Antigen-binding molecules comprising unpaired variable domains

Abstract

To provide single variable domain antibodies that can be used as pharmaceutical products.SOLUTION: The invention relates to antibodies comprising unpaired variable domains, e.g., heavy chain variable (VH) domains, for binding antigen. The invention relates also to antibodies comprising two immunoglobulin (Ig) chains, where a first Ig chain comprises a variable domain and a constant domain, and a second Ig chain comprises a constant domain, and the second Ig chain lacks a variable domain thereby leaving the variable domain of the first Ig chain unpaired.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to polypeptides comprising unpaired antibody variable regions, e.g., unpaired VH domains, for binding antigens. The present invention also relates to animals, e.g., mice, expressing antibodies comprising one or more heavy chains or heavy chain variable regions, which lack a light chain variable domain that is paired with the heavy chain variable domain to form a paired antigen binding site. [Background technology]

[0002] The antigen-binding region of a natural human immunoglobulin is composed of two variable domains, a heavy chain variable (VH) domain and a light chain variable (VL) domain, which pair together to form the Fv region. The Fv region has an antigen-binding site provided by six loops of variable amino acid sequences (complementarity determining regions (CDRs)). The VH domain contains HCDR1, HCDR2 and HCDR3 interspersed with framework regions (FRs), while the VL domain contains LCDR1, LCDR2 and LCDR3 interspersed with FRs. One or more CDRs of the VH domain and / or of the VL domain bind to the antigen. Binding can be mediated by the CDRs of both the VH and VL domains or by the CDRs of one domain alone. HCDR3 of the VH domain often plays the major role in antigen binding, although the other CDRs of both domains can and often do contribute. Even if an antigen binds exclusively or primarily to the CDRs of the VH domain, the presence of a VL domain in the Fv can stabilize the VH in a functional binding conformation.

[0003] Antibody variable domains are generated in vivo through combinatorial rearrangement of gene segments at the immunoglobulin (Ig) locus in cells of the B-lymphocyte lineage, resulting in a repertoire of encoded amino acid sequences capable of binding to the diverse immunogenic stimuli encountered by the immune system. The human Ig heavy chain locus has approximately 41 functional V gene segments, 27 functional D gene segments, and 6 functional J gene segments, depending on the haplotype. Nucleic acids encoding VH domains are generated by recombination of VDJ gene segments. The V gene segments encode the N-terminal region of the polypeptide chain, including FR1, HCDR1, FR2, HCDR2, FR3, and the start of HCDR3, while the D gene segment is encompassed by HCDR3 and the J gene segment provides the end of HCDR3 and the C-terminal framework region FR4. The highly variable nature of HCDR3 sequences in the antibody repertoire reflects the combinatorial diversity generated by the rearrangement of many different V, D, and J segments. Humans have two Ig light chain loci: kappa (κ) and lambda (λ). Human Ig light chain loci have approximately 40 functional Vκ segments, 5 functional Jκ segments, 29 functional Vλ segments, and 4 functional Jλ segments, depending on the haplotype. Nucleic acids encoding VL domains are produced by recombination of two gene segments, V and J, at either the kappa (κ) or lambda (λ) locus. The v gene segment encodes FR1, LCDR1, FR2, LCDR2, FR3, and the first part of LCDR3, while the J gene segment forms the second part of LCDR3 and FR4. In addition to the combinatorial diversity resulting from VDJ and VJ recombination and from VH / VL pairing, further antibody sequence diversity is generated by junctional mutations at the junctions of gene segments and by the in vivo process of somatic hypermutation in response to antigen binding.

[0004] WO90 / 05144 (MRC) discloses that a VH domain, when isolated from a complete antibody comprising a heavy and light chain, can bind to an antigen in a 1:1 ratio and with a binding constant comparable in magnitude to that of the complete antibody molecule.

[0005] Camelids (a family of animals that includes camels and llamas) naturally produce antibodies that bind antigen with unpaired VH domains in the absence of a VL. Such antibodies are bivalent binders that are completely devoid of immunoglobulin light chains and are composed of homodimeric heavy chains, each of which contains an antigen-binding single VH or "VHH" domain, a hinge region, and a dimerized constant region containing CH2 and CH3 domains. Although homologous to the heavy chains of classical mammalian antibodies, such "heavy chain antibodies" ("HCAbs") lack the first domain of the constant region (CH1), which is excised during mRNA processing (WO94 / 04678 Casterman & Hamers; WO96 / 34103 Hamers & Muyldermans).

[0006] Similar mutations occur in a human pathological condition known as heavy chain disease. Immunoglobulin heavy chains are expressed containing the VH, CH2 and CH3 domains, but lacking the CH1 domain. In patients with this disease, heavy chains are found to accumulate instead of pairing with light chains to form normal antibodies.

[0007] Cartilaginous fish such as sharks also have antibodies composed of heavy chains and lacking any light chains. These antibodies are called IgNARs (immunoglobulin new antigen receptors) and have been used as a source of single domain antibodies called VNAR fragments.

[0008] Researchers have been working to develop single variable domain antibodies into pharmaceuticals. These molecules, known as domain antibodies (dAbs), are bioactive as monomers, and their small size and inherent stability also make them highly suitable for assembly into larger molecules to create drugs with extended serum half-life and / or other pharmacological activity. Antigen-binding molecules that contain antibody single variable domains include immunoconjugates (e.g., dAb toxins) and chimeric antigen receptors (CARs). To this end, antibody single variable domain binders have been cloned and expressed in recombinant systems, and in vitro libraries of such binders have been generated for selection and screening using systems such as phage display.

[0009] Experimental animals such as mice have been genetically engineered to express heavy chain antibodies as an additional source of single antigen-binding variable domains. One can start by knocking out (deleting or inactivating) the animal's endogenous Ig light chain locus (WO92 / 03918 Genpharm; WO03 / 000737 The Babraham Institute). Then, only the heavy chain is expressed and homodimerized (Figure 1a). However, the CH1 domain of the heavy chain is intrinsically disordered and adopts a normal immunoglobulin fold only when interacting with its cognate partner light chain Cκ or Cλ domain. Expression of Ig heavy chains has been reported to be unproductive due to misfolding and aggregation of the CH1 domain. Nevertheless, in light chain-deficient mice, some functional HCAbs are spontaneously produced through a variant of the class switching mechanism. In class switching, the nucleic acid encoding the VH is normally linked to the nucleic acid encoding the CH1-CH2-CH3 constant region, but in an unusual mechanism, the VH is joined only to CH2-CH3. The resulting HCAbs, like those in camelids, lack the CH1 domain and can be expressed and selected for antigen binding, but functional HCAbs are only generated with low efficiency by this method because normal mechanisms of class switching dominate, producing complete Ig heavy chains, including CH1.

[0010] Thus, mouse strains used to produce heavy chain antibodies have been engineered to have genetic deletions of the CH1 domain of the immunoglobulin heavy chain and knockouts of the light chain. Such mice produce antibodies that contain dimeric heavy chains and lack light chains, with each heavy chain having a variable (VH) domain and constant regions CH2 and CH3. The constant regions dimerize to form an Fc region, while the two VH domains are available for bivalent antigen binding (Figure 1b). After immunization of mice with a target antigen, heavy chain antibodies specific for the antigen can be generated, selected, affinity matured in vivo, and isolated. Nucleic acids encoding the VH domains can then be recombinantly expressed and optionally cloned to provide polypeptides comprising the VH domains, optionally in the context of larger molecules such as CARs or other products for therapeutic or diagnostic use.

[0011] Erasmus Universiteit Rotterdam has described transgenic mice containing exons from camelid VHH domains or "camelized" VH domains in their genome and heavy chain constant region genes that did not express a functional CH1 domain (WO02 / 085944, WO02 / 085945, WO2006 / 008548, WO2010 / 109165). "Camelized" VHs are human (or other non-camelid) VH sequences that have been mutated to resemble camelid VHHs. In such transgenic platforms, the heavy chain genes have been engineered to eliminate a functional CH1 domain. As reported in WO02 / 085944, in the absence of CH1, heavy chain antibodies could not associate with light chains to form "conventional" antibodies, since CH1 is the natural partner of the light chain constant domain. WO2006 / 008548 reported that normal B cell maturation and antibody production in these mice depended on the complete absence of CH1 sequences from each heavy chain constant region present in the transgenic locus. WO2004 / 049794 (The Babraham Institute) described the production of HCAbs from YAC transgenes in mice, where the CH1 domain of the heavy chain is excised during mRNA processing. The resulting mice could then be crossed with mice in which the endogenous heavy and light chain genes were knocked out to generate mice expressing only the desired HCAbs.

[0012] Unfortunately, "heavy chain only" mice carrying CH1 deletions in their Ig heavy chains do not have normal B cell populations. Mice lacking the DNA encoding the CH1 domain from both the μ and γ heavy chain constant region genes produced IgM heavy chains lacking CH1 (CH1Δμ) and IgG heavy chains lacking CH1 (CH1Δγ), but these mice had an increased proportion of immature B cells and impaired differentiation into follicular and marginal zone B cells. Nevertheless, the CH1 deletion was essential for the expression of both IgM and IgG antibodies in these mice, because inclusion of the CH1 domain in either the μ or γ heavy chains resulted in nonproductive expression [1].

[0013] Human VH domains are desirable for administration to humans because they are associated with lower immunogenic side effects in patients compared to administration of polypeptides of non-human origin, such as camelid VHHs. Transgenic mice expressing human antibody heavy chains are a source of human VH domains that undergo in vivo selection for antigen binding. However, human antibodies naturally contain both heavy and light chains, and in the absence of light chains, only a subset of heavy chain variable regions can generate functional heavy chain antibodies. Thus, human VH domains from heavy chain antibodies produced in heavy chain-only mice lack the sequence diversity of VH domains from intact immunoglobulins. The limited repertoire of human VH domains in such mice is exacerbated when human Ig heavy chain genes are introduced at random insertion points in the mouse genome, because the ectopic transgenic loci produce VH domains with HCDR3 sequences that are shorter than in humans (believed to result from limited N-addition during VDJ recombination) and undergo limited hypermutation, resulting in an already low diversity of VH domains. This appears to be an inherent limitation of known random insertion transgenic platforms for human antibody generation. The latter problem can be solved by integrating human immunoglobulin genes into the endogenous immunoglobulin loci of the host animal, rather than at random positions in the genome [2]. Recombination and somatic hypermutation at the native locus in such mice generate a more diverse array, which allows the evolution of the resulting less soluble VH domains into domains with improved biophysical properties (e.g., solubility) in vivo.

[0014] WO2011 / 072204 (Regeneron Pharmaceuticals) described mice expressing heavy chain antibodies comprising a human VH domain and a mouse constant region with a deleted CH1 domain. These mice were said to contain a germline deletion of the CH1-encoding sequence of the endogenous Ig constant region genes, thus rendering the mice incapable of expressing IgG mRNA comprising the sequence encoding the CH1 domain, but because the CH1 domain of the IgM isotype constant domain was not deleted, the mice retained the ability to express normal functional IgM antibodies. WO2013 / 171505 (Kymab Limited) described mice engineered to express normal IgM and CH1-deleted IgG antibodies, where stage-specific class switching from IgM to IgG in lymphocytes involved inactivation of the endogenous Ig light chain locus and genetic deletion of CH1 from the IgG constant region, resulting in IgG antibodies being expressed by the cells in the absence of light chain expression. This modification allowed the development of antibodies and the B cell compartment to proceed through the preferred four chain (H2L2) endogenous IgM stage followed by a subsequent IgG stage that selected for only heavy chain-only (H2) antibodies derived from the favorable pool of heavy chain VDJ recombinations provided by the earlier four chain IgM stage, but essentially eliminated the possibility of four chain antibodies at this subsequent stage.

[0015] WO2018 / 039180 (Teneobio, Inc.) reported that HCAbs with reduced aggregation propensity could be prepared by replacing the natural amino acid residue at the first position of FR4 of the HCAb with another amino acid residue to disrupt the surface-exposed hydrophobic patch that is buried at the VH-VL interface in normal Fvs. Exposure of the hydrophobic patch was identified as a contributing factor in undesired aggregation of heavy chains in the absence of light chains as well as in VH-VL domain pairing in the presence of light chains. Rats were engineered to express HCAbs containing the identified VH residue mutations and heavy chain homodimerization was performed by inactivation of the endogenous light chain locus.

[0016] Although less common than heavy chain-only antibodies, the art has also described the production of antibodies comprising unpaired VL domains. WO2009 / 143472 (Aliva Biopharmaceuticals) and WO2015 / 143414 (Regeneron Pharmaceuticals) described a method for generating antibodies with unpaired VL domains in transgenic animals by linking a genetic sequence encoding a human VL domain to a heavy chain constant region with a deletion of the CH1 domain. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] WO90 / 05144 (MRC) [Patent Document 2] WO94 / 04678 Casterman & Hamers [Patent Document 3] WO96 / 34103 Hamers & Muyldermans [Patent Document 4] WO92 / 03918 Genpharm [Patent Document 5] WO03 / 000737 The Babraham Institute [Patent Document 6] WO02 / 085944 [Patent Document 7] WO02 / 085945 [Patent Document 8] WO2006 / 008548 [Patent Document 9] WO2010 / 109165 [Patent Document 10] WO2004 / 049794 (The Babraham Institute) [Patent Document 11] WO2011 / 072204 (Regeneron Pharmaceuticals) [Patent Document 12] WO2013 / 171505 (Kymab Limited) [Patent Document 13] WO2018 / 039180 (Teneobio, Inc.) [Patent Document 14] WO2009 / 143472 (Aliva Biopharmaceuticals) [Patent Document 15] WO2015 / 143414 (Regeneron Pharmaceuticals) [Patent Document 16] WO2011 / 004192 (Genome Research Limited) [Patent Document 17] U.S. Patent No. 5,731,168 [Patent Document 18] WO2006 / 106905 [Patent Document 19] WO2013 / 157954 [Patent Document 20] WO2011 / 143545 [Patent Document 21] WO2007 / 110205 [Patent Document 22] WO2010 / 151792 [Patent Document 23] WO2013 / 061078 Summary of the Invention [Problem to be solved by the invention]

[0018] In the field of antibodies, including unpaired variable domains and transgenic animals to produce them, the present invention represents a move away from "heavy chain only" antibodies and "heavy chain only mice", which have been the subject of the art to date. The inventors have realized that neither CH1 deletion nor the absence of light chains is necessary to produce an antibody that binds antigen via its VH domain alone. In the present invention, the VL domain of the antibody is deleted and the CL domain is retained. This produces an antibody that comprises a heavy chain and a light chain, the heavy chain comprising an unpaired VH domain for binding antigen and a constant region comprising a CH1 domain, and the light chain comprising a CL domain and no VL domain (Figure 2). The absence of the VL domain renders the VH domain unpaired. The retained CL domain binds to the CH1 of the heavy chain, stabilizing the antibody molecule and inhibiting heavy chain aggregation. Thus, the heavy chain CH1 domain can be optionally retained as part of a complete heavy chain constant region (e.g., CH1-CH2-CH3 or CH1-CH2-CH3-CH4). The heavy and (residual) light chains are paired through the association of CH1 and CL.

[0019] Similar modifications may be used to generate antibodies that bind antigen through a VL domain in the absence of an associated VH domain. Antibodies comprising unpaired VH and / or VL domains can optionally include further modifications, such as changes to the format and design of the antibody.

[0020] Transgenic animals such as mice can be engineered to produce antibodies according to the invention for the in vivo generation and selection of antigen-specific variable domains capable of binding antigen outside the context of a VH-VL pair (Fv). By retaining the CH1 and allowing its pairing and stabilization with the shielding domain, the antibody repertoire that can be generated in animals expressing the antibodies of the invention can be significantly larger than CH1-deleted antibody platforms, where the in vivo immune response has been limited in some respects. When immunized with a target antigen of interest, animals according to the invention generate antibodies against the target, the antibodies comprising an unpaired variable domain that binds the target. Such antibodies and / or nucleic acids encoding them can then be recovered from the animals, and DNA encoding the variable domains can then be recombinantly expressed and, optionally, the unpaired variable domains can be incorporated into binding molecules (e.g., antibodies or chimeric antigen receptors) comprising the variable domain and one or more further protein domains. The unpaired variable domains, antibodies and other binding molecules comprising them, nucleic acids encoding them, cells and transgenic animals containing such nucleic acids, as well as methods of generating and using the foregoing, are all aspects of the invention. [Means for solving the problem]

[0021] In a first aspect, the invention provides an antibody comprising an unpaired variable domain (VH domain or VL domain) linked to a constant region, the constant region comprising a CH1 domain and a domain paired to the CH1 domain. The domain paired to the CH1 domain is referred to herein as a "shielding domain", and the interface between the heavy chain CH1 domain and the shielding domain may act to stabilize the CH1 domain, thereby increasing the solubility of the antibody and / or inhibiting aggregation. The shielding domain may be a polypeptide domain, such as an immunoglobulin domain, i.e. a polypeptide domain comprising an immunoglobulin fold. The shielding domain may be a CL domain (Cκ or Cλ) or an Ig domain of a surrogate light chain λ5 protein. An example of a λ5 protein may be a human λ5 domain lacking the 50 amino acid unique region at the N-terminus of human λ5, or an example of a λ5 protein may be a non-human λ5 domain lacking a region corresponding to the 50 amino acid unique region at the N-terminus of human λ5. Preferably, the shielding domain is a CL domain, e.g., Cκ, such that the constant region comprises a CH1:CL domain pair. The unpaired variable domain can be linked to either the CH1 domain or the shielding domain, e.g., as a fusion protein. This core structure of an unpaired variable domain linked to a constant region comprising a CH1:CL or CH1:λ5 domain pair may be provided in isolation (a protein consisting of that structure) or as part of a larger polypeptide molecule, optionally comprising further antibody constant domains and / or functional moieties. Although such further domains and moieties may be included, the antibodies of the invention characteristically comprise an unpaired variable domain for binding antigen, and thus lack any polypeptide domain that pairs with said variable domain to provide an antigen-binding site.

[0022] The constant region can comprise two polypeptide chains, one containing the CH1 domain and one containing the shield domain. Thus, an antibody can have a first polypeptide comprising unpaired variable domains; A second polypeptide; may include the first polypeptide comprises a CH1 domain and the second polypeptide comprises a shield domain paired with the CH1 domain; or The first polypeptide comprises a shield domain and the second polypeptide comprises a CH1 domain.

[0023] For example, the antibody may be a first polypeptide comprising unpaired variable domains (e.g., VH domains) and a CH1 domain; a second polypeptide comprising a shield domain (e.g., CL or λ5) paired with a CH1 domain; may include. In the first polypeptide, the variable domain is preferably the N-terminal domain, followed preferably by an adjacent CH1 domain. The second polypeptide lacks the N-terminal variable domain, thereby leaving the variable domain of the first polypeptide unpaired. The second polypeptide may comprise the shielding domain and may be devoid of further domains, or may consist of the shielding domain and a C-terminal extension and / or functional portion of one or more further domains (e.g., an Ig constant domain). Optionally, the second polypeptide consists of the shielding domain.

[0024] Alternatively, the antibody is a first polypeptide comprising a variable domain (e.g., a VH domain) and a shield domain; a second polypeptide comprising a CH1 domain paired with the shield domain; may include The second polypeptide lacks a variable domain, thereby leaving the variable domain of the first polypeptide unpaired. In the first polypeptide, the variable domain is preferably the N-terminal domain followed by an adjacent shield domain. The second polypeptide lacks the N-terminal variable domain, thereby leaving the variable domain of the first polypeptide unpaired. The second polypeptide may comprise the CH1 domain and be free of further domains, or may consist of the CH1 domain and a C-terminal extension and / or functional portion of one or more further domains (e.g., Ig constant domains). Optionally, the second polypeptide consists of the CH1 domain.

