Screening platform for bispecific antibodies

Abstract

The present disclosure provides a system for producing bispecific antibodies. In some embodiments, the system may comprise (a) a first library of nucleic acids, wherein the members of the first library encode a light chain fusion polypeptide comprising: (i) a common light chain comprising a light chain C region and a light chain V region; and (ii) the antigen binding region of a single chain antibody that binds to a first antigen, wherein: the sequence of the antigen binding region of the single chain antibody varies and the sequence of the common light chain does not vary; and (b) a second library of nucleic acids, wherein the members of the second library encode antibody heavy chain comprising a heavy chain V region and a heavy chain C region, where the sequence of the heavy chain V region varies. Methods of screening are also provided.

Description

[0001] Atty. Dkt: OMN1-005WO

[0002] SCREENING PLATFORM FOR BISPECIFIC ANTIBODIES

[0003] CROSS-REFERENCING

[0004] This application claims the benefit of U.S. provisional application serial number 63 / 738,366, filed on December 23, 2024, which application is incorporated by referenced for all purposes.

[0005] BACKGROUND

[0006] Conventional methods of screening for bispecific antibodies often involve introducing three separate nucleic acid molecules into cells. In these methods, one nucleic acid encodes a common light chain, another nucleic acid encodes a first heavy chain that binds to a first antigen when it pairs with the common light chain, and another nucleic acid encodes a second heavy chain that binds to a second antigen when it pairs with the common light chain. Because introducing three separate nucleic acids into cells can be inefficient, such methods can be impractical and / or limited in their throughput. Specifically, if the transfection rate is inefficient then only a small number of cells may receive all three nucleic acids.

[0007] This disclosure provides a potential solution to this problem.

[0008] SUMMARY

[0009] The present disclosure provides a method of screening for a bispecific antibody, as well as a system for performing the same. In this system, heavy chain sequences that recognize a first antigen (target A) may be combined in a pairwise manner with the antigen binding regions of single chain antibodies (e.g., VHH, scFv or picobody) that recognize a second antigen via a soluble common light chain that is fused to each single chain antibody antigen binding region. In this context, the common light chain can be thought of as a ‘chaperone linker’ since it simultaneously acts as both a chaperone for the heavy chain (i.c., a passive binding partner for the heavy chain) while, at the same, also acting as a linker for the single chain antibody. This system, because it only relies on introducing two nucleic acids (one for the heavy chain and one for the common light chain-single chain antibody fusion) into cells, is much more efficient than convention systems and, consequently, can be used to produce and test large panels of test bispecific antibodies.

[0010] In some embodiments, the two component system may comprise (a) a first library of nucleic acids, wherein the members of the first library encode a light chain fusion polypeptide comprising: (i) a common light chain comprising a light chain C region and a light chain V region; and (ii) the antigenAtty. Dkt: OMN1-005WO binding region of a single chain antibody that binds to a first antigen, wherein: the sequence of the antigen binding region of the single chain antibody varies and the sequence of the common light chain does not vary; and (b) a second library of nucleic acids, wherein the members of the second library encode an antibody heavy chain comprising a heavy chain V region and a heavy chain C region, where the sequence of the heavy chain V region varies. The first and second libraries are capable of producing bispecific antibodies when they are transfected into the same cells, without the addition of any further nucleic acids.

[0011] In comparison to other systems (which, in many cases, require three nucleic acids: one encoding a common light chain, and two others that encode heavy chains that binds to the first and second antigens when they pairs with the common light chain) the present platform is much more efficient because the only two nucleic acids need to be paired. As such, the present system provides a much higher number of candidate antibodies, even if the efficiency of transfection is lower. Also, because the present system relies on the common light chain acting as a chaperone linker, the present library does not need to be as complex in order to obtain pairs of polypeptides that bind to the desired antigens. In other words, without the common light chain one would need as many light chains as heavy chains. Instead, in the present strategy the common light chain becomes non- variable and, as such, does not need to be diversified. This allows for a much greater number of pairs to produce which, in turn, provides candidates even if the integration efficiency is lower.

[0012] In addition, because the present screening method only requires the expression of two polypeptides in a cell, the method should be more efficient than other methods and, in addition, may reduce the noise that is often associated with other systems.

[0013] Moreover, because the present system does not rely on different heavy chains pairing with each another in a cell (which is conventional in traditional bi-specific antibody screening platforms), there is no need for any “knob-into-hole” amino acid substitutions to promote heavy chain heterodimerization.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Fig. 1 illustrates a conventional method for assembling bispecific antibodies.

