgenic mouse" refers to a mouse that contains cells having a transgene as defined herein. The transgene can be present in all or some of the cells of the mouse.

[0097] The invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, patents, and published patent applications cited throughout this application are hereby expressly incorporated by reference.

[0098] Examples

[0099] Example 1 : Preparation of a human κ binary common light chain construct

[0100] In this example, the preparation of a transgene construct for expressing one of two alternative "fixed" human κ light chains is described. The fixed nature of the light chain means that the VK and J segments to be expressed have been rearranged in a manner that precludes normal and random VK-J recombination that occurs naturally during early B cell development.

[0101] Conceptual Framework

[0102] The intention behind the binary common light chain concept is to give transgenic mice one of two alternatives in terms of the human κ LC that they will express. By doing so, this helps to implement the common light chain (CLC) approach that can be used for the bispecific antibody (bsAb) platform. One of the more successful bsAb approaches is to utilize a CLC that is shared between two unrelated heavy chains, where two unrelated heavy chains encode two different antigen specificities together with their CLC while utilizing the same LC, as Figure 1 illustrated schematically in. In this bispecific antibody format, Ab1 and Ab2 are from transgenic mice with a normal and unrearranged human or murine VH repertoire but with a fixed light chain with known HC promiscuity. This format is known to exhibit low immunogenicity, expression levels similar to conventional antibodies, and the bispecific antibody product has normal Fc-receptor interactions. In addition, in terms of HC:LC pairing combinations, this format is not affected by some of the other more complex bispecific antibody schemes and is not affected by the engineering required to enforce correct pairing or to selectively purify correctly paired species (for a review of such schemes, see, for example, Kontermann and Brinkmann (2015) Drug Discovery Today 20(7)).

[0103] An advantageous feature of the binary common light chain constructs described herein is to provide two different CLCs for the mouse to "choose" from; thus, in those cases where a particular HC:LC pair is excluded or non-reactive to the antigen, another alternative is available. The selection of the fixed or common allelic κ light chain is typically based on: (i) the expression frequency in the normal population; (ii) the ability to pair with multiple human HC families (where known); and (iii) the ability to pair with known J segments.

[0104] Dual Light Chain Construct Design

[0105] A representative schematic of the binary light chain transgenic construct is shown in Figure 2 . A binary transgenic enhancer recombination signal sequence (RSS) and recombination activating gene (RAG) gene activation system is used to present silent loci prior to recombination and to cause functional expression after recombination, resulting in a random selection between two different pre-rearranged κ light chains. Prior to the action of RAG, both cassettes are inactive; an artificial splice / pA termination signal prevents functional light chain expression of the upstream and correctly oriented sequences. The RSSs are removed from their normal context as they are not linked to coding sequences; instead, they are located within non-coding introns so as not to interfere with the coding sequences or the RNA splicing behavior (thus, in this case, non-homologous end joining should not have an impact).

[0106] After RAG-mediated recombination: (i) V1-J1 becomes active by excision of the SA / pA termination cassette and the V2-J2 cassette, and the operable linkage of the V1-J1 promoter and coding sequence to downstream enhancer and constant region sequences, or (ii) V2-J2 becomes active by inversion of the V2-J2 cassette, resulting in the operable linkage of the V2-J2 promoter and coding sequence to downstream enhancer and constant region sequences.

[0107] As Figure 3 schematically illustrated in, the binary fixed light chain method, which confers the ability to express two possible light chains, should result in an increased hit frequency, since having two light chain options increases the odds of successful pairing with any given heavy chain, providing a solution for more binding to two antigens of interest. Ultimately, the mouse "decides" on the best light chain fit.

[0108] Light Chain V Region Selection

[0109] The expression frequency of VK regions in the population was used as a starting point for light chain V region selection. The IMGT® database provides information on VK frequencies, identifying VK 1-39 and 3-20 as the most frequently used, but 4-1, 3-11, 3-15, 3-28, and 1-5 were also observed at high frequencies. VH / VK pairings were also analyzed based on the data published by 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 different VH pairings across most VH families.

[0110] Further BLAST analysis was performed to identify the commonly used LCDR3s from VK 1-39 + JK2 and VK 4-1 + JK4. For VK 1-39 + JK2, the common LCDR3 was identified as having the sequence CQQSYSTPYTF (SEQ ID NO: 1). For VK 4-1 + JK4, the common LCDR3 was identified as having the sequence CQQYYSTPLTF (SEQ ID NO: 2). The optimized alleles of VK 1-39 + JK2 and VK 4-1 + JK4 were then prepared using these most common LCDR3s, which have the amino acid sequences shown in SEQ ID NOs: 3 and 4, respectively.