[0025] The pairing of the CH1 domain with the shielding domain forms a constant region, which may additionally comprise further domains such as CH2, CH3 and / or CH4, typically as C-terminal domains. The first or second polypeptide may comprise CH2-CH3 linked to the C-terminus of the CH1 or shielding domain, respectively. The first polypeptide may comprise (in the N-to-C direction) an unpaired variable domain linked to CH1-CH2-CH3 (e.g., VH-CH1-CH2-CH3), and the second polypeptide may lack an N-terminal variable domain and comprise or consist of a shielding domain (e.g., CL or λ5). As another example, the first polypeptide may comprise (in the N-to-C direction) an unpaired variable domain (e.g., VH-CH1) linked to CH1, and the second polypeptide may comprise (in the N-to-C direction) a shielding domain linked to CH2-CH3 (e.g., CL or λ5 linked to CH2-CH3). The domains can be directly linked by a peptide bond or a peptide linker. The hinge region is usually located immediately upstream of the CH2 domain, such that the polypeptide can include a CH1 domain or a shield domain linked to the CH2-CH3 domains via the antibody hinge region. The constant region of the antibody according to the invention can be a human constant region. It can comprise or consist of the amino acid sequence of the constant region of a natural human IgG, for example of human IgG1. Alternatively, the constant region can be derived from a non-human animal, such as a rodent (e.g., a mouse constant region or a rat constant region). In the non-human animals described herein, the constant region can be an endogenous constant region encoded by an endogenous immunoglobulin locus in the genome of the non-human animal.

[0026] Exemplary pairings of first and second polypeptides in the antibodies of the present invention (domains shown in the N to C direction) are shown in Table 1 below.

[0027] [Table 1]

[0028] As shown in.

[0029] Polypeptides that include CH2-CH3 can include additional domains, for example, some antibody isotypes include a CH4.

[0030] In a given CH1:shielding domain pair, any further constant domain (e.g., CH2-CH3) is usually linked to only one of the CH1 and shielding domains. However, the CH2-CH3 constant domain naturally dimerizes to form an antibody Fc region. A polypeptide comprising CH2-CH3 can associate with a second polypeptide comprising CH2-CH3 through interchain pairing between the CH2 and / or CH3 regions to form an Fc region comprising dimerized CH2-CH3. Interchain disulfide bonds can form, which are usually present in natural antibodies. Such dimerization can produce antibodies that comprise multiple antigen-binding sites. For example, an antibody can comprise two unpaired variable domains, each linked to a constant region comprising CH2 and / or CH3.

[0031] The antibody can comprise two first polypeptides and two second polypeptides. The pairs of first and second polypeptides can be independently selected from those shown in Table 1 above. The antibody can comprise two first polypeptides and two second polypeptides, each first polypeptide comprises unpaired variable domains and a CH1 domain; each second polypeptide comprises a shield domain paired with a CH1 domain; One or both of the second polypeptides lack a variable domain.

[0032] Preferably, the antibody comprises two unpaired variable domains. For example, a four-chain antibody can comprise two unpaired variable domains (e.g., two VH domains), each linked to a CH1:shield domain pair, and an Fc region. Optionally, the four-chain antibody comprises two first polypeptides and two second polypeptides, both of which have the same domain structure, e.g., two first polypeptides VH-CH1-CH2-CH3 and two second polypeptides CL. Thus, a four-chain antibody can comprise two first polypeptides, the first polypeptides being any of the first polypeptides shown in Table 1, and two second polypeptides, the second polypeptides being the corresponding second polypeptides shown in Table 1. The two first polypeptides can have the same amino acid sequence, i.e., the antibody can comprise two copies of the same first polypeptide. The two second polypeptides can have identical amino acid sequences, i.e., the antibody can comprise two copies of the same second polypeptide. Alternatively, the sequences of the two first polypeptides can differ from each other and / or the sequences of the two second polypeptides can differ from each other. The sequence difference is optionally within (e.g., only within) the variable domains, e.g., the antibody can comprise two different unpaired variable domains.

[0033] With reference to the structure of a complete four-chain immunoglobulin comprising two heavy-light chain pairs, each heavy chain comprises VH-CH1-CH2-CH3 and each light chain comprises VL-CL, and antibodies with unpaired VH domains can be produced by removing one or both VL domains and leaving the CL domain in place. An antibody according to the invention can comprise two heavy chains and two light chains, each heavy chain comprising a VH domain and a constant region comprising a CH1 domain, each light chain comprising a CL domain, and one or both light chains lacking a VL domain, thereby leaving one or both VH domains unpaired. Conversely, antibodies with unpaired VL domains can be produced by removing one or both VH domains and leaving the CH1 domain in place. Variations can be illustrated with reference to other antibody formats, some of which are discussed herein. Selection for expression and antigen binding in transgenic animals is facilitated when the four-chain antibody is composed of two identical first polypeptides (e.g., two identical heavy chains) and two identical second polypeptides (e.g., two identical light chains), because this affects the natural mode of antibody expression, assembly, and display on the surface of antibody-producing cells in animals such as mice and humans.

[0034] In a preferred embodiment, both VL domains of an antibody are deleted, thereby producing a four-chain immunoglobulin that has the natural structure of an immunoglobulin (e.g., IgG or IgM) except for the absence of the VL domain (Figure 2). Similarly, antibodies with unpaired VL domains can be generated by deleting the VH domain and retaining the paired CH1:CL domains.

[0035] The antibody according to the invention may comprise two heavy chains and two light chains, Each heavy chain comprises an unpaired VH domain for binding a target antigen and a heavy chain constant region comprising a CH1 domain; Each light chain contains a shield domain (eg, a CL domain), and the light chain lacks a VL domain.

[0036] The light chain may comprise a shielding domain and no further domains, or may consist of a shielding domain and a C-terminal extension (e.g., an Ig constant domain) and / or functional portion of one or more further domains. Optionally, the light chain consists of a shielding domain. Antibodies lack a VL domain or other domain that pairs with a VH domain to form an antigen-binding site, such that the unpaired VH domain provides the binding site for the target antigen.

[0037] Optionally, the positions of the CH1 domain and the shielding domain (e.g., CL) can be exchanged relative to their positions in natural human antibodies. In a four-chain antibody containing two binding arms, the positions of the CH1 domain and the shielding domain can be exchanged in only one arm or in both arms. Thus, the overall format of the molecule can be symmetric or asymmetric.

[0038] Similarly, the natural positions of the VH and VL domains are interchangeable, such that, optionally, the VH domain is linked to the shield domain or the VL domain is linked to the CH1 domain.

[0039] Different antibody chain formats can be combined to form heterodimers (e.g., each half of a heterodimer comprising unpaired variable domains and two-chain constant regions), which are optionally bispecific with respect to antigen binding. An antibody can comprise one unpaired variable domain specific for one antigen or epitope and a second unpaired variable domain specific for a different antigen or epitope. Bispecific antibodies comprising unpaired variable domains capable of binding a first and a second antigen or epitope, respectively, can also be assembled by dimerization of polypeptides of the same format (each half of the dimer comprising unpaired variable domains and two-chain constant regions). Thus, optionally, only the unpaired variable domains differ in amino acid sequence, and the antibody molecules are otherwise symmetrical. Additional antigen binding regions can be optionally incorporated to provide trispecific or further order multispecific binding.

[0040] The polypeptide domains and antibodies according to the invention are preferably human. The unpaired variable domains may be human. The antibodies may be fully human.

[0041] The genome of non-human animals can be engineered to produce antibodies according to the invention. In the case of the production of antibodies with unpaired human variable domains, these will be transgenic animals with genomes into which human immunoglobulin genes have been integrated. The non-human animals can have genomes that include immunoglobulin loci engineered to express antibodies with unpaired variable domains according to the invention. B-lymphocytes of such animals can express antibodies according to the invention in response to antigenic stimulation, and antibodies with unpaired variable domains can be generated by administering a target antigen to the animal. Non-human animals and their cells for the production of antibodies with unpaired variable domains, methods of producing them by genetic engineering, and the use of the animals or cells to generate antibodies all represent further aspects of the invention. Suitable non-human animals include laboratory animals such as rodents, e.g. mice and rats.

[0042] In one embodiment, an animal, e.g. a mouse, according to the invention expresses an antibody comprising one or more heavy chains or VH domains, the antibody being characterized in that it lacks a VL domain which pairs with the VH domain to form an Fv region, the heavy chain comprises a CH1 domain (or the VH domain is linked to a CH1 domain), and the antibody comprises a shielding domain which is paired with the CH1 domain.

[0043] The genome of a non-human animal or a non-human animal cell can be engineered for expression of an antibody according to the invention, for example by A plurality of variable region gene segments that can be rearranged to encode variable domains upstream of DNA encoding an immunoglobulin constant region that includes a CH1 domain; and Genes encoding shielding domains, e.g., CL domains, that lack functional expression of variable region gene segments can be manipulated to include

[0044] In another embodiment, an animal, e.g., a mouse, according to the present invention expresses an antibody comprising one or more heavy chains or VH domains, wherein the antibody is characterized in that it lacks a VL domain which pairs with the VH domain to form an Fv region, the heavy chain comprises a shielding domain (or the VH domain is linked to a shielding domain), and the antibody comprises a CH1 domain which is paired with the shielding domain.

[0045] The genome of a non-human animal or a non-human animal cell, a plurality of variable region gene segments that can be rearranged to encode a variable domain upstream of DNA encoding a shielding domain (e.g., a CL domain) that lacks functional expression of the variable region gene segments; and Genes encoding immunoglobulin constant regions containing CH1 domains that lack functional expression of variable region gene segments can be manipulated to include

[0046] In the case of expression of an antibody comprising an unpaired VH domain for binding an antigen, the functional expression of the endogenous light chain variable domain can be inactivated in the animal, for example by knocking out the endogenous expression of the variable region gene segments from the lambda and / or kappa loci. Preferably, VJ rearrangement of the light chain gene segments does not occur or is non-productive in the B lymphocytes of the animal according to the invention. The functional expression of VpreB can optionally be inactivated in the animal, for example by deleting or mutating the endogenous VpreB gene to render it non-functional, so that the animal does not functionally express the VpreB polypeptide. The animals, cells (e.g., B lymphocytes) and antibodies according to the invention may be devoid of VL domains and VpreB. Alternatively, the expression of the VL domain and / or VpreB in the cells or animals (e.g., B lymphocytes of the animal) can be minimal, for example less than 10%, optionally less than 5%, compared to that of an antibody comprising an unpaired VH domain.

[0047] The animals according to the invention represent a departure from prior art platforms for the production of antibodies with unpaired variable domains. Up until now, binding molecules comprising or consisting of single binding domains were generated in the context of heavy chain only (or light chain only) antibodies, and therefore the heavy chain (or light chain) was isolated, expressed and the molecule engineered to counter the loss of stability, solubility and / or other properties compared to antibodies in which the binding site is provided by a VH-VL pair. The aggregation problem caused by the heavy chain CH1 domain was previously solved by deleting that domain, and a variety of different antibody discovery platforms were generated based on the CH1 deletion approach. The present invention uniquely provides an antibody discovery platform in which the CH1 domain is retained and stabilized through pairing with a shield domain, thereby allowing the in vivo selection of single binding domains from a repertoire of immunoglobulins that mirrors the CL:CH1 pairing of natural four-chain antibodies in the context of an otherwise natural antibody structure, and that provides unpaired antibody variable domains. Antibodies of interest can be selected directly from the repertoire of antibodies generated in the transgenic animals according to the invention. The unpaired variable domains of such antibodies can also be selected and used as binders in the form of single domain binding molecules, or they can be incorporated into larger binding molecules such as chimeric antigen receptors (CARs) in which the unpaired variable domains provide the antigen binding site.

[0048] The genome of a non-human animal or a non-human animal cell, a first immunoglobulin locus capable of expressing a heavy chain or a first polypeptide according to the invention, the heavy chain or the first polypeptide comprising a variable domain (e.g. a VH domain) and a CH1 domain; and A second immunoglobulin locus capable of expressing a light chain or a second polypeptide according to the invention, wherein the light chain or the second polypeptide comprises a shielding domain (e.g. a Cκ domain) paired with said CH1 domain, and wherein the light chain or the second polypeptide lacks a variable domain. can be manipulated to include The heavy chain or first polypeptide and the light chain or second polypeptide expressed from the respective loci can pair through the association of the CH1 domain and the shielding domain, and the absence of a variable domain in the light chain or second polypeptide renders the variable domain of the heavy chain or first polypeptide unpaired.

[0049] For example, the genome of a non-human animal or a non-human animal cell may be an immunoglobulin heavy chain locus that encodes, or can be rearranged to encode, an immunoglobulin heavy chain comprising a human VH domain and a CH1 domain; and An immunoglobulin locus that has been engineered to express a polypeptide that includes a shielding domain (e.g., a lambda or kappa CL domain, or λ5) paired with a CH1, wherein the polypeptide does not include a variable domain. The nucleic acid encoding the VL domain of the light chain can be absent and / or VJ rearrangement at the locus can be inactivated. Preferably, the animal completely lacks functional expression of a light chain comprising a VL domain, and thus the endogenous light chain locus can be modified to prevent functional expression of the VL domain.

[0050] Preferably, the immunoglobulin heavy and / or light chain loci are endogenous immunoglobulin loci of the animal. Thus, the endogenous immunoglobulin heavy chain locus (or loci) on mouse chromosome 12 can be engineered to contain DNA of a human heavy chain locus, thereby expressing a heavy chain containing a human VH. The immunoglobulin light chain locus (or loci) can be an endogenous immunoglobulin kappa light chain locus on mouse chromosome 6 and / or an endogenous immunoglobulin lambda light chain locus (or loci) on mouse chromosome 16. An immunoglobulin domain for pairing with CH1 (shield domain) can be expressed at the endogenous immunoglobulin locus of the animal under the control of a human or endogenous transcriptional control element (promoter / enhancer), for example, a polypeptide containing a CL domain can be expressed at the endogenous light chain locus. The light chain locus can be lambda or kappa. Optionally, both the lambda and kappa loci are engineered to express a light chain that contains a CL domain and lacks a VL domain.

[0051] The non-human animal according to the invention may comprise B lymphocytes expressing antibodies comprising unpaired VH domains for binding an antigen, the genome of the animal comprising: A plurality of variable region gene segments that can be rearranged to encode variable domains upstream of DNA encoding an immunoglobulin constant region that includes a CH1 domain; and Genes encoding CL domains that lack functional expression of variable region gene segments Includes.

[0052] In animal B lymphocytes, the genome is an antigen-binding variable domain linked to a CH1 domain, and a light chain comprising a CL domain and lacking a VL domain, thereby leaving the antigen-binding variable domain unpaired; The antibody is functional to express an antibody comprising:

[0053] For the generation of non-human animals containing B lymphocytes expressing antibodies that contain unpaired VH domains for binding antigen, a suitable method is to A plurality of variable region gene segments that can be rearranged to encode variable domains upstream of DNA encoding an immunoglobulin constant region that includes a CH1 domain; and Genes encoding CL domains that lack functional expression of variable region gene segments manipulating the genome of a non-human animal cell (e.g., an embryonic stem cell or a zygote) to comprise generating an animal from said cell or from a population of cells comprising said cell; Includes.

[0054] The variable region gene segments can be rearranged to encode a VH domain or a VL domain for binding a target antigen. A VH domain is generated by rearrangement of one V gene segment, one D gene segment, and one J gene segment, and the genome can be engineered to include one or more V gene segments, one or more D gene segments, and one or more J genes to rearrange to encode a VH domain. The minimum is one V, one D, and one J, but the inclusion of a larger number of V, D, and / or J gene segments results in a greater variety of codable VH domains. Alternatively, if the unpaired variable domain of an antibody is a VL domain, the genome can be engineered to include one or more V gene segments and one or more J gene segments to rearrange to encode a VL domain. The minimum is one V and one J, but the inclusion of a larger number of V and J gene segments results in a greater variety of codable VL domains.

[0055] Preferably, the gene segments are human. For example, a full set of human heavy chain V, D and J gene segments may be included, or a full set of human light chain V and J gene segments may be included. The genome may include 41 human heavy chain V gene segments. The genome may include a human heavy chain D gene segment. The genome may include 6 human heavy chain J gene segments. The genome may include 38 light chain κ V gene segments. The genome may include 5 human light chain κ J gene segments. Preferably, the CL domain is a human CL domain, e.g., human Cκ or human Cλ.

[0056] The gene encoding the CL domain can comprise an insert DNA of a human Ig light chain locus, comprising an exon encoding a light chain variable region leader sequence (e.g., human Vκ leader sequence) and an exon encoding a CL domain (e.g., human Cκ domain) separated by an intron containing a JC intron enhancer element. Transcription of the DNA results in excision of the intron, thereby joining the two exons, thus resulting in a nucleic acid encoding the CL domain (e.g., comprising the nucleotide sequence of SEQ ID NO:5) linked to an upstream (5') leader sequence encoding a signal peptide. The human light chain variable region gene segment is not functionally expressed, may be absent (deleted or not included in the animal genome), or may be inactivated. The encoded CL domain can be a human Cκ domain comprising SEQ ID NO:4. The encoded sequence can comprise or consist of SEQ ID NO:6, which includes an N-terminal signal peptide SEQ ID NO:2, which can be post-translationally cleaved to leave the Cκ domain SEQ ID NO:4. FIG. 3.

[0057] The human variable region gene segment can be inserted at the endogenous immunoglobulin heavy chain locus of the genome. The gene encoding the human CL domain can be inserted at the endogenous light chain locus of the genome (e.g., the Igκ locus). In one embodiment, human transcriptional control elements are included with the coding sequence. However, optionally, the inserted human DNA can be under the control of endogenous transcriptional control elements in the animal genome. In one embodiment, the inserted human DNA is (i) endogenous control elements; (ii) rodent control elements; (iii) mouse regulatory elements; (iv) rat regulatory elements; (v) primate regulatory elements; (vi) non-human primate (e.g., monkey) regulatory elements; (vii) a human regulatory element; or (viii) A mixture of two of (i) to (vii), including both human and rodent (e.g., mouse or rat) elements (e.g., a human promoter and a rodent intronic enhancer and / or a 3' locus enhancer). The gene is under the control of one or more control elements (e.g., a promoter and / or an enhancer (such as an intron enhancer and / or a 3' locus enhancer)) selected from:

[0058] In another example, the control elements can be mammalian.

[0059] Expression of endogenous Ig heavy and / or light chains can be inactivated, for example, by deletion or inactivation of variable region gene segments. In one example, the endogenous lambda locus comprises a deletion of at least 100, 150, or 200 kb of DNA to inactivate endogenous lambda variable domain expression (optionally in combination with an endogenous kappa locus modified to encode a shielding domain as described herein). In one example, the endogenous kappa locus comprises a deletion of at least 100, 150, or 200 kb of DNA to inactivate endogenous kappa variable domain expression (optionally in combination with an endogenous lambda locus modified to encode a shielding domain as described herein).

[0060] The genome of a mouse or mouse cell can be modified by insertion of multiple human variable region gene segments, which can be rearranged to encode human variable domains, upstream of human DNA encoding an immunoglobulin constant region comprising a human CH1 domain, at an endogenous immunoglobulin heavy chain locus on mouse chromosome 12. Similarly, DNA encoding a human CL domain (e.g., Cκ) can be inserted at the endogenous Igκ light chain locus on mouse chromosome 6. DNA encoding a human CL domain (e.g., Cλ) can alternatively or additionally be inserted at the endogenous IgA locus on mouse chromosome 16. Expression of the mouse heavy and light chains (κ and / or λ) can be inactivated, for example, by deleting the encoding DNA or by rendering its expression non-functional.