[0016] Fig. 2 illustrates an embodiment of the present method.

[0017] In Figures 1 and 2, “cmLC” refers to “common light chain”, “HC” refers to “heavy chain” and “LC” refers to “light chain”.Atty. Dkt: OMN1-005WO

[0018] DETAILED DESCRIPTION

[0019] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0020] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0022] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a binding domain” includes a plurality of binding domain. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0023] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.Atty. Dkt: OMN1-005WO

[0024] Systems

[0025] In some embodiments, a two component system may comprise: (a) a first library of nucleic acids, wherein the members of the first library encode a light chain fusion polypeptide comprising: (i) a common light chain comprising a light chain C region and a light chain V region and (ii) the antigen binding region of a single chain antibody that binds to a first antigen, wherein: the sequence of the antigen binding region of the single chain antibody varies in the first library; and the sequence of the common light chain does not vary in the first library; and (b) a second library of nucleic acids, wherein the members of the second library encode antibody heavy chain comprising a heavy chain V region and a heavy chain C region, wherein: the sequence of the heavy chain V region varies in the second library, and the antibody heavy chain binds to a second antigen when it is paired with the common light chain of (a). In this system, expression of a member of the first library and a member of the second library in a cell results in a multi-specific antibody comprising: a first binding domain that binds to the first antigen; and a second binding domain that binds to the second antigen. This is a two-component system in the sense that the first and second libraries sufficient to produce bispecific antibodies when they are transfected into the same cells (i.e., without the addition of any further nucleic acids).

[0026] Examples of such an antibody are shown at the bottom of Fig. 2. As shown, the first binding domain (which binds to target B in Fig. 2) may comprise the antigen binding region of a single chain antibody and the second binding domain (which binds to largel A in Fig. 2) may comprise a light chain V region from a common light chain and a heavy chain V region.

[0027] In this library, the light chain fusion polypeptide and antibody heavy chain are expressed as separate polypeptides. As such, the antigen binding region of the single chain antibody and the heavy chain V region are expressed on separate polypeptides, not the same polypeptide as they would be if they were “pre -paired”. As such, in the present system the first and second libraries are distinct molecules and may be in separate containers. For example, in some embodiments, the members of the first library of nucleic acids may be in the wells of one or more first multi-well plates and the members of the first library of nucleic acids may be in the wells of one or more second multi-well plates. Because the system relies on only two libraries, the system does not need a third library of nucleic acids or a separate vector comprising a common light chain. In any embodiment, the members of the first library may be housed in containers that are separate to the containers that house the members of the second library.

[0028] As illustrated, the bispecific antibody may comprise two arms, wherein each arm comprises a first binding domain and a second binding domain. Such an antibody is illustrated at the bottom of Fig. 2. As shown, this antibody is considered “tetravalent” because it has a total of four binding domains,Atty. Dkt: OMN1-005WO “symmetrical” in that both arms bind to the same antigens and “2+2” because each arm binds to two antigens. As such, this antibody can be referred to as a “symmetrical 2+2 tetravalent” bispecific antibody.

[0029] As illustrated, the common light chain may be tethered at the N-terminal or C-terminal of the antigen binding region of the single chain antibody.

[0030] In any embodiment, the first library may comprise at least 10 members (e.g., at least 50, at least 100, at least 1,000, at least 10,000, at least 50,000 or at least 100,000 members) and, independently, the second library comprises at least 10 members (e.g., at least 50, at least 100, at least 1,000, at least 10,000, at least 50,000 or at least 100,000 members).

[0031] Some members of the first library may bind to the same epitope in a first antigen and other members of the first library may bind to a different epitope in the first antigen. The epitopes that are bound by the polypeptides encoded by the first library may or may not overlap with one another and, in some embodiments, may include a combination of overlapping and non-overlapping epitopes. Likewise, some members of the second library may bind to the same epitope in a second antigen and other members of the second library may bind to a different epitope in the second antigen. The epitopes that are bound by the polypeptides encoded by the second library may or may not overlap with one another and, in some embodiments, may include a combination of overlapping and non-overlapping epitopes.

[0032] The members of the first library are in separate containers and the members of the second library are in separate containers. In some embodiments, the containers may be wells of a multi-well plate (e.g., a 96- or 384-well plate.