[0111] Further design of the light chain sequence can include alteration of somatic hypermutation (SHM) sites and codon optimization for expression. When balancing considerations of sequence modification, optimization for good expression is chosen over alteration of SHM sites.

[0112] Transgenic Construct and Mouse Preparation

[0113] The construction of the binary fixed light chain transgenic construct is schematically illustrated in Figure 4 . The RSS(12) elements are derived from the huVK1-39*01 allele (in two opposite orientations). The RSS(23) element is from the huIGKJ1*01 allele. The cassette incorporating the splice acceptor (SA) and polyadenylation (pA) sequences is derived from the human IGλ 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 to provide various genetic "buffers", but also includes 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 binary switch mechanism, this should always be "protected" by the 1-39 5' segment. Additionally, approximately 2.8 kb of information is present 5' of the human IGKC coding region included in this construct, which remains intact regardless of which VK segment functionally recombines. The starting point of this region is defined by the position of the RSS(23) site. Further, approximately 560 bp of information is present 3' of the human IGKC segment, intended to align with the coordinates of the landing pad of the CRISPR-edited murine genomic IGKC locus.

[0114] The binary fixed light chain transgenic is further schematically illustrated 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 κ light chain containing the κC exon. Additionally, the 4-1 cassette in the antisense orientation is also blocked from functional splicing. The action of RAG proteins on the construct during B cell development affects RSS recombination and randomly activates either the 1-39 cassette or the 4-1 cassette. The nucleotide sequence of a representative binary fixed light chain transgenic, as illustrated in Figure 5 , is shown in SEQ ID NO: 5.

[0115] Using standard techniques known in the art, the binary fixed light chain transgenic construct was knocked into the murine κ locus via cre / lox-mediated RMCE (recombinase-mediated cassette exchange), as previously depleted of all murine Vk, Jk, and kC information, as schematically illustrated in Figure 6 . Thus, the resulting mice express the end products of the fully human 1-39 / J2 / kC light chain or 4-1 / J4 / kC light chain. Twenty-three ES clones were successfully knocked in and 4 were sequence verified by targeted locus amplification (TLA) technology. Mice containing the binary fixed light chain transgenic can be bred to homozygosity. The mice can be crossed with fully diverse heavy chain mice (e.g., humanized or human HC transgenic mice).

[0116] Final plasmid-based transgenic constructs, including appropriate vector sequences, are schematically illustrated in Figure 7 as shown.

[0117] Functional validation of the dual-fixed light chain mice can include genomic DNA analysis showing recombination occurring in B cells to generate two different light chain alleles, RNA analysis showing that in-frame, spliced transcripts can be generated in mouse B cells, and examination of the protein levels of the κ light chain. The titers and immunoreactivities against test antigens can also be examined and compared with wild-type mice.

[0118] Characterization of Transgenic Mice

[0119] To demonstrate that recombination occurs in B cells to generate RSS-recombined fixed light chain alleles, genomic DNA was prepared from ear or spleen biopsies, under the assumption that a higher frequency of B cells (and thus recombination) can be found in the latter compared to ear tissue. As Figure 8 seen, standard PCR was performed using gene-specific primers (see the direction arrows above the transgenic schematic). PCR amplicons of the appropriate size for the two recombinant alleles were found only in the spleen samples (as expected; see the arrows on the agarose gel photograph), and not in the ear DNA samples, where few or no B cells should be found. In addition, the amplicons indicated by the arrows were gel-purified and DNA sequenced according to the sequence chromatogram inset; the results showed that the junction sequences generated by RSS-mediated recombination were heterogeneous. This is the expected result for such junctions ultimately resolved via the non-homologous end joining (NHEJ) process.

[0120] To demonstrate that such rearranged fixed light chain loci are capable of correctly expressing spliced mRNA transcripts, RT-PCR was performed on spleen RNA samples from the transgenic common light chain mice. As Figure 9 seen, sequence analysis of individual cDNA clones showed that in-frame transcripts could be recovered when compared to a computer-generated hypothetical reference sequence, corresponding to the two fixed light chain alleles of the fully human 1-39 / J2 / kC light chain or the fully human 4-1 / J4 / kC light chain. Many of these sequences corresponded 100% to the reference sequence, while other sequences may vary due to accidental somatic hypermutation events.