[0061] Antibodies according to the invention can be produced in an animal of the invention. The antibodies will generally be expressed in the B lymphocytes of the animal. A naive repertoire of unpaired variable domains is obtained from the animal prior to immunization with the antigen. An antigen-specific repertoire is obtained after immunization with the antigen. At least 50% (e.g., 75% or more, or all antibody-expressing B lymphocytes) in the animal can express antibodies comprising unpaired variable domains according to the invention. After immunogenic exposure of the animal to a target antigen, B lymphocytes expressing antibodies comprising unpaired variable domains that bind the target antigen will be positively selected by the immune system and undergo proliferation and somatic hypermutation, thereby generating a repertoire of variable domains that recognize the target antigen. One or more such antibodies, or their antigen-binding variable domains, or their encoded nucleic acids, can then be recovered from the animal and used in downstream processes such as recombinant expression, incorporation into larger antigen-binding polypeptides, and / or therapeutic applications, examples of which are described herein.

[0062] A method for generating an antibody comprising an unpaired variable domain for binding an antigen can include exposing a non-human animal of the invention to immunogenic stimulation by a target antigen. The antibody and / or the nucleic acid encoding it can then be isolated from the animal (e.g., by isolating B lymphocytes from blood, bone marrow and / or spleen), allowing identification of the variable domain sequence (nucleotide and / or amino acid sequence). Optionally, mutations can be introduced into the sequence, e.g., in one or more CDRs or in one or more FRs, such as reversion of non-germline framework residues to germline, insertion, substitution or deletion of residues within the variable domain, to refine the properties of the variable domain, such as binding affinity, or physical properties, such as stability and solubility. DNA encoding the variable domain comprising one or more mutations can then be provided in a vector, e.g., a plasmid, an expression vector, a transfection vector or a cloning vector. The sequence encoding the variable domain can be provided as part of a larger sequence encoding a polypeptide comprising the variable domain and one or more additional domains. Examples of such polypeptides include antibodies and CARs, which are described in detail elsewhere herein. Preferably, the variable domain is the N-terminal domain of such a polypeptide, since this is its natural location in immunoglobulins and exposes the CDRs of the variable domain binding site. However, other formats are possible, and the variable domain can be optionally connected to the N-terminal domain via a peptide linker, and the variable domain can still retain its antigen binding ability. The encoding nucleic acid can be introduced into the genome of a host cell, and the cells containing the recombinant DNA can then be cultured for expression of the antibody, isolated variable domain, or polypeptide comprising the unpaired variable domain. The antibody, isolated variable domain, or polypeptide can then be purified from the cells or from the cell culture medium, formulated into a composition comprising a pharma- ceutically acceptable excipient, and used therapeutically in the treatment (optionally prophylactic treatment) of diseases and conditions compatible with treatment by binding the target antigen recognized by the unpaired variable domain.The encoding nucleic acid and / or cells containing it also represent potential therapeutic products themselves. For example, T lymphocytes can be engineered to express a CAR that contains an unpaired variable domain, and the T lymphocytes can be used for immunotherapy by targeting the antigen recognized by the unpaired variable domain.

[0063] The antibodies according to the invention can also be used in vitro, for example in diagnostic methods, to bind antigens. Antibodies that bind a target antigen can be used in methods to detect whether the target antigen is present in a sample, optionally to quantify the target antigen, or to select the target antigen from a mixture (optionally in solution) containing the antigen surrounded by other molecules. The antibody can be optionally immobilized on a surface, for example on beads or a matrix, optionally in a column, to facilitate isolation of the antibody:antigen complex. Conversely, the target antigen (optionally immobilized on a surface, for example on beads or a matrix, optionally in a column) can be used to select antibodies of the invention that are capable of binding the target antigen by contacting a mixture (e.g., a solution) containing the antibody surrounded by other molecules (e.g., other antibodies with different unpaired variable domains). Binding of the unpaired variable domains of the antibody to the target antigen results in the formation of an antibody:antigen complex, which can then be isolated (e.g., separated from the solution). One or more washing steps can be performed to remove unbound antibody or unbound antigen. Overall, the method for preparing an antibody:antigen complex comprises: exposing the target antigen to an antibody according to the invention in vitro; allowing binding of the unpaired variable domains to the target antigen, thereby forming an antibody:antigen complex; and Isolating the antibody:antigen complex may include.

[0064] The method may further comprise determining the sequence or identity of the antibody or antigen in the antibody:antigen complex, and / or quantifying the number of antibody:antigen complexes formed.

[0065] The present invention will now be described in more detail with reference to the attached drawings. Headings within this document are included merely to aid navigation and should not be interpreted as limiting. Embodiments of the invention described separately may be combined, unless the context dictates otherwise. Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many additional equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to fall within the scope of protection of the appended claims. [Brief description of the drawings]

[0066] [Figure 1A] (a) Heavy-chain-only antibodies (HCAbs) comprise two heavy chains and lack light chains. Each heavy chain comprises (from N- to C-terminus) a VH domain (1) and a constant region comprising domains CH1 (11), CH2 and CH3. The CH2-CH3 regions of the two heavy chains dimerize to form the Fc region (3), while the VH domain (1) and the CH1 domain (11) are unpaired. [Figure 1B] (b) CH1-deleted HCAb comprising two heavy chains and lacking a light chain. Each heavy chain has a VH domain (1) fused to a constant region comprising (N- to C-terminally) domains CH2 and CH3 but lacking domain CH1. The constant regions of the two heavy chains dimerize to form an Fc region (3), while the two unpaired VH domains (1) are available for bivalent antigen binding. [Diagram 2]1 shows an embodiment of an antibody according to the invention. The antibody comprises two heavy chains and two light chains. Each heavy chain comprises (from N-terminal to C-terminal) a VH domain (1) and a constant region comprising domains CH1 (11), CH2 and CH3. The CH2-CH3 regions of the two heavy chains dimerize to form an Fc region (3). Each light chain comprises a CL domain (4) and lacks any further domains. The CL domain (4) pairs with the CH1 domain (11), such that the antibody comprises two heavy:light chain pairs and dimerizes via the CH2-CH3 of the Fc region (3) into a four-chain antibody molecule. The VH domain (1) of each heavy chain is unpaired, and the two VH domains of the antibody are available for bivalent antigen binding. [Figure 3A] FIG. 1 shows the construction of an immunoglobulin locus for expression of a Cκ-shielding domain and the resulting Cκ sequence according to the present invention: a) Generation of a gene encoding a Cκ-shielding domain by humanization of the mouse κ locus with human κ locus DNA containing a 90 kb deletion. [Figure 3B] Figure 1 shows the construction of an immunoglobulin locus for expression of a Cκ-shielding domain and the resulting Cκ sequence according to the present invention. a) Diagram of a human Cκ-shielding domain cDNA with a signal peptide. Nucleic acid encoding the signal peptide, SEQ ID NO: 1. Nucleic acid encoding an isolated Cκ-shielding domain, SEQ ID NO: 3. Nucleic acid encoding a Cκ with a signal peptide, SEQ ID NO: 5. [Figure 3C] Figure 2 shows the construction of an immunoglobulin locus for expression of a Cκ-shielding domain and the resulting Cκ sequence according to the present invention. c) Diagram of the translated Cκ-shielding domain. Signal peptide, SEQ ID NO: 2. Isolated Cκ sequence after predicted cleavage of the signal peptide, SEQ ID NO: 4. Encoded Cκ sequence including the signal peptide, SEQ ID NO: 6. [Figure 4A] Figure 1 shows human Vκ1-5 sequences in the unmodified human κ locus: a) Vκ1-5 cDNA SEQ ID NO: 9. Vκ1-5 leader sequence nucleic acid SEQ ID NO: 11. [Figure 4B]Figure 1 shows human Vκ1-5 sequence at the unmodified human κ locus. b) Translated Vκ1-5. Encoded Vκ1-5 with signal peptide SEQ ID NO: 10. Signal peptide SEQ ID NO: 12. [Diagram 5] 1 shows a prediction of signal peptide cleavage in the Cκ-shield domain according to the invention, made using SignalP software. The N-terminal sequence is a predicted signal peptide with a cleavage site between residues 22 and 23. [Figure 6A] FIG. 1 shows the construction of an immunoglobulin locus for expression of a λ5 shielding domain according to the present invention. [Figure 6B] FIG. 1 shows the construction of an immunoglobulin locus for expression of a λ5 shielding domain according to the present invention. [Figure 7] Schematic representation of the mouse Ig heavy chain locus on mouse chromosome 12, containing a large fragment of the human Ig heavy chain locus. Human DNA contains (in 5' to 3' order) a set of 41 V gene segments, a set of D gene segments, and a set of 6 J gene segments upstream of the gene encoding the set of heavy chain constant genes M (Cμ) through A2 (Cα). This locus can be present in a transgenic animal or transgenic animal cell according to the invention. [Figure 8] Schematic representation of the mouse Igκ chain locus at its endogenous locus on mouse chromosome 6, containing a large fragment of the human Igκ light chain locus including a deletion of Vκ1-5 to jκ5. The human DNA comprises (in 5' to 3' order) a set of Vκ gene segments and the leader sequence of Vκ1-5 encoding the signal peptide SP, upstream of the Cκ gene. This locus can be present in a transgenic animal or transgenic animal cell according to the invention. [Figure 9] FIG. 1 shows a schematic of the inactivated mouse λ locus on mouse chromosome 16. [Figure 10]FIG. 1 shows a more detailed scheme for inactivation of the endogenous mouse λ locus by deletion of a large (200 kb) genomic fragment from chromosome 16. Inactivation was achieved by a large deletion that removed Vλ2, Vλ3, Vλ1 as well as the Jλ2 / Cλ2 cluster, leaving the Jλ3 / Cλ3 and Jλ1 / Cλ1 clusters. [Figure 11] FIG. 1 shows primers used for RT-PCR amplification of kappa light chain (KLC) and / or truncated kappa light chain fragment (KCF) from mouse lymphocyte RNA. [Figure 12] Figure 3 shows RT-PCR analysis of the human kappa fragment locus (KCF), the unmodified humanized kappa locus (KLC), plus a WT control. PCR 1 uses forward and reverse oligos for human Cκ (primers HCP428 / HCP431), thus giving the same product from both loci. PCR 2 uses a forward oligo for human Vκ1-5 5'UTR and a reverse oligo for human Cκ 3'UTR (primers HCP446 / HCP451). PCR 2 shows a product of the expected smaller size for KCF compared to the full length kappa light chain from the KLC locus. [Figure 13] FIG. 1 shows sequence alignment of RT-PCR products (PCR 2) from multiple KCF animals with the predicted KCF transcript reference sequence, demonstrating the presence of the correct Vκ1-5 exon 1 / Cκ splice junction. [Figure 14] FIG. 1 shows the modification of the WT mouse kappa locus to introduce a human λ5 transgene. The vector contains the mouse 3' and 5' homology arms of lkb, the human Vκ promoter (white box), human intron and intronic enhancer (vertical hashed box) and a truncated human λ5 (diagonal hashed box). The vector can also contain a deleteable positive / negative selection cassette that is removed from the final targeted locus. Targeting of this vector replaces the endogenous mouse Jκ1-Jκ5, intron and intronic enhancer, and Cκ with the vector insert. [Figure 15]FIG. 1 shows transcripts and proteins produced by the mouse kappa / human λ5 locus. [Figure 16a] (a) Mouse kappa locus after successful rearrangement. [Figure 16b] (b) Mouse kappa locus after deletion to generate the kappa fragment locus. [Figure 17A] FIG. 1 shows the spliced ​​coding sequences and proteins of the Vκ3-2 and Vκ3-4 versions of the kappa fragment locus. [Figure 17B] See Figure 17A. [Figure 18] FIG. 1 shows the modification of the mouse kappa locus with a targeting vector to express a truncated mouse K fragment. The vector contains mouse 3' and 5' homology arms of approximately lkb, a mouse Vκ promoter (vertical hashed box), a mouse Vκ leader containing a mouse Vκ intron (black box), and a partial mouse Jκ5 (white box). The vector can also contain a deleteable positive / negative selection cassette that is removed from the final targeted locus. Targeting of this vector replaces only the endogenous mouse Jκ1-Jκ5, leaving the remainder of the mouse kappa locus intact, including the mouse k intron enhancer. [Figure 19A] FIG. 1 shows the spliced ​​coding sequences and protein products from the mouse loci targeted by truncated kappa Vκ6-17 or truncated kappa Vκ10-96 fragments. [Figure 19B] See Figure 19A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] Polypeptides containing unpaired variable domains Pairing between polypeptide domains refers to molecular association at the interface between spatially adjacent domains. Pairing generally involves non-covalent bonds (e.g., hydrophobic and / or electrostatic interactions), but domains can additionally be covalently linked, for example, via one or more disulfide bonds or other molecular linkers. Paired domains can optionally be part of the same polypeptide and thus covalently linked, or non-covalently paired as well, and can be adjacent in the primary polypeptide sequence or separated by one or more other domains. Examples of paired domains are the VH:VL pair of an antibody Fv region (whether formed by separate polypeptides each comprising a VH and a VL domain, or by a single chain Fv (scFv)), the CH1:CL pair between the heavy and light chains of an antibody, and the (CH2-CH3):(CH2-CH3) pair between the constant regions of an antibody Fc region. Paired domains can be heterodimers or homodimers. By extension, the concept of paired polypeptides includes a molecular association between two polypeptides, for example, through pairing of domains of the respective polypeptides. For example, an antibody may be referred to as comprising paired heavy and light chains (or a "heavy chain:light chain pair"), where, for example, the CH1 domain of the heavy chain is paired with the CL domain of the light chain.

[0068] An unpaired domain is one that is not paired with another domain. An unpaired variable domain corresponds to an antibody Fv region with either the VH or VL domain removed. The unpaired variable domain may be either VH or VL. An unpaired variable domain may be capable of binding antigen outside the context of an Fv. Thus, a VH domain may not require the presence of a paired VL domain for antigen binding. Conversely, a VL domain may not require the presence of a paired VH domain for antigen binding. Indeed, in many cases, especially when the variable domain is selected for antigen binding in its unpaired state (as described in the methods herein, such as the method of generating antigen-specific antibodies in vivo by immunization of transgenic mice), an unpaired variable domain can specifically bind its target antigen with an affinity comparable to that of an Fv region from a "normal" antibody raised against that target. Moreover, for at least certain categories of antigens, including those with clefts or deep binding pockets in their three-dimensional structure, antibodies comprising unpaired variable domains according to the invention are preferred over whole Fv regions. This is because the more compact structure of the unpaired variable domains presents a smaller binding site that can reach epitopes buried within the antigen, whereas such epitopes may be inaccessible or less accessible to binding by the intact Fv region. Exemplary target antigens and the generation of antibodies thereto are described in detail elsewhere herein.

[0069] Although association refers to a non-covalent interaction (optionally supplemented by a covalent bond between the domains), references to linkage herein generally refer to covalent attachment. Domains of a polypeptide may be linked via peptide bonds or peptide linkers. Moieties may also be linked by other suitable covalent attachments, the scope of which will be apparent to a protein chemist.

[0070] A domain of a polypeptide comprises a sequence that folds into a structure, e.g., an immunoglobulin fold or other stable conformation. When a polypeptide comprises a domain as part of a longer polypeptide sequence, the domain will generally have a tertiary structure that is separate or distinct from the rest of the polypeptide. Generally, domains are responsible for distinct functional properties of the protein and can often be added, removed, or transferred to other proteins without loss of function of the remainder of the protein and / or the function of the domain.

[0071] Antibodies contain immunoglobulin domains, which have an "immunoglobulin fold" of two beta sheets of antiparallel beta strands linked by disulfide bonds and hydrophobic interactions. The constant domain referred to herein may be an immunoglobulin constant domain, e.g., a CH1, CH2, CH3 or CL domain. A constant region may comprise one or more constant domains linked and / or paired with each other. Antibody constant regions are described in detail elsewhere herein.

[0072] When multiple domains of a polypeptide are described herein, they will generally be arranged in a standard N-terminal to C-terminal direction, unless the context dictates otherwise. For example, unless otherwise indicated, a polypeptide comprising a VH domain and a CH1 domain will comprise the VH domain "upstream" of the CH1 domain. A polypeptide can be represented by its domain structure, such as VH-CH1, where the domains are shown in the N-terminal to C-terminal direction from left to right.

[0073] Optionally, the antibodies or polypeptide chains according to the invention can be fused or conjugated to further polypeptide sequences and / or to labels, tags, toxins or other molecules, for example to form immunocytokines. For example, the constant region or shielding domain of an antibody can be linked to a cytokine such as IL-2. Links between polypeptide or peptide sequences can be conveniently made by generating a fusion protein that contains the two (or more) sequences in tandem.

[0074] Antibodies, variable domains and specific antigen binding The term "antibody" as used herein includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies), and single chain molecules, as well as antibody fragments. Antibodies comprising unpaired variable domains according to the present invention may comprise naturally occurring or known antibody structures, subject to the modification that one or more Fv regions of the antibody are replaced with unpaired variable domains. This may be envisaged as removing either the VL (or VH) domain from the Fv, so that the remaining VH (or VL) domain is unpaired.

[0075] The antibodies and polypeptide domains herein can be human. Human variable domains are preferred due to their lower immunogenicity in compositions intended for administration to humans. Although the constant region of the antibody can be human (particularly in the context of antibodies for administration to humans, as discussed above), the constant region can be generated in a transgenic non-human animal as a chimeric antibody comprising a human variable region and a non-human animal constant region, followed by replacement of the non-human animal constant region with a human constant region to provide a fully human antibody. Alternatively, fully human antibodies can be generated directly from a transgenic animal whose genome has been engineered to contain human variable region gene segments and human constant region genes. Thus, the antibody of the invention can be a human antibody or a chimeric antibody comprising one or more human variable regions and one or more non-human (e.g., murine) constant regions. The antibody of the invention can comprise at least one unpaired human variable region linked to a human constant region.

[0076] An antibody variable domain (e.g., an unpaired variable domain as described herein) provides the binding site for an antigen. The recognition between an antibody and its cognate antigen can be referred to as specific binding, as opposed to nonspecific binding, in which antibodies or other polypeptides bind non-target molecules through relatively low affinity interactions. Antigen binding of the variable domain is via contacts between the antigen and one or (usually) multiple residues of the CDRs (HCDRs or LCDRs). One or more FR residues of the variable domain may also contact the antigen. The region of an antigen to which an antibody binds is called its epitope. The region of an antibody that binds an antigen is called its paratope.

[0077] A variable domain or binding site that "specifically binds" or is "specific" for a particular antigen or epitope can be one that binds to that particular antigen or epitope without substantially binding to other antigens or epitopes. For example, binding to an antigen or epitope is specific if the antibody binds with a KD of 1 mM or less, e.g., 100 μM or less, 10 μM or less, 1 μM or less, 100 nM or less, e.g., 10 nM or less, 1 nM or less, 500 pM or less, 100 pM or less, or 10 pM or less. Binding affinity (KD) can be determined using standard procedures known to those of skill in the art, such as binding in an ELISA and / or affinity determination using surface plasmon resonance (SPR) (e.g., Biacore™, Proteon™, or KinExA™ liquid phase affinity assays (Sapidyne Instruments, Idaho) that allow detection down to fM affinity). In one embodiment, SPR is performed at 25° C. In another embodiment, SPR is performed at 37°C. In one embodiment, SPR is performed at physiological pH, such as at about pH 7 or pH 7.6 (e.g., using HEPES-buffered saline (also called HBS-EP) at pH 7.6). In one embodiment, SPR is performed at physiological salt levels, e.g., 150 mM NaCl. In one embodiment, SPR is performed at detergent levels of 0.05% by volume or less, e.g., in the presence of P20 (polysorbate 20; e.g., Tween-20™) at 0.05% and EDTA at 3 mM. SPR can be performed at 25°C or 37°C in a buffer at pH 7.6 that is 150 mM NaCl, 0.05% detergent (e.g., P20) and 3 mM EDTA. The buffer can contain 10 mM Hepes. In one example, SPR is performed at 25°C or 37°C in HBS-EP. HBS-EP is available from Teknova Inc., California, catalog number H8022.