[0033] As would be apparent, the first and second libraries may contain additional sequences (e.g.. promoters and terminators, etc.) that provide for expression of the light chain fusion polypeptide and the antibody heavy chain (as separate polypeptides) in a cell. In these embodiments, the coding sequence for the light chain fusion polypeptide and the coding sequence for the antibody heavy chain may be each operably linked to a promoter and terminator. In the special case of a bicistronic vector, the two libraries could be combined into a single vector before introducing them into cells. In these embodiments, the coding sequences for the light chain fusion polypeptide and the antibody heavy chain could be added to a bicistronic vector before transfection. In these embodiments, the light chain fusion polypeptide and the antibody heavy chain will still be expressed as separate polypeptides.

[0034] In any embodiment, the light chain V region of (a), the antigen binding region of the single chain antibody of (a) and / or the heavy chain V region of (b) are humanized or were made by diversifying human sequences.Atty. Dkt: OMN1-005WO In any embodiment, the C region of the antibody heavy chain should contain at least a CHI domain, since this domain is responsible for pairing with the light chain C region.

[0035] The single chain antibody can be, for example, a VIIII antibody, a picobody, or a scTv.

[0036] Common light chain antibodies are antibodies that have different heavy chain variable domains but the same light chain variable domain. In such antibodies, this light chain (which may be referred to as a “common light chain” or “fixed light chain” provides the proper structure for assembly of an antibody molecule but is a passive partner for antigen binding and may be considered a ‘chaperone’ partner for the variable heavy chain. Transgenic animals that produce common light chain animals include rats (see, e.g.. Harris et al (Front Immunol. 201824:9:889)) and chickens (Ching et al (MAbs. 2021; 13(1):

[0037] 1862451)), among others.

[0038] In any embodiment, the heavy chain V regions used in the method may be obtained by immunizing an animal that already expresses the common light chain with an antigen, where the animal has a diminished capacity to diversify the light chain. In these embodiments, the diversified heavy chain V regions may be amplified from cDNA made from the animal and used in conjunction with the light chain V region of the common light chain (which was expressed by the animal prior to immunization).

[0039] In any embodiment, the common light chain used in the system or method pairs with all of the antibody heavy chains used in the system or method to produce antibodies that specifically bind to an antigen. As noted above, in these antibodies the light chain is passive partner for antigen binding and the heavy chain variable domain is responsible for the specificity of binding.

[0040] Common light chains find particular use in producing multispecific antibodies, since it is advantageous if the light chain is common to all branches of the mullispecilic antibody, with the binding specificity determined solely by the heavy chain. Common light chain antibodies can be produced in animals which have reduced capacity to diversify the light chain. In these animals the heavy chain sequence is diversified and capable of high-affinity antigen-specific binding and broad epitope diversity when paired with a fixed light chain (which, in many cases, will be a human light chain). Such animals may produce diversified population of antibodies that have a “common light chain”, i.e., a diversified population of antibodies that all have the same or almost the same light chain variable region, where the light chain light chain variable regions of such antibodies play a passive role in determining binding specificity of the antibodies but nevertheless need to be present for correct folding and secretion. In these cases, the light chain for an antibody can be pre-selected prior to making the transgenic animals. For example, in some cases, the animal may be engineered to produce a diversified population of antibodies that have a common light chain variable region encoded by the human germline, thereby ensuring that at least the light chain of an antibody that contains the common light chain variable region should be wellAtty. Dkt: OMN1-005WO tolerated immunologically when it is administered to a human. In particular, such light chains can be used in bispecific antibodies have two binding specificities. In these embodiments, both arms of a bispecific antibody have the same light chain (i.e., the common light chain) and different heavy chains (which largely determine the binding specificity of the arm). Common light chain antibodies have a conventional “VH” structure and have both a heavy chain sequence and a light chain sequence. Both the heavy and light chains of a common light chain antibody have the following structure: FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4.

[0041] In the context of the present system and method, the common light chain encoded by the first library should pair with all the antibody heavy chains in the same library (even though they have different sequences and, in some embodiments, bind to different epitopes), thereby allowing the expression of active antibodies in the cells.