[0121] To confirm that the rearranged and expressed fixed light chain alleles can participate in normal immune responses, transgenic mice were immunized with the COVID-19 spike protein and serum titers were measured by ELISA. Prior to immunization, the mice were bled to determine the baseline level of human k light chain expression. Three different mouse genotypes were analyzed. The first was a compound heterozygote consisting of one fixed light chain transgenic allele and one mouse κLC null allele (so-called KlaP, lacking all Vk and Jk sequences); the second was a compound heterozygote consisting of one wild-type mouse κ light chain allele and one mouse κLC null allele (KLaP); the third was normal wild-type mice. As Figures 10A - 10C shown in the results of Figures 10A - 10C , using a non-species-specific anti-κ detection reagent, the serum Igκ levels of the naive mice before immunization were indistinguishable. After immunization, a two-stage detection was used with COVID spike capture in the first stage followed by detection of all κ light chains (non-species-specific) in the second stage, and robust titers against the COVID spike protein were detected via sandwich ELISA. Determination of the midpoint of the serum titer dilution (EC50) showed that the mice containing the fixed light chain showed robust titers and differed from the wild-type response by only about 2-3-fold. Hybridoma fusion of the immunized mice followed by selection of antigen-specific clones yielded 200+ antigen-specific hits; a subset of 48 randomly selected clones was then sequenced for the light chain component, revealing that 45 of these were specific for the 1-39JK2 allele and 3 were specific for the 4-1JK4 allele. From the entire group of 48 clones, 13 different mouse VH heavy chain partners were identified. Together, these results demonstrate that the binary transgenic system is fully functional and can serve as a source of common light chains to facilitate bispecific antibody construction and therapeutic development.

[0122] Example 2: Preparation of Human λ Dual Common Light Chain Construct

[0123] In this example, the preparation of a transgenic construct for expressing one of two alternative "fixed" human λ light chains is described. Following the method for generating fixed light chain mice for human κ light chain described in Example 1, a similar procedure was carried out to design and generate fixed human λ light chain transgenes. The process has several steps:

[0124] (i) Determine the frequency of VL usage in humans, with the idea that highly expressed alleles are well tolerated and support pairing with a large number of potential heavy chain variable domains;

[0125] (ii) Combine VL usage data with JL usage data to define specific VL-JL conjugates available for preparing transgenes for knock-in;

[0126] (iii) Generate a computational version of the fixed or pre-rearranged human λ allele (cassette) in the context of their human genomic sequences; and

[0127] (iv) Assemble the cassette into a functional transgenic for gene targeting (i.e., also containing a constant region, an enhancer (if required), and engineered sites for site-specific gene delivery or knock-in).

[0128] λ Light Chain V Region Selection

[0129] Use the expression frequency of λV regions in the population as a starting point for λ light chain V region selection. IMGT ® The database provides information on Vλ frequencies, identifying that Vλ 2-14 and 3-19 are the most frequently used, but 3-21, 3-1, and 1-51 are also observed at high frequencies. IGVL frequencies as reported in human SARS2 patients were also examined (J. Exp. Med. (2022) Vol. 219, No. 9 e20220367). Additionally, Vλ and Jλ frequencies in the human naive repertoire were analyzed based on data published by DeKosky et al. (2015) Nat. Med. 21:86-91. Analysis of the data reported by DeKosky et al. led to the identification of the top three Vλ-Jλ pairs for each of the three patients studied. The dataset was sorted by frequency for the Vλ segment and pivot analysis was performed. Based on this analysis, two candidate VL-JL pairs were identified as VL2-14 / JL2 and VL1-40 / JL2. However, using the J2 segment for the two VL-JL pairs of the binary construct would result in nearly identical CDR3 domains from each. Since one of the characteristics of the binary fixed light chain method is that the transgenic provides a "choice" between two different λ light chains, sequence divergence is an important consideration, and a second VL-JL selection was made based on the divergence parameter. For this reason, non-J2 segments, such as the JL1 segment, can be used in one of the two VL-JL pairs of the final construct. Thus, the VL-JL pairs selected for the binary fixed λ light chain transgenic are VL2-14 / JL2 + VL1-40 / JL1.

[0130] Selection of VL2-14 / JL2 DNA sequence: Since there are many possible junctions between the VL-JL segments, it is helpful to rank different junction pairs in order to select a specific VL2-14 / JL2 DNA sequence for use in a transgenic construct. Based on the DeKosky dataset, the identified CDR3 regions were analyzed because it contains the VL-JL junction within it. Among the thousands of VL2-14 / J2 LCDR3 sequences returned from each of the three donors, the most frequently recovered LCDR3 was identified and an alignment was run using commercially available Geneious Prime software to generate a consensus sequence. Additionally, the CDR3 sequences were translated to obtain the amino acid sequences and an alignment was run to determine the most common amino acid sequence of CDR3. The LCDR3 sequence analysis yielded the following consensus sequence (where the sequence extends to the end of the J segment): CSSYTSSSTLVVFGGGTKLTVL (SEQ ID NO: 6). A pBLAST of this sequence confirmed that it is commonly found in the NCBI database. A pBLAST was also performed on the full-length variable domain of VL2-14 / JL2, which revealed that the full variable domain sequence also returned a BLAST hit. Overall, the sequence analysis of the VL2-14 / J2 combination confirmed that it is found in GenBank and is likely to be expressed and functional.