[0078] In one example, the affinity is 1. Coupling anti-human or anti-mouse (or other relevant non-human vertebrate, e.g. matching C region of antibody) IgG (e.g. Biacore BR-1008-38) to a biosensor chip (e.g. GLM chip), such as by primary amine coupling; 2. Exposing the IgG to a test antibody (or its heavy chain) containing a constant region to capture the test antibody (or its heavy chain) on the chip; 3. Passing test antigen across the capture surface of the chip at 1024 nM, 256 nM, 64 nM, 16 nM, 4 nM, along with a control of 0 nM (i.e., buffer alone); and 4. Determining the binding affinity of the test antibody / chain to the test antigen using surface plasmon resonance, e.g., under the SPR conditions discussed above (e.g., in physiological buffer at 25° C.); Determined using SPR by. SPR can be performed using any standard SPR instrument, such as with Biacore™ or using the ProteOn XPR36™ (Bio-Rad®).

[0079] Regeneration of the capture surface can be accomplished with 10 mM glycine at pH 1.7, which removes the captured antibody and makes the surface available for another interaction. Binding data can be fitted to a specific 1:1 model using standard techniques, for example, using the model specific to the ProteOn XPR36™ analysis software.

[0080] Antibody constant region The constant region of the antibody can comprise one or more human constant domains or can be a human constant region. For example, the unpaired variable domain (e.g., VL domain) can be attached at its C-terminus to an antibody light chain kappa constant domain or lambda constant domain. The unpaired variable domain (e.g., VH domain) can be attached at its C-terminus to all or a portion of an immunoglobulin heavy chain constant region (e.g., CH1 domain or Fc region) from any antibody isotype, such as IgG, IgA, IgE, and IgM, as well as isotype subclasses such as IgG1 and IgG4.

[0081] The antibody constant region may comprise the constant region of human IgG, IgM, IgA, IgD, or IgE. The antibody heavy chain constant region may comprise the constant region of human heavy chain IgG, IgM, IgA, IgD, or IgE.

[0082] Exemplary human constant region sequences are provided in attached Table A.

[0083] Alternatively, the constant regions of the antibodies of the invention may be non-human constant regions. For example, when antibodies are produced in transgenic animals (examples of which are described elsewhere herein), chimeric antibodies can be produced comprising human variable regions and non-human (host animal) constant regions. Some transgenic animals produce fully human antibodies. Others have been engineered to produce antibodies comprising chimeric heavy chains and fully human light chains. When an antibody comprises one or more non-human constant regions, these can be replaced with human constant regions to provide antibodies more suitable for administration to humans as therapeutic compositions, since this reduces the immunogenicity of the antibodies.

[0084] Digestion of whole immunoglobulins with the enzyme papain results in two identical antigen-binding fragments, also known as "Fab" fragments, and an "Fc" fragment, which has no antigen-binding activity but has the ability to crystallize. As used herein, "Fab" refers to a fragment of an antibody that contains one constant domain and one variable domain of each of the heavy and light chains. The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. "Fc fragment" refers to the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of an antibody are determined by the sequence of the Fc region, which is also recognized by Fc receptors (FcRs) found on certain types of cells.

[0085] Transgenic animals containing human immunoglobulin loci The non-human animal genome can be engineered to contain one or more human heavy chain V gene segments, one or more human heavy chain D gene segments and one or more human heavy chain J gene segments for expression of a human VH domain. The non-human animal genome can contain a full set of all human heavy chain V, D and J gene segments. A human VDJ gene segment can be inserted upstream of the constant region for production of a VH domain linked to the constant region.

[0086] Similarly, the non-human animal genome can be engineered to contain one or more human light chain V gene segments and one or more human light chain J gene segments for expression of a human VL domain. The gene segments can be kappa or lambda. The non-human animal genome can contain a full set of all human kappa or lambda gene segments. A human VJ gene segment can be inserted upstream of the constant region for production of a VL domain linked to the constant region.

[0087] Because the antibodies of the present invention comprise a VH domain or a VL domain in unpaired form, the genome can comprise a heavy chain gene segment in the absence of a light chain gene segment (or when expression of the light chain gene segment is inactivated), or the genome can comprise a light chain gene segment in the absence of a heavy chain gene segment (or when expression of the heavy chain gene segment is inactivated).

[0088] Recombination of V(D)J gene segments generates combinatorial diversity in each variable domain. By including a full set of human heavy (or light) chain gene segments, the complete combinatorial diversity of human variable domains can be incorporated into the transgenic animal platform. Affinity maturation of these variable domains then proceeds through the natural in vivo process of somatic hypermutation and selection, thereby providing a broad and diverse repertoire of sequences from which antigen-specific variable domains with desirable properties can be identified and retrieved. The antibody production platform described herein utilizes the natural function of CH1 pairing with the shielding domain, thereby allowing the maintenance of a complete human heavy chain repertoire and avoiding the need to re-engineer human heavy chains. Preferably, the heavy chain immunoglobulin locus is or comprises an unmodified human heavy chain locus and / or expresses human immunoglobulin heavy chains comparable in all respects to those generated in humans. Such a platform provides a complete repertoire of antibodies that includes unpaired human VH domains for binding antigens, thereby allowing the selection of desired molecules of interest suitable for development into pharmaceuticals.

[0089] Methods for producing transgenic animals with genomes containing all or part of the human immunoglobulin loci are well known in the art. Such animals have been used to discover and produce several antibodies containing human variable domains that are currently on the market as pharmaceuticals, with many more in the development pipeline. Methods for engineering non-human animal genomes to contain human immunoglobulin gene segments are described, for example, in WO2011 / 004192 (Genome Research Limited), which is incorporated herein by reference. Examples of transgenic non-human animals include Kymouse™ (e.g., as described in WO2011 / 004192), VelociMouse®, OmniMouse®, Omnirat®, XenoMouse®, HuMabmouse®, and MeMo Mouse®.

[0090] In addition to the variable regions, the genome of the non-human animal encodes the constant regions of the antibodies described herein. The locus containing the variable region gene segments further comprises a constant region gene or genes, for example, a heavy chain constant region containing CH1 (e.g., containing domains CH1, CH2 and CH3), or a constant region containing CL (e.g., containing domains CL, CH2 and CH3). The constant region at the locus can be a human constant region, i.e., in this case, the domains are of human origin. The non-human animal genome can be engineered to contain one or more human constant region genes. The non-human animal genome can contain a complete repertoire of human constant region genes: M, D, G3, G1, A1, G2, G4, E and A2, respectively, optionally by inserting fragments of human genomic DNA containing these genes.

[0091] The constant region can comprise a fragment of the human IgH locus from Cμ (and optionally including) the 5'-most γ1 exon; optionally including all γ1 CH1-3 or CH1-M2 exons. In another embodiment, the fragment is from and includes Sμ or Eμ. In another embodiment, the fragment is from a point within the first 400, 500, 600, 700 800 or 900 nucleotides of the IgH JC intron, and includes the intronic DNA 5' and adjacent intronic DNA 5' of Eμ and Cμ.

[0092] The constant region can also include at least one IgH C gene segment, e.g., a Cmu gene segment, and optionally one or more of an alpha, delta, epsilon, and gamma (e.g., gamma-1) C gene segment. In one embodiment, the constant region includes a Cmu and a Cgamma (e.g., a gamma-1, human gamma-1, mouse gamma-1, or rat gamma-1 C segment). One or both of the Cmu and Cgamma can be endogenous to the non-human animal; e.g., Cmu is endogenous and Cgamma is endogenous or human. In one example, the gene segments of the C region are in the germline order of the C segments found in the IgH or IgL locus of the human, rodent, rat, or mouse genome. In one example, the gene segments of the C region are in the germline order of the C segments found in the IgH or IgL locus of the mouse genome. This order is known to those skilled in the art.

[0093] In one example, the antibody C gene segment is an endogenous segment of the non-human animal, and optionally the constant region is an endogenous heavy chain constant region at the endogenous heavy chain locus, or an endogenous light chain (kappa or lambda) constant region at the endogenous light chain locus. Thus, when the animal is a mouse, the C segment is on chromosome 12 (for an IgH C segment), chromosome 6 (for an Igκ C segment), or chromosome 16 (for an Igλ C segment).

[0094] The non-human animal genome can be engineered to contain a first and a second locus, the first locus engineered to express a first polypeptide or heavy chain according to the invention, and the second locus engineered to express a second polypeptide or light chain according to the invention. The first and second loci can be on the same chromosome or on different chromosomes.

[0095] The first locus can be engineered to comprise a fragment of a human immunoglobulin heavy chain locus, including a VDJ segment and constant region genes, along with intergenic regions and regulatory elements. Alternatively, the first locus can be engineered to comprise a fragment of a human immunoglobulin kappa light chain locus or a lambda light chain locus, including a VJ segment and constant region genes, along with intergenic regions and regulatory elements.

[0096] The V(D)J gene segments can be linked to a constant region that is an endogenous non-human animal constant region. This can be done by inserting human variable region gene segments into the non-human animal genome upstream of the endogenous constant region at the endogenous immunoglobulin locus. Alternatively, the constant region can be human, and human DNA comprising the V(D)J gene segments and a human constant region can be inserted into the endogenous locus, thereby completely humanizing the locus. Preferably, intergenic and regulatory elements of the human immunoglobulin locus are also included.

[0097] A human heavy chain gene can be inserted into the mouse heavy chain locus on chromosome 12 to provide a first locus encoding a first polypeptide, or a heavy chain locus, as described herein.

[0098] A second locus can be engineered for expression of the shielding domain. The second locus can include DNA of the human kappa light chain locus, including the human kappa constant region gene and optionally associated human intergenic and regulatory elements. Thus, the non-human animal genome can be engineered to include a fragment of the human kappa locus, including the constant region gene and regulatory elements. Productive rearrangement of the variable region gene segments is inactivated, for example, by deletion of all Jkappa gene segments. A human Ckappa gene can be inserted at the mouse kappa locus on chromosome 6. Figure 8.

[0099] Expression of endogenous variable region gene segments from an endogenous Ig locus or loci in a non-human animal can be inactivated, for example, by deletion or inversion of a stretch of DNA that includes the endogenous V(D)J region. Optionally, the endogenous λ locus is unmodified. However, the endogenous λ locus can also be inactivated in some cases (Figure 9).

[0100] In one example, human heavy chain variable region DNA and / or constant region DNA is integrated at an endogenous antibody locus of a non-human animal (e.g., a mouse). Optionally, the donor locus and recipient locus can be matched such that human heavy chain locus DNA can be integrated at an endogenous heavy chain locus, human κ light chain locus DNA can be integrated at an endogenous λκ light chain locus, and human λ light chain locus DNA can be integrated at an endogenous λ light chain locus, although such matching is not required.

[0101] The constant region can comprise an endogenous (e.g., murine) antibody constant gene segment. The constant region can additionally comprise one or more human constant gene segments. The antibody locus can be an IgH locus, and the constant region comprises an endogenous Cμ constant gene segment and a human Cγ constant gene segment. The antibody locus can be an IgH locus, and comprises an endogenous Sμ operably linked upstream of a Cμ (e.g., an endogenous Cμ). For example, the locus can comprise an endogenous intronic enhancer (e.g., Eμ if the antibody locus is IgH; or iEκ if the antibody locus is Igκ). The locus can comprise an endogenous 3′ enhancer of the antibody locus.

[0102] Alternatives to targeted integration of human DNA at the endogenous immunoglobulin loci of a non-human animal include random integration of human DNA into the animal genome, or targeted integration of human DNA at a locus separate from the endogenous immunoglobulin loci (optionally on a different chromosome). The Rosa26 locus can be a convenient target. Optionally, the first and / or second locus is therefore not an endogenous antibody locus, e.g., DNA is randomly inserted into the germline genome of the animal. In one embodiment, the constant region comprises an exogenous antibody constant gene segment (and thus the antibody does not comprise an endogenous antibody constant gene segment). The exogenous constant gene segment may be of the same species as the animal or of a different species, e.g., human constant gene segments or rodent (e.g., mouse or rat) constant gene segments.

[0103] The transgenic non-human animal and non-human animal cell genome preferably comprises a fully humanized heavy chain locus containing a full repertoire of human V, D and J gene segments and all human constant region genes plus human enhancers. In contrast to random insertion, in situ targeting of human immunoglobulin locus DNA to the endogenous immunoglobulin locus of the non-human animal facilitates accurate and precise regulation of the immune response. Thus, the transgenic animal of the present invention can present a robust immune response, including somatic hypermutation and affinity maturation of antibody variable domains, after exposure to an immunogenic composition containing a target antigen. Expression of mouse heavy chain from the endogenous mouse heavy chain locus can be inactivated by inversion of the variable region and replacement of the mouse constant region with a human heavy chain constant region genomic fragment. Deletion of mouse variable region gene segments is an alternative form of replacement and / or inversion of endogenous DNA. For example, mouse heavy chain V and / or J gene segments can be deleted.

[0104] Well-validated methods are now available for genome engineering in animals, and available techniques are continually being improved. Engineering of ES cells can be performed by homologous recombination or recombinase cassette exchange [2; WO2011 / 004192], and chromosome engineering can also be performed in zygotes [3].

[0105] In one embodiment, a first locus is created by inserting a variable region gene segment into an endogenous IgH locus of an embryonic stem cell (ES cell) or an iPS cell (e.g., a mouse cell) of the animal species. A second locus can then be introduced as a transgene into the genome of the cell (or its progeny cell or zygote), and the transgene is inserted into the genome of the cell or zygote. A non-human animal is then generated from the cell, zygote, or their progeny. Alternatively, as known in the art, the loci can be engineered in separate non-human animal cells (e.g., ES cells, iPS cells, or zygotes), which are developed into animals that contain the engineered loci in their germline DNA, and the individual animals are bred together to combine the loci in the germline genome of the progeny animal. Thus, a first animal whose germline genome contains the first locus is mated with a second animal whose germline genome contains the second locus to produce a progeny animal whose germline genome contains both loci. The progeny animals can be observed to produce lymphocytes that express antibodies as described herein.

[0106] The animals according to the invention can be homozygous for the first locus or heavy chain locus and / or homozygous for the second locus or light chain locus as described herein. In another form, the vertebrate is heterozygous for the locus or each of such loci. Preferably, the animals are homozygous at the humanized heavy and light chain loci. The animals can be homozygous at all three immunoglobulin loci, i.e., at the heavy chain locus, the lambda locus and the kappa locus. As described above, heterozygous animals are first generated for each locus and bred to generate double or triple homozygous animals (depending on the number of modified loci), thus creating a stable breeding population. Breeding colonies of transgenic animals can be housed in animal houses, optionally under germ-free or specific pathogen free (SPF) conditions. Male and female mice can be grouped separately or together.

[0107] In one example, a mouse comprising a humanized heavy chain locus as described herein, an animal comprising a humanized κ locus as described herein, and an animal comprising an optionally inactivated λ locus as described herein are separately generated and then bred together to create a breed in which a fully human heavy chain is expressed at the endogenous Ig heavy chain locus, the VJ rearrangement at the endogenous κ locus is inactivated by deletion of the J region, and the locus expresses a Cκ fragment (e.g., SEQ ID NO: 4). Figure 3c. Cκ does not contain variable domain sequences. Cκ functions as a shielding domain to stabilize the CH1 of the heavy chain of the expressed antibody.

[0108] The endogenous λ locus of a mouse can be inactivated (e.g., deleted in whole or in part) so that the locus does not express a light chain comprising a VL domain. There may be no functional polypeptide expression from the λ locus. However, in other embodiments, the λ locus of a mouse may be unmodified. Expression from the λ locus naturally occurs only at low levels in mice. Furthermore, if the κ locus is active, for example if the κ locus encodes and expresses a shielding domain (e.g., a CL domain or a λ5 domain), the λ locus can be silenced through the natural process of allelic exclusion in B lymphocytes. Thus, whether naturally by allelic exclusion, by genome engineering, or by any other method, expression from the λ locus can be silenced in B lymphocytes of an animal (e.g., a mouse) according to the invention. Inactivation of a locus by deletion is illustrated in Example 3.

[0109] Aspects of the present specification may be expressed in terms of the non-human animal being a mouse, but it is understood that other laboratory or livestock animals or any other non-human vertebrate may be suitable for carrying out the present invention. The vertebrate may be a rodent, such as a mouse or a rat. In other examples, the vertebrate is a bird (e.g., chicken), a fish (e.g., shark or zebrafish), a mammal (e.g., rabbit), a livestock animal (e.g., cow, sheep, pig or goat), or a camelid (e.g., llama, alpaca or camel). In one embodiment, the mouse strain is 129 (or 129 hybrid), C57BL6 (or C57BL6 hybrid), or derived from AB2.1, AB2.2, JM8, BALB / c, or F1H4 ES cell line.

[0110] Knockouts can be used to provide access to human / non-human (e.g., human / mouse) cross-reactive antibodies. Thus, in addition to the modifications described above, the expression of endogenous targets can be inactivated in the genome of the non-human animal. The resulting knockout animals can be used for immunization with the target antigen and can generate stronger immune responses (e.g., higher antibody titers and / or greater antibody diversity) against the target compared to animals expressing the endogenous target. This is because, if the target antigen (e.g., human protein X) has homology with the endogenous target of the non-human animal (e.g., mouse protein X), the immune repertoire of the non-human animal will have been negatively selected against the self-antigen, such that immunization with the human protein will generate predominantly antibodies that are selective for the human protein and that are not cross-reactive with the endogenous protein from the non-human animal. Thus, the use of knockout animals can increase the diversity of antibodies obtained from immunization, including those that recognize epitopes conserved between species, and generate potentially useful cross-reactive antibodies.

[0111] λ5 and B cell development B lymphocyte (B cell) development is characterized by the ordered rearrangement of immunoglobulin variable region genes. After VDJ rearrangement of heavy chain gene segments, precursor B cells (pre-B cells) are generated. After VJ rearrangement of light chain gene segments, pre-B cells develop into mature B cells that carry IgM at the cell surface and can be presented to antigens. Assembled antibodies, including heavy and light chains, are transported to the B cell surface, while free heavy chains are retained in the endoplasmic reticulum (ER) in association with the 70 kDa heat shock protein chaperone BiP.

[0112] A crucial step in B cell differentiation is the selective expansion of cells with functional μ heavy chains resulting from productive rearrangement of heavy chain gene segments. This is achieved by the association of μ heavy chains with surrogate light chain proteins λ5 and VpreB and the signaling heterodimer Igαβ, resulting in the formation of the pre-B cell receptor (pre-BCR). The surrogate light chain has the entire structure of a light chain, but is a non-covalent heterodimer of VpreB (homologous to VL) and λ5 (homologous to CL). The N-terminal region of λ5 represents an extra β-chain that is not part of the normal Ig domain, while the Ig domain of VpreB lacks one of the canonical β-chains. Complementation of the incomplete Ig domain of VpreB by the extra β-chain in λ5 is necessary and sufficient for folding and assembly of these proteins to generate the surrogate light chain [4]. λ5 can disulfide bond to the μ heavy chain of pre-B cells. The high-resolution structure of the pre-BCR Fab-like fragment was published in 2007 and showed that unique regions of VpreB and λ5 interact with each other and with the heavy chain CDR3, potentially influencing antibody repertoire selection [5].