[0042] VHH antibodies are known in the art and have a heavy chain variable domain that can fold and bind to epitopes autonomously, i.e., without an associated light chain and without significantly aggregating. These antibodies may be referred to as “heavy chain-only”, “HCO”, or “single domain” antibodies (sdAbs), shark antibodies, camelid antibodies, single domain antibodies and nanobodies in other publications. VHH antibodies occur naturally in shark, camel and llama. However, there are several strategies for producing such antibodies from VH antibodies. See, e.g., lanssens et al (Proc. Natl. Acad. Sci. 2006 103:15130-5), Briiggemann et al (Crit. Rev. Immunol. 200626:377-90), Zou et al (J. Immunol. 2005 175:3769-79) and Nguyen et al (Immunology 2003 109: 93-101), for example. A VHH variable domain can be made by introducing substitutions into the variable domain of a VH antibody. For example, VHH antibodies were made by chickens that have been genetically engineered to produce VHH antibodies (see, e.g., Vuong et al MAbs. 2024 16 :2435476). Advantageous features of VHH antibodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo. VHH antibodies can readily be linked genetically to, e.g., Fc-domains, other nanobodies or single chain antibodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles, etc. The binding domain of a VHH antibody does not require a light chain for correct folding or binding to an antigen. See, e.g., Bever et al (Anal Bioanal Chem. 2016 Sep; 408: 5985-6002). As with conventional VH antibodies, VHH antibodies have three CDRs (CDR1, CDR2 and CDR3) that are flanked by framework. The structure of the binding domain (which may be referred to as a ‘variable domain’) of a VHH antibody is as follows: FW1-CDR1- FW2-CDR2-FW3-CDR3-FW4 (see, e.g., Noel et al, Biochimie 2016 131:11-19 for a detailed description of VHH structure).

[0043] In some embodiments, the VHH may be humanized, i.e., modified so that it becomes more like aAtty. Dkt: OMN1-005WO human antibody and therefore less immunogenic, methods for which are known. In some embodiments, the VHH may have been diversified using a ‘camelized’ human antibody using human sequences. Picobodies are derived from “stalk and knob” domain antibodies. Cattle (i.e., cows or bovines) produce antibodies that have a cysteine-rich ultralong CDR H3 that can be in the range of 30 to 70 amino acids in length and folds into a unique “stalk and knob” domain, with the knob protruding far out of the antibody surface (see, e.g., Huang et al Proc. Natl. Acad. Sci. 2023 120: e2303455120). These knobs retain their ability to bind to the antigen when they are expressed on their own, i.e., independently from the rest of the antibody. In these embodiments, the knob can be expressed as a fusion protein which is then cleaved to release the knob (see, e.g., Huang et al Proc. Natl. Acad. Sci. 2023 120: e2303455120). Alternatively, the knob can be cleaved from an antibody or Fab scaffold (Macpherson et al. PLoS Biol 2020 18: e3000821) or synthesized chemically (Macpherson et al ACS Chem. Biol. 2021 16, 9, 1757— 1769). These knobs (which are referred to as “knob domains” or “picobodies”) are the smallest known fragment of an antibody that can bind to an antigen independently. A knob domain may be expressed on its own or as a fusion with another protein (referred to as a “fusion partner” herein), wherein the knob domain may be at the N-terminus, C -terminus or within the fusion partner. If the fusion partner is an antibody, the knob domain may be fused to the N- or C- terminus of the heavy or light chain (in which case the antibody could potentially be bispecific since the variable domain may be intact), or it could positioned in the variable domain of the antibody (e.g., in the heavy chain CDR3). In embodiments in which the knob domain is fused to another protein, the knob domain may in some cases additionally comprise a stalk and / or a flexible linker that connects the knob domain to the protein. For example, if the knob domain is in the heavy chain CDR3, then it may contain a stalk to allow it to protrude from the rest of the antibody. If the knob domain is expressed in conjunction with a stalk, then an almost unlimited number of sequences are available for the ascending and descending regions of the stalk. Specifically, in the context of bovine antibodies the stalk has a purely structural function (i.e., it does not participate in binding). In bovine antibodies, the stalk is a beta sheet (composed of two anti-parallel beta strands that arc hydrogen bonded). Stalk sequences should be interchangeable between antibodies because, functionally, they only serve to distance the knob from the rest of the antibody while keeping the ends of the knob in proximity with one another. Since the sequences of thousands of bovine antibodies are publicly available or could be readily obtained, a large number of options would be available. Moreover, methods for designing beta sheets (composed of two anti-parallel beta strands) de novo are well known (see, e.g., Hecht et al (Proc Natl Acad Sci U S A. 199491: 8729-8730), Marcos (Nat Struct Mol Biol. 201825: 1028-1034) and Pan et al (J. Biol. Chem. 2021 296, 100558), among many others)) and, as such, stalks can be readily designed.Atty. Dkt: OMN1-005WO As such, a picobody is typically in at least 15 amino acids in length, e.g. in the range of 15 to 50 amino acids in length, cysteine-rich (i.e., may contain 4-10 cysteines, e.g., 4, 6, 8 or 10 cysteines) and may be based on an ultralong-CDR3 antibody (which, e.g., may be made by a cow or another species that naturally makes such antibodies, or by another species that has been engineered to produce such antibodies (e.g., a chicken)). Picobodies have the potential to bind antigens with concave epitopes and, as such, may have particular value for certain targets. Further details of picobodies may be found in, e.g., Svilenov et al (Nature Com. 2021 12: 6737), Huang et al (Proc. Natl. Acad. Sci 2023