[0131] Selection of VL2-1-40 / JL1 DNA sequence: The 1-40 / J1 pair was analyzed in a similar manner to the 2-14 / J2 pair. The nucleotide sequences from the DeKosky dataset were aligned to determine the "consensus" LCDR3 from the VL1-40 / J1 transcript. The nucleotide sequences were also translated and an alignment of the translated sequences was prepared. The LCDR3 sequence analysis yielded the following consensus sequence (where the sequence extends to the end of the J segment): CQSYDSSLSGYVFGTGTKVTVLG (SEQ ID NO: 7). A pBLAST of the LCDR3 sequence and the sequence incorporated into the full variable domain confirmed that these sequences are readily found in the NCBI database. Overall, similar to the VL2-14 / J2 analysis, the sequence analysis of the VL1-40 / J1 combination indicates that these are likely to be well tolerated and are commonly found in the human lambda light chain repertoire.

[0132] Construction of Fixed Light Chain Transgene

[0133] In terms of the overall strategy, the "binary" method of fixed light chain mice has been verified with human kappa light chains, as described in Example 1 and Figure 2described in the general overview. Based on the success of the κ fixed light chain model, a λ fixed light chain model was created by replacing the previous κ-encoding sequence with the coding sequences identified in the above λ light chain V region selection. This results in a λ binary fixed light chain construct containing two λ variable region cassettes (λFLC1 and λFLC2) arranged in opposite orientations, where both cassettes are inactive in the germline and one of the two cassettes becomes active upon RAG recombination, as Figure 11 schematically illustrated in. As in the κ construct described in Example 1, the green and yellow triangles represent recombination signal sequences (RSSs), which typically function during recombination to join a given light chain V segment to a given light chain J segment. In this case, the RSS sites are used to join the coding sequences to each other; in this transgenic case, they are placed in non-coding regions but are still intended to perform the same recombination / joining function.

[0134] The two λ fixed light chain cassettes are designed to "pre-rearrange" the Vλ and Jλ segments, so there is no CDR3 variability or junctional diversity in this region. Additionally, given the success of the existing κ methods, many of the original κ light chain non-coding transgenic sequences were used to generate the λ transgene. Additionally, as described herein (e.g., in Example 1 and Figure 6 herein), the λ transgene can be site-specifically delivered to the murine κ light chain locus, such as by recombinase-mediated cassette exchange (RMCE). Alternatively, CRISPR-based recombination methods can be used to deliver the λ transgene to an endogenous light chain locus, such as the κ locus.

[0135] Mice containing the λ binary fixed light chain transgene can be bred to homozygosity. The mice can be crossed with fully diverse heavy chain mice (e.g., humanized or human HC transgenic mice).

[0136] Functional validation of the binary fixed light chain mice can include genomic DNA analysis showing recombination occurs in B cells to generate two different light chain alleles, RNA analysis showing in-frame, spliced transcripts can be generated in murine B cells, and examination of the protein levels of the λ light chain. The titer and immunoreactivity to a test antigen can also be examined and compared to wild-type mice.

[0137] Sequence Listing Summary

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

Claims

1. A transgenic construct, the transgenic construct comprising: (a) A first immunoglobulin light chain variable cassette (VL1) and a second immunoglobulin light chain variable cassette (VL2), wherein VL1 and VL2 each comprise a promoter, a light chain V region, a light chain J region, and a splice donor site; (b) A termination cassette (SC) comprising a splice acceptor site and a polyadenylation signal; and (c) A first recombination signal sequence (RSS) 12mer (RSS1), a second recombination signal sequence 12mer (RSS2), and an RSS23mer (RSS3); wherein the transgenic construct comprises from 5' to 3' VL1 - RSS1 - SC - RSS2 - VL2 - RSS3 and wherein VL2 is in an antisense orientation relative to VL1.

2. The transgenic construct according to claim 1, wherein in B cells carrying the transgenic construct, VL1 and VL2 are inactive before RAG - mediated recombination and VL1 or VL2 is active after RAG - mediated recombination.