[0113] The surrogate light chain acts as a chaperone, displacing ER-resident BiP from its CH1 domain and escorting the heavy chain μ together with Igαβ to the cell surface [6]. Expression and formation of the pre-BCR dramatically improves the efficiency of pre-B and B cell production by signaling increased proliferative activity of pre-B cells. Surface presentation of the membrane-bound μ chain is essential for the clonal expansion of these cells and the initiation of light chain gene rearrangement.

[0114] The surrogate light chain is then suppressed while the light chain gene segment is rearranged. The product of the successfully rearranged light chain gene pairs with the heavy chain to form an antigen-binding BCR on the surface of the immature B cell. These cells migrate to the peripheral blood and secondary lymphoid organs and develop into mature B cells ready for subsequent antigen encounters [7]. Expression of VpreB and λ5 is silenced after the pro-B and pre-B cell stages, so these proteins are not naturally present in B lymphocytes.

[0115] In 1996, Papavasiliou, Jankovic and Nussenzweig [8] described evidence for two pathways for the induction of B cell development; one activated through surrogate light chain (lambda 5) and IgM, and the other activated through conventional light chain (kappa or lambda) and IgM. In the absence of kappa and lambda light chain expression in the mice studied, expression of lambda 5 rescued the surface presentation of IgM, which delivers the signal for B cell development. Guloglu et al. [9] also later reported that heavy chains were not expressed in B cells from RAG / lambda 5 double knockout mice transfected with heavy chain DNA, but could be expressed on the surface of mouse B cells when cotransfected with either lambda 5 or a truncated lambda 5 that eliminated the N-terminal unique region preceding the lambda 5 immunoglobulin fold.

[0116] Using VpreB knockout mice, it has been shown that VpreB is required for efficient B cell development, especially for the transition to pre-BCR-bearing cells (pre-BII stage) [7, 10]. In HEK cells expressing heavy chain mutants that did not require Igαβ for signaling, expression of the complete λ5 polypeptide without VpreB did not result in surface presentation of IgM. However, expression of truncated λ5 without VpreB allowed surface presentation of IgM. Indeed, surface presentation of IgM in cells expressing only truncated λ5 was superior to that in cells expressing the complete surrogate light chain (both VpreB and λ5). Surface presentation of IgM was even higher when Igκ light chain was expressed

[11] .

[0117] Selective deletion of CH1 domain in class-switched isotypes Co-expression of the shielding domain with a polypeptide containing a CH1 domain provides great immunological benefits and greatly facilitates the discovery of antibodies containing unpaired variable domains. However, there are situations in which it is still desirable to generate antibodies containing unpaired variable domains linked to constant regions carrying CH1 deletions. If this is desired, it is best achieved after the antibody has undergone affinity maturation (rather than having the CH1 deletion present in pre-BCR). Thus, the heavy chain constant region present in the class-switched antibody can be selectively engineered to carry the CH1 deletion.

[0118] The CH1 domain is optionally deleted from non-IgM heavy chain constant region genes and retained in the μ constant region gene. The IgM heavy chain can be expressed and combined with a shielding domain of the invention (e.g., λ5, or Cκ from an engineered κ locus detailed elsewhere herein). The CH1-deleted non-mu heavy chain (e.g., IgG or IgA heavy chain) can then be expressed after class switching in lymphocytes. In B lymphocytes containing such a combination of genomic modifications, the shielding domain pairs with the CH1 of the μ heavy chain to form an IgM antibody, and then class switching in the B lymphocyte generates heavy chain-only antibodies, e.g., CH1-deleted IgG and CH1-deleted IgA, with heavy chains comprising unpaired VH domains and constant regions lacking the CH1 domain. WO2013 / 171505 (Kymab Limited) described a non-human animal expressing normal IgM and CH1 deleted IgG antibodies, where stage specific class switching from IgM to IgG in lymphocytes was accompanied by genetic deletion of CH1 from the IgG constant region, resulting in expression of IgG heavy chain only antibodies. The methods and embodiments of WO2013 / 171505, which are incorporated herein by reference, may be used in the present invention.

[0119] In one embodiment, the region encoding CH1 is deleted from the IgA constant region gene in the heavy chain locus of the non-human animal (e.g., the humanized heavy chain locus of the transgenic animal). The CH1 domain is optionally deleted in the context of a complete human heavy chain locus inserted into the endogenous heavy chain locus of the non-human animal. IgA is abundant, and switching from IgG to IgA is frequently observed. Thus, selectively deleting CH1 in the IgA isotype while retaining the complete heavy chain of the IgM isotype allows the development of early B cells with complete heavy chains, which undergo affinity maturation in the normal manner and subsequently undergo isotype switching to IgA antibodies that contain heavy chains with CH1 deletion. The antibody can contain unpaired variable domains (e.g., VH domains) throughout this in vivo evolution, thereby ensuring selection for a repertoire of variable domains that are effective as single, isolated antigen-binding domains.

[0120] bispecific antibody The antibody according to the invention can exhibit bispecific or multispecific antigen binding. Thus, the antibody can comprise a first and a second unpaired variable domain, the first unpaired variable domain specifically binding to a first antigen or epitope and the second unpaired variable domain specifically binding to a second antigen or epitope. The first and second variable domains can have different amino acid sequences from each other to bind different first and second antigens / epitopes, respectively. The different epitopes can be epitopes of one antigen or of different antigens. Thus, for example, the first unpaired variable domain can specifically bind antigen A (but not antigen B) and the second unpaired variable domain can specifically bind antigen B (but not antigen A).

[0121] Just as "heavy chain only" antibodies of the prior art have often been the format of choice for bispecific and multispecific antibodies, the antibodies of the present invention are also suitable for this purpose.

[0122] Providing antigen-binding sites within unpaired variable domains expressible as part of a single polypeptide chain has advantages, since bispecific antibodies can be generated through association (e.g., after co-expression) of two such polypeptides comprising different variable domains (and thus different antigen-binding sites). This contrasts with the more complex generation of conventional four-chain bispecific antibodies comprising two different heavy:light chain pairs and thus four different polypeptide chains, which when expressed together can assemble into 10 different possible antibody molecules, including homodimers (homodimeric anti-A binding arms and homodimeric anti-B binding arms), molecules in which one or both light chains are swapped between the H-L pairs, and the "correct" bispecific heterodimeric structure.

[0123] The antibody formats of the present invention allow for the expression of antibodies as relatively simple two- or three-chain molecules, which, combined with the constant region design, can strongly aid assembly into the desired bispecific format.

[0124] The "knobs-into-holes" technique for making bispecific antibodies was described in

[12] and U.S. Pat. No. 5,731,168, both of which are incorporated herein by reference. The principle is to engineer paired CH3 domains of heterodimeric heavy chains such that one CH3 domain contains a "knob" and the other CH3 domain contains a "hole" in the sterically opposite position. The knob is created by replacing a small amino acid side chain at the interface between the CH3 domains, while the hole is created by replacing a large side chain with a small one. The knob is designed to insert into the hole, which favors heterodimerization of the various CH3 domains while destabilizing the formation of homodimers. In a mixture of antibody heavy and light chains that assemble to form a bispecific antibody, the proportion of IgG molecules with paired heterodimeric heavy chains is therefore increased, resulting in an increased yield and recovery of active molecules.

[0125] Mutations Y349C and / or T366W can be included to form a "knob" in the IgG CH3 domain. Mutations E356C, T366S, L368A and / or Y407V can be included to form a "hole" in the IgG CH3 domain. Knobs and holes can be introduced into any human IgG CH3 domain, for example, the CH3 domains of IgG1, IgG2, IgG3 or IgG4. A preferred example is IgG4. IgG4 can include further modifications such as "P" and / or "E" mutations. A "P" substitution at position 228 of the hinge (S228P) stabilizes the hinge region of the heavy chain. An "E" substitution in the CH2 region at position 235 (L235S) abolishes binding to FCγR. Bispecific antibodies of the invention can contain an IgG4 PE human heavy chain constant region, optionally comprising two such paired constant regions, optionally in this case one with a "knob" mutation and one with a "hole" mutation.

[0126] While the knobs-into-holes technique involves engineering amino acid side chains to create a molecular shape complementary to the interface of paired CH3 domains of bispecific heterodimers, another method to promote heterodimer formation and prevent homodimer formation is to engineer amino acid side chains to have opposite charges. The association of the CH3 domains of heavy chain heterodimers is aided by the pairing of oppositely charged residues, whereas paired positive charges or paired negative charges would make homodimer formation energetically unfavorable. WO2006 / 106905 described a method for producing heteromultimers composed of two or more types of polypeptides (such as heterodimers of two different antibody heavy chains), including substitutions in amino acid residues that form the interface between the polypeptides such that heteromultimer association is modulated, the method comprising: (a) modifying a nucleic acid encoding amino acid residues that form interfaces between polypeptides from an original nucleic acid such that association between the polypeptides forming one or more multimers is inhibited in heteromultimers capable of forming two or more types of multimers; (b) culturing the host cell so as to express the nucleic acid sequence modified by step (a); and (c) recovering the heteromultimer from the host cell culture, The modification of step (a) is Substituting one or more amino acid residues at an interface such that two or more amino acid residues, including the mutated residues, forming the interface carry the same positive or negative charge; This is the process of modifying the original nucleic acid.

[0127] One example of this is the inhibition of association between heavy chains by introducing electrostatic repulsion at the interface of the heavy chain homodimer, for example by modifying amino acid residues that contact each other at the interface of the CH3 domains, including: Residue numbering follows the EU numbering system, but 356th and 439th place, 357th and 370th place, 399th and 409th place Examples include:

[0128] By modifying one or more of these pairs of residues in the CH3 domain of a first heavy chain to have similar charges (both positive or both negative), heavy chain homodimer pairing is inhibited by electrostatic repulsion. By engineering the same pair or pairs of residues in the CH3 domain of a second (different) heavy chain to have opposite charges compared to the corresponding residues in the first heavy chain, heterodimer pairing of the first and second heavy chains is promoted by electrostatic attraction.

[0129] In one example, amino acids at the CH3 junction of the heavy chain constant region are modified to introduce charge pairs, and the mutations are listed in Table 1 of WO2006 / 106905. It has been reported that modifying the amino acids at positions 356, 357, 370, 399, 409, and 439 of the heavy chain to introduce charge-induced molecular repulsion at the CH3 junction has the effect of increasing the efficiency of formation of the intended bispecific antibody. WO2006 / 106905 also exemplified a bispecific IgG antibody in which the CH3 domain of IgG4 was engineered with knob-into-hole mutations.

[0130] Further examples of charge pairs are disclosed in WO2013 / 157954, which describes a method for producing a molecule comprising a heterodimeric CH3 domain from a single cell, the molecule comprising two CH3 domains capable of forming a face. The method comprises the steps of: (a) a first nucleic acid molecule encoding a polypeptide chain comprising a first CH3 domain, the first CH3 domain comprising a K residue at position 366 according to the EU numbering system; and (b) a second nucleic acid molecule encoding a polypeptide chain comprising a second CH3 domain, the second CH3 domain comprising a D residue at position 351 according to the EU numbering system; providing The method further included culturing the host cells, allowing expression of the two nucleic acid molecules, and harvesting the molecule comprising the heterodimeric CH3 domain from the culture.

[0131] A further method of manipulating electrostatic interactions in polypeptide chains to favour heterodimer formation over homodimer formation was described in WO2011 / 143545.

[0132] Another example of engineering at the CH3-CH3 junction is the strand-exchange engineering domain (SEED) CH3 heterodimer. The CH3 domain is composed of alternating segments of human IgA CH3 and IgG CH3 sequences, forming a pair of complementary SEED heterodimers called "SEED bodies" [13; WO2007 / 110205].

[0133] Bispecifics have also been produced with heterodimerized heavy chains that have been differentially engineered in the CH3 domain to alter their affinity for binding to purification reagents such as Protein A. WO2010 / 151792 is a first polypeptide comprising, from N-terminus to C-terminus, a first epitope-binding region that selectively binds a first epitope, an immunoglobulin constant region comprising a first CH3 region of a human IgG selected from IgG1, IgG2, and IgG4; a second polypeptide comprising from N-terminus to C-terminus a second epitope-binding region that selectively binds a second epitope, an immunoglobulin constant region comprising a second CH3 region of a human IgG selected from IgG1, IgG2, and IgG4, The second CH3 region comprises a modification that reduces or eliminates binding of the second CH3 domain to Protein A; A second polypeptide; In this study, a heterodimerized bispecific antigen-binding protein was described, comprising:

[0134] The antibodies of the present invention can utilize any of these techniques and molecular formats, as desired.

[0135] immunity A further aspect of the invention is the use of a non-human animal as described herein to produce antibodies comprising unpaired variable domains which specifically bind a target antigen. Antibodies comprising unpaired variable domains can be produced by exposing the non-human animal to an immunogenic challenge with the target antigen as described herein.

[0136] A method for producing an antibody that binds a target antigen includes providing a non-human animal having a genome as described herein; (a) immunizing an animal with a target antigen (e.g., with a cell expressing the antigen or with a purified recombinant antigen); (b) isolating the antibodies produced by the animal; (c) testing the antibody for the ability to bind the antigen; (d) selecting one or more antibodies that bind the antigen; may include.

[0137] The non-human animal can be a knockout animal in which endogenous expression of a target (eg, an ortholog of a human target antigen) has been inactivated.

[0138] The non-human animals described herein can be loaded with a target antigen, and lymphocytes (such as B cells) can then be harvested from the animals that express the antibodies. The lymphocytes are fused with a myeloma cell line to prepare immortalized hybridoma cell lines, which are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. Nucleic acids encoding the variable regions can be isolated and linked to the desired isotype constant regions. Such antibodies can be produced in cells such as CHO cells. Alternatively, nucleic acids encoding the variable domains can be isolated directly from lymphocytes.

[0139] Nucleic acids encoding the antibody heavy chain variable domain and / or the antibody light chain variable domain of a selected antibody can be isolated. Such nucleic acids can encode the heavy and / or light chain of a complete antibody, or the variable domains without the associated constant regions. As mentioned above, the encoding nucleotide sequences can be obtained from lymphocytes.

[0140] Antibody discovery is significantly facilitated by working with antibodies that contain unpaired variable domains (provided by either the heavy or light chain) rather than paired antigen-binding domains (provided by the heavy:light chain pair), since identification of correctly paired sequences is not required. Prior techniques for in vivo antibody discovery include bulk sequencing of variable domains from B cells, which in "classical" systems generates a huge number of VH and VL domain sequences for which it is highly desirable to know which VH and VL domain sequences are paired with which VL domain sequences. Pairwise tracking can add an extra layer of complexity, for example requiring single cells to be sorted into individual wells of a plate for analysis, so that VH and VL sequence information is identified from each cell simultaneously. In contrast, when variable domain sequence information is contained in a single VH or VL domain sequence per B cell, antibody sequences can be conveniently processed in bulk, rather than as individual cells. Bulk sequencing of B cells from immunized animals can be performed with or without a step of antigen-specific cell sorting.

[0141] Optionally, once the nucleic acid encoding the variable domain is obtained, it is conjugated to a nucleotide sequence encoding a desired constant region or other polypeptide domain to provide a nucleic acid encoding a polypeptide comprising an unpaired variable domain.

[0142] If the immunized mammal produces a chimeric antibody with a non-human constant region, this antibody can be replaced with a human constant region to generate an antibody that will be less immunogenic when administered to humans as a medicine.Providing a specific human isotype constant region is also important to determine the effector function of the antibody, and some suitable heavy chain constant regions are discussed herein.Alternatively, the nucleic acid encoding the variable domain can be linked to a non-antibody polypeptide domain, for example, to encode a CAR.

[0143] Other modifications to the nucleic acid encoding the antibody heavy and / or light chain variable domains can be performed, such as mutating residues and creating variants. There are many reasons why it may be desirable to create variants, including optimizing the antibody sequence for large-scale manufacturing, facilitating purification, enhancing stability, or improving suitability for inclusion in a desired pharmaceutical formulation. Protein engineering operations can be performed at one or more selected residues in the sequence, for example, to replace an amino acid with an alternative amino acid (optionally creating a variant containing all naturally occurring amino acids at this position, with the possible exception of Cys and Met), and monitoring its effect on function and expression to determine the optimal substitution. In some cases, it is not desirable to replace a residue with Cys or Met, or to introduce these residues into the sequence, as doing so may create manufacturing difficulties (e.g., due to the formation of new intramolecular or intermolecular cysteine-cysteine ​​bonds). When a lead candidate is selected and optimized for manufacturing and clinical development, it will generally be desirable to change its antigen-binding properties as little as possible, or at least to retain the affinity and potency of the parent molecule, however, variants may be generated to modulate key antibody properties such as affinity, cross-reactivity, or neutralization capacity.

[0144] The isolated (optionally mutated) nucleic acid can be introduced into a host cell, e.g., a CHO cell. The host cell is then cultured under conditions for expression of a polypeptide comprising the variable domain.

[0145] Antibodies can bind cell surface receptors, e.g., the extracellular domain of such receptors. Cells that express the antigen or a desired fragment thereof on their cell surface (e.g., cells transfected with a nucleic acid encoding the antigen or fragment and that express high levels of the antigen or fragment) can be used for immunization.

[0146] Exemplary categories of antigens include transmembrane receptors, such as seven-transmembrane receptors, e.g., G-protein coupled receptors (GPCRs). Receptors can include an extracellular (EC) domain, a transmembrane domain, and a cytoplasmic domain. It is common to use antibodies to target the EC domain of a receptor, since the EC domain is more accessible to antibodies injected into a patient. Receptors of interest can bind ligands such as hormones, neurotransmitters, cytokines, growth factors, cell adhesion molecules, or nutrients. Target antigens can be antigens of pathogens, to generate antibodies (and isolated unpaired variable domains) that bind to the pathogen or infected cells.

[0147] In many envisioned situations, the target antigen will be a human antigen.

[0148] Antibodies produced according to the invention can bind human antigens and non-human orthologues of the antigens, for example, can bind both human and rodent (e.g., mouse or rat) antigens. Antibodies produced by the methods described herein can be tested to confirm specific binding to human and non-human animal target antigens. Cross-reactive antibodies can thus be selected, which can be screened for other desirable properties as described herein.

[0149] Methods of generating antibodies against an antigen (e.g., a human antigen) through immunization of the animal with an endogenous antigen (e.g., an endogenous mouse antigen) whose expression has been knocked out in the animal can be carried out in animals capable of generating antibodies comprising human variable domains. The genome of such animals can be engineered to contain human or humanized immunoglobulin loci encoding human variable region gene segments, and optionally endogenous or human constant regions. Recombination of human variable region gene segments produces human antibodies, which can have either non-human or human constant regions. The non-human constant regions can then be replaced by human constant regions if the antibody is intended for in vivo use in humans. Such methods and knockout transgenic animals are described elsewhere herein and in WO2013 / 061078.

[0150] Encoding Nucleic Acids and Expression Methods An isolated nucleic acid encoding an antibody according to the invention can be provided. The nucleic acid can be DNA and / or RNA. Genomic DNA, cDNA, mRNA, or other RNA, or any combination thereof, of synthetic origin, can encode the antibody.

[0151] The present invention provides constructs in the form of plasmids, vectors, transcription cassettes or expression cassettes comprising at least one polynucleotide above. Exemplary nucleotide sequences are included in the sequence listing. Reference to a nucleotide sequence described herein encompasses DNA molecules having the particular sequence, and RNA molecules having the particular sequence in which U replaces T, unless the context requires otherwise.

[0152] The present invention also provides a recombinant host cell comprising one or more nucleic acids encoding the antibody. A method of producing the encoded antibody can include expression from the nucleic acid, for example, by culturing a recombinant host cell containing the nucleic acid. The antibody can be so obtained, isolated and / or purified using any suitable technique, and then used as appropriate. The production method can include formulating the product into a composition comprising at least one further component, such as a pharma- ceutically acceptable excipient.