[0044] 120 (39) e2303455120) and Passon et al (Biotechnol Adv. 2023 :64:108120).

[0045] scFvs “single-chain variable fragments” are a fusion of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin, connected with a short linker peptide of ten to about 25 amino acids. The linker may be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa.This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. ScFvs are well known and are described in e.g., Ahmad et al, Clin Dev Immunol. 20122012: 980250 and Munoz-Lopez et al, Cancers (Basel) 2022 14: 4206.

[0046] Methods

[0047] Also provided is a method for producing a bispecific antibody. In some embodiments, the method may comprise obtaining a system as described above, introducing a member of the first library and a member of the second library into the same cell and incubating the cell to provide for expression of the bispecific antibody. In the cell, the light chain fusion polypeptide and antibody heavy chain are expressed as separate polypeptides which pair with one another.

[0048] In these embodiments, the first and second library members may be introduced into the cells using two separate vectors. However, in some embodiments, the coding sequences for the light chain fusion polypeptide and the antibody heavy chain may be pre-combined into one vector before they are introduced into a call in some circumstances. In these latter embodiments, the light chain fusion polypeptide and the antibody heavy chain will still be expressed as separate polypeptides. As such, in these cases, the vector may be bi-cistronic for example.

[0049] Depending on how the method is implemented, the bispecilic antibody could be secreted from the cell or it may be tethered to the cell surface, i.e., displayed on the cell surface. Methods for engineering antibodies to be secreted by a cell or tethered to a cell surface are well known. In some embodiments, the antibody heavy chain may have a hydrophobic N-terminal domain, allowing it to be anchored to the plasma membrane, whereas the light chain fusion polypeptide may be soluble.Atty. Dkt: OMN1-005WO In these embodiments and as illustrated in Fig. 2, the light chain fusion polypeptide and the antibody heavy chain pair together via interactions with the common light chain to produce a functional bispecific antibody, i.e., an antibody that has a first binding domain that binds to the first antigen and a second binding domain that binds to the second antigen. In these embodiments, the first binding domain comprises a light chain V region from a common light chain and a heavy chain V region, and the second binding domain comprises the antigen binding region of a single chain antibody.

[0050] In some embodiments, the method may comprise testing the bispecific antibody for binding to the first antigen and the second antigen where the testing may in some embodiments comprise detecting (e.g. measuring) binding of the bispecific antigen to the first antigen and the second antigen at the same time.

[0051] In any embodiment, the method may comprise introducing pairwise combinations of members of the first and second libraries into the same cells and incubating the cells to provide for expression of multiple bispecific antibodies. These embodiments may involve independently testing the multiple bispecific antibodies for binding to the first antigen and the second antigen.

[0052] The combinations may be tested systematically or randomly and, in some embodiments, the antibodies may be tested in pools and then deconvoluted. The number of combinations tested may be at least 100, at least 1,000, at least 10,000, at least 100,000, at least IM or at least 10M. As noted above screening of IM combinations should only require 2,000 constructs and, as such, the screening can be done very efficiently.

[0053] Because the present system relies on expressing two polypeptides in a cell, the pairing should be highly efficient relative to other systems that require the expression of three polypeptides in the cell. In some embodiments, at least 10%, at least 30%, at least 50%, at least 70% of the populations of cells into which the constructs are introduced will produce a bi-specific antibody that is detectable by FACs or ELISA, for example.