3. The transgenic construct according to claim 1, wherein the light chain V region and J region are human κ sequences.

4. The transgenic construct according to claim 3, wherein VL1 or VL2 comprises the Vκ 1 - 39 region.

5. The transgenic construct according to claim 3, wherein VL1 or VL2 comprises the Jκ JK2 region.

6. The transgenic construct according to claim 3, wherein VL1 or VL2 comprises the Vκ 1 - 39 region and the Jκ JK2 region.

7. The transgenic construct according to claim 3, wherein VL1 or VL2 comprises the Vκ 4 - 1 region.

8. The transgenic construct according to claim 3, wherein VL1 or VL2 comprises the Jκ JK4 region.

9. The transgenic construct according to claim 3, wherein VL1 or VL2 comprises the Vκ 4 - 1 region and the Jκ JK4 region.

10. The transgenic construct according to claim 3, wherein VL1 comprises the Vκ 1 - 39 region and VL2 comprises the Vκ 4 - 1 region.

11. The transgenic construct according to claim 3, wherein VL1 comprises the Vκ 1 - 39 region and the Jκ JK2 region and VL2 comprises the Vκ 4 - 1 region and the Jκ JK4 region.

12. The transgenic construct according to claim 3, wherein VL1 comprises the Vκ 4 - 1 region and VL2 comprises the Vκ 1 - 39 region.

13. The transgenic construct according to claim 3, wherein VL1 comprises the Vκ 4 - 1 region and the Jκ JK4 region and VL2 comprises the Vκ 1 - 39 region and the Jκ JK2 region.

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

15. The transgenic construct according to claim 14, wherein the light chain constant region is a human κ constant region.

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

17. The transgenic construct according to claim 16, wherein the enhancer comprises the intronic human kappa enhancer (mEKi).

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

1.

19. The transgenic construct according to any one of claims 1-17, wherein VL1 or VL2 comprises a CDR3 containing the sequence shown in SEQ ID NO:

2.

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

3.

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

4.

22. The transgenic construct according to claim 1, wherein the transgenic construct comprises the sequence shown in SEQ ID NO:

5.

23. A transgenic animal comprising the transgenic construct according to any one of claims 1-22.

24. The transgenic animal according to claim 23, wherein the transgenic animal is a mouse.

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

26. A method of generating an antibody against an antigen of interest, the method comprising administering the antigen of interest to the transgenic mouse according to claim 25 such that an antibody that binds to the antigen of interest is produced.

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

28. The transgenic construct according to claim 1, wherein the light chain V region and J region are human lambda sequences.

29. The transgenic construct according to claim 28, wherein VL1 or VL2 comprises the V lambda 2-14 region.

30. The transgenic construct according to claim 28, wherein VL1 or VL2 comprises the J lambda JL2 region.

31. The transgenic construct according to claim 28, wherein VL1 or VL2 comprises the V lambda 2-14 region and the J lambda JL2 region.

32. The transgenic construct according to claim 28, wherein VL1 or VL2 comprises the V lambda 1-40 region.

33. The transgenic construct according to claim 28, wherein VL1 or VL2 comprises the J lambda JL1 region.

34. The transgenic construct according to claim 28, wherein VL1 or VL2 comprises the V lambda 1-40 region and the J lambda JL1 region.

35. The transgenic construct according to claim 28, wherein VL1 comprises the V lambda 2-14 region and VL2 comprises the V lambda 1-40 region.

36. The transgenic construct according to claim 28, wherein VL1 comprises a Vλ 2-14 region and a Jλ JL2 region and VL2 comprises a Vλ 1-40 region and a Jλ JL1 region.

37. The transgenic construct according to claim 28, wherein VL1 comprises a Vλ 1-40 region and VL2 comprises a Vλ 2-14 region.

38. The transgenic construct according to claim 28, wherein VL1 comprises a Vλ 1-40 region and a Jλ JL1 region and VL2 comprises a Vλ 2-14 region and a Jλ JL2 region.

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

40. The transgenic construct according to any one of claims 1 to 39, wherein the light chain constant region is a human κ constant region or a human λ constant region.

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

6.

42. The transgenic construct according to any one of claims 28-40, wherein VL1 or VL2 comprises a CDR3 containing the sequence shown in SEQ ID NO:

7.

43. A transgenic animal comprising the transgenic construct according to any one of claims 28-42.

44. The transgenic animal according to claim 43, wherein the transgenic animal is a mouse.

45. The transgenic mouse according to claim 44, further comprising a transgenic 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 VL1 or VL2.

46. A method of generating an antibody against an antigen of interest, the method comprising administering the antigen of interest to the transgenic mouse according to claim 45 such that an antibody that binds to the antigen of interest is produced.

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

Images