[0153] Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems, as well as transgenic plants and animals.

[0154] Expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. A common bacterial host is Escherichia coli (E. coli). Expression in eukaryotic cells in culture is also available to those of skill in the art as an option for production. Mammalian cell lines available in the art for expression of heterologous polypeptides include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2 / 0 rat myeloma cells, human embryonic kidney cells, human embryonic retina cells, and many others.

[0155] The vector may contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as appropriate. Nucleic acids encoding antibodies may be introduced into host cells. Nucleic acids may be introduced into eukaryotic cells by a variety of methods, including calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia or, in the case of insect cells, baculovirus. For the introduction of nucleic acids in host cells, particularly eukaryotic cells, virus-based or plasmid-based systems may be used. Plasmid systems may be maintained episomally or may be inserted into the host cell or into an artificial chromosome. Insertion may be by either random or targeted integration of one or more copies at single or multiple loci. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation, and transfection using bacteriophage. Following the introduction, the nucleic acid may be expressed, for example, by culturing host cells under conditions for expression of the gene and then, optionally, isolating or purifying the antibody.

[0156] The nucleic acids of the invention can be integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by the inclusion of sequences that facilitate recombination with the genome, according to standard techniques.

[0157] The invention also provides methods comprising using the nucleic acids described herein in an expression system to express an antibody.

[0158] composition The antibodies according to the invention and nucleic acids encoding same may be provided in isolated form and / or in solution, for example an aqueous solution.

[0159] The present invention further provides a composition (e.g. a pharmaceutical composition or a composition for medical use) comprising an antibody, a bispecific antibody, a polypeptide, an antibody heavy or light chain, a VH domain, a VL domain, or a nucleotide sequence thereof, obtained or obtainable by the invention of the invention disclosed herein.

[0160] Antibodies may be monoclonal or polyclonal, but are preferably provided as monoclonal antibodies for therapeutic use. They may be provided as part of a mixture of other antibodies, optionally including antibodies of different binding specificities.

[0161] The antibodies according to the invention, and the encoding nucleic acids, will usually be provided in isolated form. Thus, the antibodies, VH domains and / or VL domains, and nucleic acids can be provided purified from their natural environment or from the environment in which they are produced. Isolated antibodies and isolated nucleic acids are free or substantially free from materials with which they are naturally associated, such as other polypeptides or nucleic acids with which they are found in vivo, or the environment in which they are prepared (e.g. cell culture), if such preparation is by in vitro recombinant DNA techniques. Optionally, an isolated antibody or nucleic acid is (1) free of at least some other proteins with which it would normally be found; (2) essentially free of other proteins from the same source, e.g., from the same species; (3) expressed by cells from a different species; (4) isolated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated; (5) operably associated (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated; or (6) not found in nature.

[0162] The antibodies or nucleic acids can be formulated with a diluent or adjuvant and still be isolated for practical purposes, for example, they can be mixed with a carrier when used to coat microtiter plates for use in immunoassays, or mixed with a pharma- ceutically acceptable carrier or diluent when used in therapy. Other active ingredients can be included in the therapeutic preparation, as described elsewhere herein. The antibodies can be glycosylated naturally in vivo or by a heterologous eukaryotic system such as CHO cells, or can be non-glycosylated (e.g., when produced by expression in prokaryotic cells). The present invention encompasses antibodies with modified glycosylation patterns. In some applications, modifications to remove undesirable glycosylation sites or, for example, removal of fucose moieties to improve ADCC function can be useful

[14] . In other applications, glycosylation modifications can be made to modify CDC.

[0163] Typically, an isolated product constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. The antibody can be substantially free of proteins or polypeptides or other contaminants found in its natural or produced environment that would interfere with its therapeutic, diagnostic, prophylactic, research, or other uses.

[0164] An antibody can be one that has been identified, isolated, and / or recovered (e.g., naturally or recombinantly) from components of its production environment. An isolated antibody can be free of association with all other components from its production environment, such as, for example, that the antibody has been isolated to a standard that is likely to be approved or has been approved by the FDA. Contaminating components of the production environment, such as those resulting from recombinant transfected cells, are substances that would normally interfere with research, diagnostic, or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, an antibody will be purified: (1) to greater than 95% antibody by weight, and in some embodiments, greater than 99% antibody by weight, as determined, for example, by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver staining. An isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody or nucleic acid encoding it will be prepared by at least one purification step.

[0165] A polypeptide (e.g., an antibody) comprising an unpaired variable domain, or a nucleic acid encoding same, may be formulated for the desired route of administration to a patient, e.g., in a liquid (optionally an aqueous solution) for injection. The composition may include the polypeptide or nucleic acid in combination with a medical injection buffer and / or with an adjuvant. A variety of delivery systems are known and can be used to administer the pharmaceutical composition of the invention. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.

[0166] The composition may include a diluent, excipient, or carrier. If the composition is a pharmaceutical composition or a composition for medical use, the diluent, excipient, or carrier is pharmaceutically acceptable. "Pharmaceutically acceptable" refers to one approved or expected to be approved by a regulatory agency of the USA Federal or state government, or listed in the US Pharmacopeia or other pharmacopoeias generally accepted for use in animals, including humans. "Pharmaceutically acceptable carrier, excipient, or adjuvant" refers to a carrier, excipient, or adjuvant that can be administered to a subject together with a drug, for example, together with any antibody, VL, or antibody chain described herein, and that does not destroy the pharmacological activity and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the drug.

[0167] The composition comprising the polypeptide or nucleic acid described herein can be contained in a sterile container in vitro. The composition can be present in a bag or other medical container connected to an IV syringe. The composition can be present in a vial, syringe or injection device. In one example, a kit is provided that includes the antibody, polypeptide or nucleic acid as well as a package and instructions for use in the therapeutic method described herein.

[0168] The present invention provides therapeutic compositions comprising a polypeptide comprising an unpaired variable domain as described herein. Therapeutic compositions comprising a nucleic acid encoding such a polypeptide are also provided. The encoding nucleic acid is described in more detail elsewhere herein and includes DNA and RNA, e.g., mRNA. In the therapeutic methods described herein, the use of nucleic acid encoding an antibody and / or the use of cells containing such nucleic acid can be used as an alternative (or in addition) to compositions comprising the antibody itself. Cells (e.g., human cells, e.g., human lymphocytes) containing nucleic acid encoding an antibody, optionally with the nucleic acid stably integrated into the genome, thus represent medicines for therapeutic use in patients. Cells expressing a CAR, e.g., CAR-T cells, are an example. Alternatively, nucleic acid encoding an antibody of the invention can be introduced into human B lymphocytes, optionally into ex vivo modified B lymphocytes derived from the intended patient. Optionally, memory B cells are used. Administration of cells containing the encoding nucleic acid to a patient provides a reservoir of cells capable of expressing the antibody, allowing for a longer therapeutic effect compared to administration of an isolated nucleic acid or an isolated antibody.

[0169] Chimeric antigen receptors (CARs) The antibodies and non-human animals according to the invention represent a source of unpaired antigen-binding variable domains that can be incorporated into various modular molecular designs, one of which is the chimeric antigen receptor (CAR). CARs contain an antigen-binding moiety fused to a T cell activation moiety, typically in a transmembrane receptor that also contains a T cell activation cytoplasmic domain. CAR-T structures have been described that contain a VH domain for binding the target antigen [15, 16].

[0170] Thus, the unpaired variable domains can be linked to a T cell activation moiety to provide a CAR. Optionally, T lymphocytes are engineered to express the CAR on their surface. The CAR and cells are preferably human.

[0171] It may be desirable to engineer CAR-T cells to express IL-7 and CCL19, as these factors have been reported to be important for maintaining T cell zones in lymphoid organs

[17] .

[0172] The unpaired variable domains produced as described herein can be used to construct a nucleic acid encoding a human CAR, which can then be transfected into human T cells and / or integrated into the T cell genome. The activity of the CAR can be assessed, for example, by introducing the CAR-T cells into syngeneic tumor-bearing animals and / or human cell lines and observing the effect on the target cells.

[0173] Unless otherwise specified herein or the context permits, any unpaired variable domain herein may be a VH (heavy chain variable domain, e.g. a human, canine, feline, equine, fish or avian VH domain), a VHH (e.g. a camelid variable domain, whether humanized or not) or a VL (light chain variable domain, such as a kappa VL or lambda VL, e.g. a human, canine, feline, equine, fish or avian VL domain).

[0174] Unless otherwise specified herein or the context permits, any unpaired variable domains may be human, humanized, chimeric (e.g., mouse-human chimera or rat-human chimera), rodent (e.g., mouse or rat), canine, feline, equine, fish or avian variable domains.

[0175] Unless otherwise specified herein or the context permits, any inserted DNA (e.g., human variable region DNA at a heavy chain locus) and / or DNA encoding a shielding domain can be human, humanized, chimeric (e.g., mouse-human or rat-human chimeric), rodent (e.g., mouse or rat), canine, feline, equine, fish or avian DNA, preferably human DNA. For example, any heavy chain locus herein can comprise one or more variable region gene segments disclosed in Table C(a) (Table 4, Table 5), or can comprise at least 50%, 60%, or 90% of such gene segments, or can comprise all of such gene segments.

[0176] An example of a shielding domain is Cκ, encoded by the gene segment shown in Table C(b).

[0177] An example of a suitable antigen (e.g., an antigen with which an animal of the invention may be immunized or to which an antibody or variable domain of the invention may bind) is: ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA; AWl; AIG1; AKAP1; AKAP2; AIYIH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1; BMP2; BMP3B (GDF1O); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (Plectin); BRCA1; Cl9orflO (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6 / JAB61); CCL1 (I-309); CCL11 (Eotaxin); CCL13 (MCP-4); CCL15 (MIP-id); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL2O (MIP-3a); CCL21 (MIP-2); SLC; Exodus-2; CCL22 (MDC / STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2 / Eotaxin-2); CCL25 (TECT); CCL26 (Eotaxin-3); CCL27 (CTACK / ILC); CCL28; CCL3 (MIP-1a); CCL4 (MIP-1b); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1 / HM145); CCR2 (mcp-1RB / RA); CCR3 (CKR3 / CMKBR3); CCR4; CCR5 (CMKBR5 / ChemR13); CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6); CCR7 (CKR7 / EBI1); CCR8 (CMKBR8 / TER1 / CKR-L1); CCR9 (GPR-9-6);CCRL1(VSHK1); CCRL2(L-CCR); CD164; CD19; CD1C; CD2O; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD4O; CD4OL; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD8O; CD81; CD83; CD86; CDH1(E-カドヘリン); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A(p2lWap1 / Cip1); CDKN1B(p27Kip1); CDKNIC; CDKN2A(p16lNK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB;キチナーゼ; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7(クラウジン-7); CLN3; CLU(クラステリン); CMKLR1; CMKOR1(RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSF1(M-CSF); CSF2(GM-CSF); CSF3(GCSF); CTLA4; CTNNB1(b-カテニン); CTSB(カテプシンB); CX3CL1(SCYDi); CX3CR1(V28); CXCL1(GRO1); CXCL1O(IP-10); CXCL11(I-TAC / IP-9); CXCL12(SDF1); CXCL13; CXCL14; CXCL16; CXCL2(GRO2); CXCL3(GRO3); CXCL5(ENA-78 I LIX); CXCL6(GCP-2); CXCL9(MIG); CXCR3(GPR9 / CKR-L2); CXCR4; CXCR6(TYMSTR ISTRL33 I Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP; DES;DKFZp451J0118; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNAI; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EPHB4; F3(TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FGF; FGF1(aFGF); FGF1O; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF22; FGF23; FGF3(int-2); FGF4(HST); FGF5; FGF6(HST-2); FGF7(KGF); FGF8; FGF9; FGFR3; FIGF(VEGFD); FIL1(EPSILON); FIL1(ZETA); FLJ12584; FLJ25530; FLRT1(fibronectin); FLT1; FOS; FOSL1(FRA-l); FY(DARC); GABRP(GABAa); GAGEB1; GAGEC1; GALNAC4S-65T; GATA3; GDF5; GFI1; GGT1; GM-CSF; GNAS1; GNRH1; GPR2(CCR1O); GPR31; GPR44; GPR81(FKSG8O); GRCC10(C10); GRP; GSN(gelsolin); GSTP1; HAVCR2; HDAC4; EDAC5; HDAC7A; HDAC9; HGF; HIF1A; HIP1; histamine and histamine receptor; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; 1D2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; TFNW1; IGBP1; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A;IL12B; IL12RB1; IL12RB2; 1L13; IL13RA1; IL13RA2; 1L14; 1L15; IL15RA; IL16; 1L17; IL17B; IL17C; IL17R; 1L18; IL18BP; IL1F1O; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2 IL1RN; 1L2; 1L20; IL2ORA; IL21R; 1L22; 1L22R; 1L22RA2; 1L23; 1L24; 1L25; 1L26; 1L27; 1L28A; 1L28B; 1L29; IL2RA; IL2RB; IL2RG; 1L3; 1L30; IL3RA; 1L6; IL6R; IL6ST (glycoprotein 130); 1L7; TL7R; 1L8; IL8RA; IL8RB; IL8RB; 1L9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAK1; IRAK2; ITGA1; ITGA2; 1TGA3; ITGB4 (b 4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; MTLG; KLF5 (GC box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (keratin 19); KRT2A; KRTHB6 (hair-specific type II keratin); LAMA5; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB1; midkine; MIF; MIP-2; MK167(Ki-67); MMP2; MMP9; MS4A1; MSMB;MT3 (metallothionectin-ifi); MTSS 1; MUC 1 (mucin); MYC; MYD88; NCK2; neurocan; NFKB 1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NROB1; NROB2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAM1; PLAU(uPA); PLG; PLXDC1; PPBP(CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2(COX-2); PTN; RAC2(p2lRac2); RARB; RGS1; RNF11O(ZNF144); ROBO2; S100A2; SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial monocyte-activating cytokine); SDF2; SERPINA1; SERPINIA3; SERPINB5 (maspin); SERPINE1 (PAT-i); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B (Spri); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; TB4R2; TBX21; TCP1O; TDGF1; TEK; TGFA; TGFB1; TGFB1I1; TGFB2; TGFB3;TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1(thrombospondin-1); THBS2; THBS4; THPO; TIE(Tie-i); TIMP3; tissue factor; TLR1O; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TNFAIP2(B94); TNFAIP3; TNFRSF1 1A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6(Fas); TNFRSF7; TNFSF13(April); TNFSF13B; TNFSF14(HVEM-L); TNFSF1 5(VEGI); TNFSF1 8; TNFSF4(OX40(0X40) ligand); TNFSF5(CD4O ligand); TNFSF6(FasL); TNFSF7(CD27 ligand); TNFSF8(CD3O ligand); TNFSF9(4-1BB ligand); TOLLIP; Toll-like receptor; TOP2A(topoisomerase lia); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC;versican; VHL C5; VLA-4; XCL1(lymphotactin); XCL2(SCM-lb); XCR1(GPR5 / CCXCR1); YY1; and ZFPM2.

[0178] For example, the antigen is selected from the following list (e.g., the antibody or variable domain is for administration to a human or animal subject to treat cancer or an autoimmune condition): immune checkpoint inhibitors (e.g., PD-L1, PD-1, CTLA-4, TIGIT, TIM-3, LAG-3, and VISTA, e.g., TIGIT, TIM-3, and LAG-3), immunomodulators (e.g., BTLA, hHVEM, CSF1R, CCR4, CD39, CD40, CD73, CD 96, CXCR2, CXCR4, CD200, GARP, SIRPα, CXCL9, CXCL10, CXCL11 and CD155, e.g., GARP, SIRPα, CXCR4, BTLA, hVEM and CSF1R), immunostimulants (e.g., CD137, GITR, OX40, CD40, CXCR3 (e.g., agonistic anti-CXCR3 antibodies), CD27, CD3, ICOS (e.g., agonistic anti-ICOS antibodies), e.g., ICOS, CD137, GITR and OX40).

[0179] In one example, the λ5 shielding domain herein comprises or consists of the amino acid sequence encoded by SEQ ID NO:77.

[0180] In the examples herein, the animals of the invention comprise a light chain locus (either a kappa or lambda locus) comprising SEQ ID NO: 77, 78, 81, 83, 85, 87, 89, 91, 93 or 100.

[0181] In one example herein, an animal of the invention expresses SEQ ID NO:82, 84, 86, 88, 90 or 101.

[0182] In one example, the animals of the invention comprise a kappa light chain locus (heterozygous or homozygous state) as described herein. Optionally, the locus comprises a deletion between the endogenous Vκ gene and the endogenous Jκ gene, but retains the endogenous splice junction at the 5' end of Vκ exon 2 and the 3' end of the J, in addition to Vκ exon 1. In another example, the animals of the invention comprise a replacement of the endogenous Jκ gene with a targeting vector encoding a variable region promoter, a mouse or human Vκ leader (exon 1, intron, partial exon 2), and a fragment of Jκ that retains the endogenous splice junction at the 5' end of Vκ exon 2 and the 3' end of the J. The result of both of these strategies is a locus that expresses a transcript under the control of the endogenous or human Vκ promoter, which is spliced ​​to generate a Cκ with a Vκ leader sequence and a partial J sequence. According to this strategy, any Vκ and J of the mouse kappa repertoire can be utilized. Strategy 1 uses Vκ3-2 or Vκ3-4 with Jκ5 because Vκ3-2 or Vκ3-4 are closer to the 3′ end of the locus, thus reducing the size of the deletion required, and because literature has shown that they are highly utilized in mice. See FIG. 16 for an overview of the kappa locus structure and FIG. 4 for predicted expression sequences from the two example modified loci. Vκ6-17 or Vκ10-96 can be used with Jκ5, as these Vs have been shown to be the most highly utilized in the mouse repertoire (see reference 18). See FIG. 18 for an example of a targeting strategy / locus overview and FIG. 19 for predicted expression sequences using Vκ6-17 and Vκ10-96.

[0183] In one example, all non-coding regulatory elements of the modified locus are endogenous. In the case of the modified kappa locus, this includes the specific promoter of the Vκ / Jκ / Cκ fragment, the endogenous κ intronic enhancer (located between Jκ5 and Cκ) and the endogenous κ 3' enhancer. The interaction of these endogenous promoters / enhancers with endogenous (e.g., mouse) effectors responsible for expression from the κ locus may be more effective than the interaction of the endogenous effectors with human regulatory sequences, thus resulting in a more active locus.

[0184] term The following numbered sections describing embodiments of the present invention are part of this specification. 1. A composition comprising an isolated antibody in solution, the antibody comprising an unpaired variable domain for binding a target antigen, the unpaired variable domain linked to a constant region, the constant region comprising a CH1 domain and a shielding domain linking the CH1 domain. 2. The composition of paragraph 1, wherein the unpaired variable domains are linked to the CH1 domain of the constant region. 3. A first polypeptide comprising a human variable domain and a CH1 domain; a second polypeptide comprising a shielding domain paired with the CH1 domain, the second polypeptide lacking a variable domain, thereby leaving the variable domain of the first polypeptide unpaired; A composition comprising: 4. The composition of paragraph 3, wherein the first polypeptide is an immunoglobulin heavy chain comprising VH-CH1-CH2-CH3. 5. The composition of paragraph 3 or 4, wherein the second polypeptide consists of a shield domain. 6. The composition of any one of paragraphs 1 to 5, wherein the shield domain is a CL domain. 7. The composition according to item 6, wherein CL is Cκ. 8. The composition according to item 6, wherein CL is Cλ. 9. The composition of any one of clauses 1 to 5, wherein the shielding domain is a λ5 immunoglobulin domain. 10. The composition of any one of paragraphs 1 to 9, comprising an Fc region. 11. The composition of any one of clauses 1 to 10, wherein the unpaired variable domains are VH domains. 12. The composition of any one of the preceding claims, which is a four-chain antibody comprising two of the unpaired variable domains. 13. A composition comprising an isolated antibody in solution, the antibody comprising two first polypeptides and two second polypeptides; each first polypeptide comprises a human variable domain and a CH1 domain; each second polypeptide comprises a shield domain paired with a CH1 domain of the first polypeptide; A composition wherein one or both of said second polypeptides lack a variable domain, thereby rendering one or both variable domains of the first polypeptide unpaired. 14. The composition of paragraph 13, wherein the variable domains of both first polypeptides are unpaired variable domains. 15. The composition of clause 13 or 14, wherein each said first polypeptide comprises a VH domain. 16. The composition of any one of clauses 13 to 15, wherein each said first polypeptide comprises a VH-CH1-CH2-CH3. 17. The composition of any one of clauses 13 to 16, wherein the two first polypeptides are identical. 18. The composition of any one of clauses 13 to 17, wherein the two second polypeptides are identical. 19. The composition of any one of clauses 13 to 18, wherein each said second polypeptide consists of a shield domain. 20. The composition of any one of clauses 13 to 19, wherein the shield domain is a CL domain. 21. The composition according to item 20, wherein CL is Cκ. 22. The composition according to item 20, wherein CL is Cλ. 23. The composition of any one of clauses 13 to 19, wherein the shielding domain is a λ5 immunoglobulin domain. 24. An antibody comprising a heavy chain and a light chain, the heavy chain comprises an unpaired human VH domain for binding a target antigen and a heavy chain constant region comprising a CH1 domain; The light chain contains a CL domain, An antibody in which the light chain lacks a VL domain, thereby leaving the VH domain unpaired. 25. Contains two heavy chains and two light chains, Each heavy chain comprises a human VH domain and a heavy chain constant region comprising a CH1 domain; Each light chain contains a CL domain, 25. The antibody of clause 24, wherein one or both light chains lack a VL domain, thereby leaving one or both VH domains unpaired. 26. Contains two heavy chains and two light chains, each heavy chain comprises an unpaired human VH domain for binding a target antigen and a heavy chain constant region comprising a CH1 domain; The antibody of clause 25, wherein each light chain comprises a CL domain, and the light chain lacks a VL domain. 27. The antibody of clause 26, wherein the two unpaired VH domains bind the same antigen or epitope. 28. The antibody of clause 26 or 27, wherein the two unpaired VH domains are identical in amino acid sequence. 29. The antibody of any one of clauses 25 to 28, wherein the two first polypeptides are identical in sequence and / or the two second polypeptides are identical in sequence. 30. The antibody of any one of clauses 24 to 29, wherein the heavy chain constant region comprises a CH1 domain and one or more additional CH domains. 31. The antibody of clause 30, wherein the heavy chain constant region comprises a CH2 domain and a CH3 domain. 32. The antibody of any one of clauses 24 to 31, wherein CL is Cκ. 33. The antibody of any one of clauses 24 to 28, wherein CL is Cλ. 34. The antibody of any one of clauses 24 to 33, wherein the light chain consists of a CL domain. 35. The antibody of any one of clauses 24 to 34, wherein the heavy chain constant region is a human heavy chain constant region. 36. The antibody of any one of clauses 24 to 35, wherein CL is human Cκ or human Cλ. 37. The antibody of clause 36, wherein CL comprises the human Cκ sequence of SEQ ID NO:4. 38. The antibody according to clause 37, wherein CL consists of the human Cκ sequence of SEQ ID NO:4. 39. The composition of any one of clauses 1 to 23 or the antibody of any one of clauses 24 to 38, wherein the antibody is an IgG. 40. The antibody of any one of clauses 24 to 38, wherein the antibody is IgM. 41. The composition or antibody of any one of clauses 1 to 40, wherein the antibody is a fully human antibody. 42. The composition or antibody of any one of clauses 1 to 41, wherein the unpaired variable domain binds a human antigen. 43. The composition or antibody of any one of clauses 1 to 42, wherein the unpaired variable domain binds the extracellular domain of a receptor. 44. A nucleic acid encoding the antibody of any one of clauses 1 to 43 or a polypeptide or unpaired variable domain thereof. 45. A heavy chain comprising a human VH domain for binding a target antigen and a heavy chain constant region comprising a CH1 domain; and Light chains lacking a variable domain and containing a CL domain 45. The nucleic acid of claim 44, comprising a nucleotide sequence encoding: 46. ​​The nucleic acid of paragraph 45, wherein the light chain comprises a signal peptide fused to a Cκ constant domain. 47. The nucleic acid of clause 46, wherein the light chain comprises SEQ ID NO:6. 48. The nucleic acid of clause 47, comprising the nucleotide sequence of SEQ ID NO:5 encoding the light chain. 49. A non-human animal or a cell thereof, comprising in its genome a nucleic acid according to any one of paragraphs 44 to 48. 50. A non-human animal comprising a B lymphocyte expressing an antibody according to any one of paragraphs 1 to 43. 51. The animal of clause 50, wherein upon immunogenic stimulation, at least 50% of the antibody-expressing B lymphocytes in the animal express an antibody of any one of clauses 1 to 43. 52. The animal of clause 50 or 51, wherein the B lymphocytes lack functional expression of a light chain comprising a VL domain. 53. A plurality of human variable region gene segments that can be rearranged to encode a variable domain upstream of DNA encoding an immunoglobulin constant region including a CH1 domain; and A gene encoding a CL domain lacking functional expression of a variable region gene segment, or a gene encoding an immunoglobulin domain of λ5 A non-human animal cell having a genome comprising: 54. A non-human animal comprising B lymphocytes expressing antibodies comprising unpaired human VH domains for binding an antigen, wherein the genome of the animal comprises A plurality of human variable region gene segments that can be rearranged to encode a variable domain upstream of DNA encoding an immunoglobulin constant region that includes a CH1 domain; and A gene encoding a CL domain lacking functional expression of a variable region gene segment, or a gene encoding an immunoglobulin domain of λ5 Non-human animals, including: The λ5 immunoglobulin domain may be a human, rodent, mouse, rat, rabbit or mammalian vertebrate domain. The λ5 immunoglobulin domain may be a domain having a truncation as described herein. 55. A method of producing a non-human animal comprising B lymphocytes expressing antibodies comprising unpaired human VH domains for binding an antigen, comprising: A plurality of variable region gene segments that can be rearranged to encode variable domains upstream of DNA encoding an immunoglobulin constant region that includes a CH1 domain; and A gene encoding a CL domain lacking functional expression of a variable region gene segment, or a gene encoding an immunoglobulin domain of λ5 and manipulating the genome of the non-human animal cell to comprise generating an animal from said cell or from a population of cells comprising said cell; The method includes: 56. The cell of paragraph 53 or the method of paragraph 55, wherein the cell is an embryonic stem cell or a zygote. 57. The animal, cell or method of any one of clauses 53 to 56, wherein the multiple variable region gene segments comprise one or more V gene segments, one or more D gene segments and one or more J gene segments that can rearrange to encode a VH domain. 58. The animal, cell or method of paragraph 57, wherein the one or more V gene segments, one or more D gene segments and one or more J gene segments comprise multiple human V gene segments, multiple human D gene segments and multiple human J gene segments. 59. The animal, cell or method of any one of clauses 53 to 58, wherein the multiple variable region gene segments are located at an endogenous immunoglobulin heavy chain locus of the animal. 60. The animal, cell or method of any one of clauses 53 to 59, wherein the CL domain is a human CL domain. 61. The animal, cell or method of clause 60, wherein the human CL domain is human Cκ. 62. The animal, cell or method of any one of clauses 53 to 61, wherein the gene encoding the CL domain comprises an exon encoding a light chain variable region leader sequence and an exon encoding the CL domain, separated by an intron containing a JC intron enhancer element, and the encoded CL domain comprises an N-terminal signal peptide. 63. The animal, cell or method of paragraph 62, wherein the gene encoding the CL domain comprises an exon encoding a human Vκ leader sequence and an exon encoding a human Cκ domain, separated by an intron containing a human J-Cκ intronic enhancer element, and the encoded CL domain is a human Cκ domain including an N-terminal signal peptide. 64. The animal, cell or method of clause 63, wherein the human Cκ domain comprises SEQ ID NO:4 or SEQ ID NO:6. 65. The animal, cell or method of clause 64, wherein transcription of the gene encoding the CL domain produces a nucleic acid comprising SEQ ID NO:5. 66. The animal, cell or method of any one of clauses 53 to 66, wherein the gene encoding the CL domain is in an endogenous immunoglobulin light chain locus of the animal. 67. The animal, cell or method of paragraph 66, wherein the endogenous immunoglobulin light chain locus is an endogenous Igκ locus. 68. The animal, cell or method of any one of clauses 53 to 67, wherein expression of endogenous immunoglobulin light chain variable region gene segments is inactivated. 69. The animal, cell or method of paragraph 69, wherein expression of endogenous immunoglobulin light chain is inactivated. 70. The animal, cell or method of any one of clauses 53 to 66, wherein expression of endogenous immunoglobulin heavy chain variable region gene segments is inactivated. 71. The animal, cell or method of clause 70, wherein expression of endogenous immunoglobulin heavy chain is inactivated. 72. The animal, cell or method of any one of clauses 53 to 71, wherein the animal is a rodent. 73. The animal, cell or method of paragraph 72, wherein the animal is a mouse or a rat. 74. The animal, cell or method of paragraph 73, wherein multiple human variable region gene segments capable of rearrangement to encode human variable domains upstream of human DNA encoding an immunoglobulin constant region comprising a human CH1 domain have been inserted at an endogenous immunoglobulin heavy chain locus on mouse chromosome 12, and expression of the mouse heavy chain has been inactivated. 75. The animal, cell or method of paragraph 73 or 74, wherein a gene encoding a human Cκ domain is inserted at the endogenous Igκ light chain locus on mouse chromosome 6, and expression of the mouse Igκ light chain is inactivated. 76. An animal produced by the method of any one of paragraphs 55 to 75. 77. The animal or method of any one of clauses 54 to 76, wherein the animal's B lymphocytes express an antibody of any one of clauses 1 to 43. 78. The animal or method of clause 77, wherein upon immunogenic stimulation, at least 50% of the antibody-expressing B lymphocytes in the animal express an antibody of any one of clauses 1 to 43. 79. A method for producing an antibody comprising an unpaired VH domain for binding an antigen, comprising exposing an animal according to any one of paragraphs 49 to 52, 54 or 57 to 78 to immunogenic stimulation with a target antigen. 80. The method of paragraph 79, comprising isolating the antibody or nucleic acid encoding it from the animal. 81. The method of paragraph 79 or 80, comprising determining the sequence of the unpaired variable domain of the antibody or the nucleic acid encoding it. 82. The method of paragraph 80 or 81, comprising introducing one or more mutations into the nucleotide sequence of the nucleic acid encoding the variable domain. 83. The method of paragraph 81 or 82, comprising the step of providing a DNA vector containing the encoding nucleic acid. 84. The method of paragraph 83, wherein the nucleic acid encodes a polypeptide comprising a variable domain and one or more further domains. 85. The method of any one of clauses 80 to 84, further comprising the step of cloning the encoding nucleic acid into a recombinant host cell. 86. The method of paragraph 85, further comprising culturing the cells for expression of a polypeptide comprising the variable domain. 87. The method of claim 86, comprising recovering and purifying the polypeptide from the cells or culture medium. 88. The method of any one of clauses 84 to 87, wherein the polypeptide is an isolated variable domain, an antibody, or a chimeric antigen receptor. 89. The method of paragraph 87 or 88, comprising formulating the polypeptide into a composition comprising a pharma- ceutically acceptable excipient. 90. The antibody of any one of clauses 1 to 43 for use in therapeutic treatment of the human body. 91. Exposing a target antigen to an antibody according to any one of clauses 1 to 43 in vitro; allowing the unpaired variable domains to bind to the target antigen, thereby forming an antibody:antigen complex; and Isolating the antibody:antigen complex 23. A method for preparing an antibody:antigen complex, comprising: EXAMPLES

[0185] Experimental Example Example 1 Mouse genome engineered to express human Ig light chains containing Cκ and lacking the VL domain Mice are engineered to express an antibody in which a truncated kappa light chain containing a Ckappa domain fragment is paired with the CH1 of a fully human heavy chain containing an unpaired VH domain.

[0186] The mice contain a humanized heavy chain locus on mouse chromosome 12 (Figure 7), a modified fully humanized κ locus on mouse chromosome 6 (Figure 8), and an active or inactivated endogenous λ locus or an inactivated humanized λ locus on chromosome 16 (Figure 9).

[0187] In these mice, the humanized κ locus has been engineered to inactivate the normal vj rearrangement and instead express a Cκ domain fragment.

[0188] A large deletion (approximately 90 kb) of the human κ locus removes a 90 kb fragment encompassing exon 2 of Jκ5 from Vκ1-5. This removes all of Vκ2-4, Vκ7-3, Vκ5-2, Vκ4-1, JκI, Jκ2, Jκ3, Jκ4, and Jκ5, but leaves exon 1 of Vκ1-5, which encodes most of the Vκ1-5 signal peptide, the κ enhancer, and the Cκ gene. Thus, the latter elements remain intact. The upstream V gene segments cannot rearrange in the absence of a complete set of J gene segments. Thus, normal κVJ recombination is inactivated. The remaining Vκ1-5 exon 1 (i.e., leader sequence) is then spliced ​​onto Cκ to create a new transcript encoding a Cκ polypeptide including the signal peptide (FIG. 3).

[0189] The nucleotide and protein sequences of the modified κ locus of the present invention (FIGS. 3b, 3c) can be compared to the unmodified human Vκ1-5 sequence (FIGS. 4a, 4b).

[0190] The sequence of the Cκ fragment transcript and the resulting protein contains the predicted cleavage site of the signal peptide (Figure 5). After cleavage of the signal peptide, Cκ can pair with the CH1 domain of the antibody heavy chain, allowing its proper folding and stabilization.

[0191] A modified κ locus can be generated by creating a double-stranded break at a defined position in the κ genomic locus and providing a repair template to facilitate the desired deletion. Gene deletion can be performed in mouse cells that contain a humanized κ locus, for example, in mouse embryonic stem cells that contain a humanized κ locus, or reagents can be injected into zygotes derived from mice that contain a humanized κ locus in their genome.

[0192] Animals with the desired Cκ locus are then mated with animals containing a fully human heavy chain locus to produce mice capable of producing fully human antibodies comprising unpaired VH domains.

[0193] The λ locus may be either unmodified or inactivated in this platform.

[0194] The data are presented in Example 4.

[0195] Example 2 Pairing of CH1 with the λ5 shield domain It is known that in the absence of light chains, Ig heavy chains are not expressed on the B cell surface. However, λ5 can rescue surface expression of Ig heavy chains when expressed beyond the early development of B cells in the bone marrow. A truncated λ5 protein, which can rescue surface IgM presentation in the absence of VpreB

[11] , appears to be particularly suitable. A unique region of 50 amino acids at the N-terminus of human λ5 is thought to limit the rate of λ5 folding in the absence of VpreB [4]. The immunoglobulin domain of λ5 lacking this N-terminal unique region binds the heavy chain constant domain CH1 and thus represents the shielding domain in the present invention. λ5 contains its native signal peptide or a non-native signal peptide, such as that encoded by the Vκ1-5 leader sequence or the λ1 leader sequence. The signal peptide can be cleaved post-translationally.

[0196] λ5 expression is placed under the control of the Igκ locus by replacing the Jκ region of the light chain κ immunoglobulin locus with a gene encoding the λ5 immunoglobulin domain. A replacement of 5 kb may be sufficient. The β-strand (J) preceding the λ5 Ig fold can be retained. Alternatively, it is omitted, including only the Ig domain itself. In the context of a surrogate light chain, this J-chain supplies the missing β-strand of the VpreB Ig domain [4, 5]. VpreB is not expressed during expression of Igκ (or Igλ) light chains, and therefore λ5 will be expressed in the absence of VpreB.

[0197] Figure 6 shows the modification of the humanized kappa locus with a targeting vector to introduce a human λ5 transgene. The vector contains mouse 3' and 5' homology arms, a human Vκ promoter, a human intron and an intronic enhancer, and a truncated human λ5. Targeting of this vector replaces the human JκI-Jκ5, intron and intronic enhancer, and Cκ with the vector insert.

[0198] The human Igκ locus containing the above modifications is inserted into the genome of a non-human animal, e.g., a mouse, to generate a transgenic animal expressing the human λ5 gene under the control of human Igκ transcriptional control elements. Human DNA is inserted into the animal's endogenous Igκ locus or into an independent (ectopic) locus in the animal's genome. Inactivation of the κ and / or λ light chain locus in the animal's genome can be combined with insertion of a transgene encoding all or part of the human λ5 gene or a mutant thereof. Here, the λ5 transgene is inserted at the inactivated κ and / or λ light chain locus and is under the control of human κ or λ gene control elements including promoter / enhancer elements.

[0199] Example 3 Inactivation of endogenous lambda light chains A large deletion (200 kb) of the lambda locus removes v2 through vl (the segment containing V2, V3, J2C2 and V1) but leaves J3C3, J1C1 and the downstream enhancer intact, thereby inactivating expression of the lambda light chain. Figures 9 and 10.

[0200] The locus is generated by creating a double-stranded break at a defined position in the λ genomic locus and providing a repair template to facilitate the desired deletion.

[0201] The inactivated λ locus is combined by breeding an animal containing this genomic modification with an animal containing a fully humanized heavy chain locus and a modified kappa locus as described herein, ensuring that no light chains are expressed from the endogenous λ locus, even in the absence of allelic exclusion.

[0202] In the absence of a lambda light chain, the Cκ domain pairs with the heavy chain CH1 of the expressed antibody.

[0203] Example 4 Performance of mice containing a truncated kappa light chain locus that includes the C kappa region Mosaic F0 animals generated by cytoplasmic injection, which contain a mixture of WT alleles and uncharacterized indel mutations in addition to the desired fully human kappa locus with a deletion allowing expression of a truncated kappa Cκ fragment, referred to as the KCF locus (see Example 1), were individually mated to segregate the mosaics, resulting in the generation of F1 animals each carrying an allele from a single F0. To generate F1 animals suitable for early analysis, each of the mosaic F0s was bred with an animal containing an inactivated kappa locus (inactivated by inserting a neomycin cassette into the kappa locus between Jκ5 and Cκ, thus preventing normal kappa recombination). The resulting heterozygous animals, which contain an inactivated mouse kappa locus in addition to the desired modified kappa locus (see Example 1), were analyzed at the transcript and protein levels.

[0204] RNA was extracted from splenocytes from the heterozygotes and from control animals with an unmodified fully human kappa locus using TRIzol® Reagent (Invitrogen™) and standard protocols. First strand DNA synthesis was performed using the SuperScript III First-Strand Synthesis SuperMix Kit (Invitrogen™) and either oligos specific for the human Cκ coding region or Oligo(dT) primers. The first strand DNA was then used as a template for PCR with primers specific for the human kappa constant coding region or 3′UTR in combination with oligos specific for the human Vκ1-5 leader sequence or 5′UTR. A single PCR product with the expected size for the predicted truncated kappa fragment was identified from the modified kappa locus with oligos for human Vκ1-5 5′UTR and human Cκ 3′UTR. Sequencing of this PCR product confirmed that this transcript contained the predicted splice junction between human Vκ1-5 exon 1 and the human Cκ gene, providing evidence that the large deletion made in the humanized kappa locus does indeed result in expression of the predicted truncated kappa fragment transcript. See Figure 11 for primer details, Figure 12 for PCR results, and Figure 13 for sequencing results.

[0205] The presence of correctly spliced ​​transcripts of the truncated human kappa-shield domain was confirmed in samples from multiple animals containing the KCF locus.

[0206] Example 5 Mice with human λ5 shield domain An embodiment of the invention provides a mouse containing an inactivated kappa and / or lambda light chain locus, either endogenous or humanized, combined with the insertion of a transgene encoding all or a portion of the human, rodent, non-human primate or other mammalian lambda 5 (λ5) gene, or a mutant thereof. The λ5 transgene is inserted into the inactivated kappa and / or lambda light chain locus and is under the control of endogenous and / or exogenous light chain promoters and enhancers. For example, a human promoter and enhancer; or a mouse promoter and enhancer; or a human variable region promoter and mouse enhancer (such as a mouse intron and 3' light chain locus enhancer) are used.

[0207] Inactivation of the kappa locus: 1) Deletion or replacement of Jκ1-Jκ5 genes (Example 6) 2) Deletion or replacement of Jκ1-Jκ5 genes and Cκ gene (Example 5) 3) Deletion or replacement of the C gene 4) Deletion or replacement of all Vκ genes 5) Deletion or replacement of all Vκ genes, Jκ1-Jκ5 genes, and Cκ gene 6) Insertion of a cassette that prevents normal splicing between the Jκ1-Jκ5 genes and the Cκ gene (kappa "KO" allele bred to mosaic animals for analysis) Therefore, this can be done.

[0208] Inactivation of the lambda locus is: 1) Deletion or replacement of Vλ2, Vλ3, Vλ1, plus the Jλ2 / Cλ2 cluster, leaving the Jλ3 / Cλ3 and Jλ2 / Cλ1 clusters (Example 3) 2) deletion or replacement of Vλ1 plus Jλ2 / Cλ2, Jλ3 / Cλ3, and Jλ1 / Cλ1 clusters, leaving Vλ2 and Vλ3; 3) deletions or substitutions of Vλ1, Vλ3, Vλ1, as well as Jλ2 / Cλ2, Jλ3 / Cλ3, and Jλ1 / Cλ1 clusters; This can be done by:

[0209] A targeting vector (complete transgene including homology arms, SEQ ID NO: 79) encoding a truncated human λ5 coding region (SEQ ID NO: 77) and flanked by 800-1 kb arms homologous to mouse genomic sequences upstream of kappa J1 and downstream of kappa Cκ, and preceding by a human kappa promoter and leader (from human Vκ1-5), encoding the human kappa intronic region (SEQ ID NO: 78) including the human kappa intronic enhancer. This sequence was synthesized as a single fragment and cloned into a pUC vector (Genscript™). See FIG. 14.

[0210] This vector can be used in one of two ways: 1) Modification of mouse embryos to introduce the λ5 transgene: This will be done by injecting mouse 2-cell embryos with the necessary reagents to create a double-stranded break at a defined location in the kappa genomic locus, and the above-mentioned plasmid vector providing a repair template to facilitate the desired deletion / insertion by homologous recombination. 2) Modification of mouse embryonic stem cells (mESCs) to introduce the λ5 transgene: This was done by transfecting mESCs with the necessary reagents to create a double-stranded break at a defined position in the kappa genomic locus and providing the above plasmid vector as a repair template to facilitate the desired deletion / insertion by homologous recombination. A positive / negative selection cassette was cloned into the vector immediately downstream of the 5' homology arm. This cassette confers puromycin resistance and fialulysin sensitivity (thymidine kinase gene) flanked by PiggyBac transposase compatible 3' and 5' inverted terminal sequences. This ensured efficient targeting of the vector under positive puromycin selection in mouse embryonic stem cells, followed by PiggyBac-induced excision of this cassette under negative fialulysin selection. This vector was designed to target the WT mouse kappa locus and was used in 129 mouse embryonic stem cells (mESCs) (AB2.1 cell line). The complete targeting vector insert sequence including the selection cassette, SEQ ID NO: 80. A diagram summarizing this modification of the mouse kappa locus is included in FIG. 14. Predicted transgene product sequences: SEQ ID NO:81 and SEQ ID NO:82. Complete targeting vector insert sequence, including selection cassette, SEQ ID NO:80. A diagram summarizing this modification of the mouse kappa locus is included in Figure 14. Predicted transgene product sequences: SEQ ID NO:81 and SEQ ID NO:82.

[0211] This vector was designed to target the kappa locus in WT mice and used in 129 mouse embryonic stem cells (mESCs) (AB2.1 cell line). A similar approach with modified vectors could be used to target a humanized kappa locus in mESCs containing such a locus. Similar vectors could be used to perform direct modifications in mouse 2-cell embryos by cytoplasmic injection, resulting in modifications of either the WT mouse lambda locus or the humanized lambda locus.

[0212] An alternative strategy would involve modifying the WT mouse kappa or lambda locus, or a humanized kappa / lambda locus, with a vector containing a truncated λ5 sequence from mouse (see SEQ ID NO:100 and SEQ ID NO:101), another rodent, non-human primate or other mammalian source.

[0213] mESCs containing an inactivated kappa locus / human λ5 transgene were cultured as RAG-1 − / − (B6.129S7-Rag1 <tm1mom>The mESC lines were microinjected into blastocysts derived from WTCB7bl / 6 mice. Mice homozygous for this RAG-1 mutation have no mature B or T cells due to failure of V(D)J recombination. This allows for an initial analysis of the functionality of the transgene locus in chimeras generated in this background, since any mature lymphocytes present in the lymphoid organs of these animals will be derived from the injected transgene-containing mESC line. The same mESC line was also injected into WTCB7bl / 6 blastocysts for the generation of stable mouse lines with germline transmission of the transgene.

[0214] An additional step was performed in some λ5 mESC lines to inactivate the endogenous mouse λ light chain locus by transfecting mESCs with the necessary reagents to create a double-stranded break at a defined position in the λ genomic locus and providing a small single-stranded DNA donor fragment as a repair template to facilitate the desired deletion. The region deleted is the same as that described in Example 3 (removing V2 to V1 (the segment including V2, V3, J2C2 and V1), but leaving J3C3, J1 C1 and the downstream enhancer intact).

[0215] Analysis of initial chimeras generated in a RAG-1 − / − background will be performed as follows: Lymphoid organs from chimeric mice generated by injection of the λ5 transgene into RAG-1 - / - mESCs with or without the mouse λ light chain knockout locus are isolated and the resulting cells are incubated with a staining panel containing markers of mouse lymphocytes together with anti-human λ5 antibodies, followed by analysis by flow cytometry. A positive result would indicate successful expression of the human λ5 transgene and assembly with the endogenous mouse heavy chain, allowing cell surface expression of a "VH only" antibody in vivo.

[0216] Mouse strains positive for λ5 expression on the surface of lymphocytes will be bred with other mouse breeds to introduce both the fully human heavy locus and the mouse lambda knockout, resulting in animals that produce fully human VH-only antibodies.

[0217] Example 6 Mouse kappa constant fragment shield domain with intrinsic regulatory control This embodiment is a mouse that contains a kappa locus that has been modified to inactivate the normal kappa light chain rearrangement and instead express a truncated kappa chain composed of a kappa constant region (Ck) plus a Vκ leader and partial J fragment. This can be done by either: 1) Creating a deletion between the mouse endogenous Vκ gene and the mouse Jκ gene, resulting in the removal of most of the Vκ and Jκ coding sequences but retaining the Vκ exon 1 plus the endogenous splice junctions at the 5' end of VK exon 2 and the 3' end of J. 2) The mouse Jκ gene was replaced with a targeting vector encoding the mouse Vκ promoter, leader (exon 1, intron, partial exon 2) plus a small fragment of the Jκ gene, again retaining the endogenous splice junctions at the 5' end of Vκ exon 2 and the 3' end of the J.

[0218] The outcome of both of these strategies is a locus that expresses a transcript under the control of the endogenous Vκ promoter, which is spliced ​​to generate a Cκ with a Vκ leader sequence and a partial J sequence. This strategy allows exploitation of any Vκ and J in the mouse kappa repertoire. Strategy 1 uses Vκ3-2 or Vκ3-4 with Jκ5 because they are closer to the 3′ end of the locus, thereby reducing the size of the deletion required, and because they have been shown in the literature to be highly utilized in mice. See FIG. 16 for an overview of the kappa locus structure and FIG. 4 for predicted expressed sequences from two modified loci. Strategy 2 will use Vκ6-17 or Vκ10-96 with Jκ5 because these Vs have been shown to be the most highly utilized in the mouse repertoire (see reference 18). See FIG. 18 for an example of a targeting strategy / locus overview and FIG. 19 for predicted expression sequences using Vκ6-17 and Vκ10-96.

[0219] This mouse differs from the version with human control elements in that, in the current example, all of the non-coding regulatory elements of the modified locus are endogenous. This mouse contains a specific promoter for the VK / JK / CK fragment, a mouse κ intronic enhancer (located between JK5 and CK), and a mouse κ 3' enhancer. The interaction of these endogenous promoters / enhancers with mouse effectors responsible for expression from the κ locus may be more effective than the interaction of the mouse effectors with the human regulatory sequences present in Examples 2 and 4, thus resulting in a more active locus.

[0220] Generation of the modified locus by strategy 1 involves a large, precise deletion of either 25 kb (for the Vκ3-2 version) or 54 kb (for the Vκ3-4 version). This mouse locus could be generated in multiple ways: 1) Direct modification of the mouse kappa locus by cytoplasmic injection: This is performed by injecting mouse one-cell zygotes or two-cell embryos with the necessary reagents to create a double-stranded break at a defined location in the kappa genomic locus and provide a repair template to facilitate the desired deletion. 2) Modification of the mouse kappa locus in mESCs: This is done by transfecting mESCs with the necessary reagents to create a double-stranded break at a defined location in the kappa genomic locus and provide a repair template to facilitate the desired deletion.

[0221] Figure 16 summarizes the modification of the kappa locus by this method, and Figure 17 contains an annotated sequence diagram of the predicted coding and translated sequences expressed by the modified locus. The predicted gene products, coding and translated protein sequences for the two versions are provided in SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86.

[0222] Generation of a modified locus by strategy 2 involves replacing a 1.5 kb region of the mouse kappa locus (Jκ1-5) with a 1.3 kb sequence encoding a partial Vκ / Jκ fragment. This could be done by: 1) Modification of the kappa locus in mouse embryos by cytoplasmic injection: This can be done by injecting mouse 2-cell embryos with the necessary reagents to create a double-stranded break at a defined location in the kappa genomic locus, and providing a plasmid vector as a repair template to facilitate the desired deletion / insertion by homologous recombination. 2) Modification of the kappa locus in mouse embryonic stem cells (mESCs): This can be done by transfecting mESCs with the necessary reagents to create a double-stranded break at a defined position in the kappa genomic locus, and a plasmid vector serving as a repair template to facilitate the desired deletion / insertion by homologous recombination. A positive / negative selection cassette was cloned into the vector immediately downstream of the 5' homology arm.

[0223] The targeting vector insert is synthesized as a single fragment and cloned into a pUC vector (Genscript™). Figure 18 summarizes the modification of the Kappa locus by this method, and Figure 19 contains an annotated sequence diagram of the predicted coding and translated sequences expressed by the modified locus. The predicted gene product, coding and translated protein sequences, in addition to the targeting vector insert sequence (minus the selection cassette) for two versions (VK6-17 and VK10-96), are provided in SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94.

[0224] Initial analysis of mESC lines generated by this method will be performed in the same manner as in Example 5, i.e., injection into RAG-1 − / − blastocysts, followed by analysis of chimera-derived lymphocytes for expression of the truncated kappa fragment. Mouse strains expressing the truncated mouse kappa fragment paired with the heavy chain on the surface of lymphocytes will be crossed with other strains to introduce both a fully human heavy locus and a mouse lambda knockout (as detailed in Example 3). This will result in animals that produce VH-only antibodies with fully human heavy chains and mouse kappa Cκ fragments.

[0225] (References) TIFF2024177226000002.tif120168 [Explanation of symbols]

[0226] 1 VH domain 3 Fc region 4. CL Domain 11 CH1 domain

[0227] SEQ ID NO:1: Nucleic acid encoding Cκ signal peptide Artificial sequence atggacatgagggtccccgctcagctcctggggctcctgctgctctggctcccaggaactgtggct SEQ ID NO:2: Cκ signal peptide Artificial sequence MDMRVPAQLLGLLLLWLPGTVA SEQ ID NO:3: Nucleic acid encoding an isolated Cκ sequence Homo sapiens gcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgt cacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgtta SEQ ID NO: 4: Isolated Cκ sequence Homo sapiens APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 5: Nucleic acid encoding a κVC fusion with a signal peptide Artificial sequence atggacatgagggtccccgctcagctcctggggctcctgctgctctggctcccaggaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataa cgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgtta SEQ ID NO: 6: κVC fusion with signal peptide Artificial sequence MDMRVPAQLLGLLLLWLPGTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 7: Nucleic acid encoding the complete Cκ domain Homo sapiens ggaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccagga gagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgtta Note: The first nucleotide g of SEQ ID NO:7 is provided by the Vκ exon that splices into the nucleic acid encoding the CL domain. SEQ ID NO: 8: Complete Cκ domain Homo sapiens GTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC NOTE: The N-terminal amino acid G of SEQ ID NO:8 is encoded by a gga codon formed by splicing of the vκ exon to the nucleic acid encoding the CL domain. The first g is provided by the 3' end of the vκ exon, while the remainder of the codon (ga) is provided by the 5' end of the Cκ exon. Under some definitions, the Cκ domain is considered to begin at the second residue of SEQ ID NO:8, i.e., TVA... SEQ ID NO: 9 cDNA of spliced ​​Vκ1-5 gene segment Homo sapiens atggacatgagggtccccgctcagctcctggggctcctgctgctctggctcccaggtgccaaatgtgacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgccgggccagtcagagtattagtagctggttggcctggtatc agcagaaaccagggaaagcccctaagctcctgatctataaggcgtctagtttagaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacagtataatagttattct SEQ ID NO: 10. Encoded segment of Vκ containing signal peptide Homo sapiens MDMRVPAQLLGLLLLWLPGAKCDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYS SEQ ID NO:11 Nucleic acid encoding the Vκ1-5 signal peptide Homo sapiens atggacatgagggtccccgctcagctcctggggctcctgctgctctggctcccaggtgccaaatgt SEQ ID NO:12 Vκ1-5 signal peptide Homo sapiens MDMRVPAQLLGLLLLWLPGAKC

[0228] [Table 2A]

[0229] [Table 2B]

[0230] [Table 2C]

[0231] [Table 2D]

[0232] [Table 2E]

[0233] [Table 2F]

[0234] [Table 2G]

[0235]

Table 2H

[0236]

Table 2I

[0237]

Table 2J

[0238]

Table 2K

[0239]

Table 2L

[0240]

Table 2M

[0241]

Table 2N

[0242]

Table 2O

[0243]

Table 2P

[0244]

Table 2Q

[0245]

Table 2R

[0246]

Table 2S

[0247]

Table 2T

[0248]

Table 2U

[0249]

Table 2V

[0250]

Table 3A

[0251]

Table 3B

[0252]

Table 3C

[0253]

Table 3D

[0254]

Table 3E

[0255]

Table 3F

[0256]

Table 3G

[0257]

Table 3H

[0258]

Table 3I

[0259]

Table 3J

[0260]

Table 3K

[0261]

Table 3L

[0262]

Table 3M

[0263]

Table 3N

[0264]

Table 3O

[0265]

Table 3P

[0266] [Table 3Q]

[0267] [Table 3R]

[0268] [Table 3S]

[0269] Table C: Examples of human alleles for inclusion in the mouse locus of the invention In (a) and (b) one, more or all of the following gene segments may be comprised by the heavy chain locus or the light chain (kappa or lambda) locus, respectively: (a) Heavy chain locus

[0270] [Table 4]

[0271] [Table 5]

[0272] (b) Kappa stationary domain shield

[0273] [Table 6]

Claims

1. A non-human animal comprising B lymphocytes expressing antibodies comprising unpaired human VH domains for binding an antigen, The genome of the animal A plurality of human variable region gene segments that can be rearranged to encode a variable domain upstream of DNA encoding an immunoglobulin constant region that includes a CH1 domain; and A gene encoding a CL domain lacking functional expression of a variable region gene segment, or a gene encoding an immunoglobulin domain of λ5 Including, the antibody comprises two heavy chains and two light chains, each heavy chain comprises an unpaired human VH domain for binding a target antigen and a heavy chain constant region comprising a CH1 domain; Each light chain contains a CL domain and each light chain lacks a VL domain, thereby leaving the VH domains unpaired. Non-human animals.

2. The non-human animal of claim 1, wherein the two unpaired VH domains have identical amino acid sequences.

3. The non-human animal of claim 1 or 2, wherein the heavy chain constant region comprises a CH1 domain and one or more further CH domains.

4. The non-human animal of claim 3, wherein the heavy chain constant region comprises a CH1 domain, a CH2 domain and a CH3 domain.

5. The non-human animal of any one of claims 1 to 4, wherein the CL is Cκ.

6. The non-human animal according to any one of claims 1 to 5, wherein the light chain consists of a CL domain.

7. 7. The non-human animal according to any one of claims 1 to 6, wherein the heavy chain constant region is a human heavy chain constant region and / or the CL is human.

8. The non-human animal of claim 7, wherein the CL comprises the human Cκ sequence of SEQ ID NO:

4.

9. The non-human animal of claim 8, wherein the shield domain consists of the human Cκ sequence of SEQ ID NO:

4.

10. The non-human animal of any one of claims 1 to 9, wherein the antibody is IgG or IgM.

11. The non-human animal according to any one of claims 1 to 10, wherein the antibody is a fully human antibody.

12. The unpaired variable domains are used to inhibit immune checkpoint inhibitors (e.g., PD-L1, PD-1, CTLA-4, TIGIT, TIM-3, LAG-3, and VISTA, e.g., TIGIT, TIM-3, and LAG-3), immunomodulators (e.g., BTLA, hHVEM, CSF1R, CCR4, CD39, CD40, CD73, CD96, CXCR2, CXCR4, CD200, GARP, SIRPα, CXCL9, CXCL10, CXCL11, and CD1 55, e.g., GARP, SIRPα, CXCR4, BTLA, hVEM and CSF1R), immunostimulants (e.g., CD137, GITR, OX40, CD40, CXCR3 (e.g., an agonistic anti-CXCR3 antibody), CD27, CD3, ICOS (e.g., an agonistic anti-ICOS antibody), e.g., ICOS, CD137, GITR and OX40).

13. The non-human animal of claim 1, wherein the two unpaired VH domains bind to the same epitope.

14. A method for producing an antibody comprising an unpaired variable (optionally VH) domain for binding the antigen, comprising exposing the animal of claim 1 to an immunogenic challenge with a target antigen and isolating the antibody or a nucleic acid encoding same from the animal; or 2. A method for producing a variable (optionally VH) domain for binding an antigen, comprising exposing the animal of claim 1 to an immunogenic challenge with a target antigen, and isolating the variable domain or a nucleic acid encoding same from the animal.

15. 15. The method of claim 14, further comprising cloning the encoding nucleic acid into a recombinant host cell, culturing the cells for expression of a polypeptide comprising the variable domain, and recovering and purifying the polypeptide from the cells or culture medium.

16. 16. The method of claim 15, wherein the polypeptide is an isolated variable domain, an antibody or a chimeric antigen receptor.

17. 17. The method of claim 16, comprising formulating the polypeptide into a composition comprising a pharma- ceutically acceptable excipient.

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