[0054] The antibodies produced by the cells may be tested using any suitable assay. In some embodiments the antibodies may be tested for binding to one or more target antigens with a label-based assay (e.g., an assay involving some form of detectable labeling of the antibody and / or a target antigen). Suitable label-based assays include, without limitation, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), enzyme immunoassays (ElAs), immunoradiometric assays (IRMAs), competition assays, and flow cytometry methods (e.g., fluorescence activated cell sorting, FACS). In some embodiments, the antibodies may be tested for binding to one or more target antigens with a label-free assay. Suitable label-free assays include, without limitation, surface plasmon resonance (SPR)Atty. Dkt: OMN1-005WO assays, bio-layer interferometry (BLI) assays, scintillation proximity assays (SPAs), and LC-MS detection methods.

[0055] In some cases, the assay may be a ‘mechanism of action’ assay that evaluates the specific intended mechanism of action of the antibody. By way of example, a bispecific antibody that is ultimately intended to induce activation of a T cell may be tested with an assay utilizing engineered T cells that express a reporter protein upon activation. The particulars of such assays are dependent on the specific type of antibody being tested and can be appropriately designed by the skilled person based on the present disclosure and the common knowledge in the art.

[0056] A variety of suitable assays, including the above, are known in the art and described, for example, in: Register, Ames C., Somayeh S. Tarighat, and Ho Young Lee. "Bioassay Development for Bispecific Antibodies — Challenges and Opportunities." International journal of molecular sciences 22.10 (2021): 5350; Segaliny, Aude I., et al. "A high throughput bispecific antibody discovery pipeline." Communications biology 6.1 (2023): 380; Hofmann, Tim, et al. "Greatest hits — innovative technologies for high throughput identification of bispecific antibodies." International Journal of Molecular Sciences 21.18 (2020): 6551; Sugiyama, Aruto, et al. "A semi high-throughput method for screening small bispecific antibodies with high cytotoxicity." Scientific Reports 7.1 (2017): 2862; and Bhatta, Pallavi, et al. "Bispecific antibody target pair discovery by high-throughput phenotypic screening using in vitro combinatorial Fab libraries." MAbs. Vol. 13. No. 1. Taylor & Francis, 2021; the disclosures of which are incorporated by reference herein.

[0057] In any embodiment, the method may comprise identifying at least one bispecific antibody based on at least one binding characteristic, e.g., the ability to bind to two antigens simultaneously or the affinity of those interactions, etc. In would be apparent, in any embodiments, the method may be done without introducing any further nucleic acids into the cells.

[0058] Bispecific antibodies

[0059] The antibodies produced by the method may be bispecific. Bispecific antibodies recognize two different epitopes. This dual specificity allows a wide range of applications, including redirecting immune cells such as NK and T cells to tumor cells, blocking two different signaling pathways simultaneously, dual targeting of different disease mediators, and delivering payloads to targeted sites. Illustrative examples of bispecific antibodies, which include BiKEs, BiTEs and several other formats and uses are described in Fan et al (Hematol Oncol. 2015 8:130), Sun et al (Acta Pharm Sin B. 2023 13: 3583-3597), Klein et al (Nature Reviews Drug Discovery 202423: 301-319 and Ma et al (Front Immunol. 2021 12: 626616).Atty. Dkt: OMN1-005WO In any embodiment, the first and second antigens may be on different proteins. For example, one of the antigens may be on a diseased cell (e.g., a cancer cell) or a marker for disease and the other antigen may be on an immune cell (e.g., a T cell or NK cell).

[0060] In other embodiments, the first and second antigens can be different epitopes in the same protein. In these embodiments, the bispecific antibodies may be biparatopic.

[0061] Bispecific antibodies may bind to the following pairs of antigens, for example:

[0062] CD19 and CD3; activated factor IXa and factor X; EGFR and MET; gplOO and CD3; VEGF-A and Angiopoietin-2; BCMA and CD3; CD20 and CD3; CD33 and CD3; CD38 and CD3; CD123 and CD3; CLEC12A and CD3; FcRH5 and CD3; FLT3 and CD3; CEA and CD3; EpCAM and CD3; PSMA and CD3; HER2 and CD3; P-cadherin and CD3; gpA33 and CD3; B7H3 and CD3; DLL3 and CD3; MUG 16 and CD3; EGFRvlll and CD3; GPC3 and CD3; SSTR2 and CD3; GD2 and CD3; HIV-1 and CD3; Env and CD3; GPRC5D and CD3; DLL3 and CD3; EGFR and VEGFR2; EGFR and PD-L1; CD 16 and CD30; CD 16 and CD33; CD47 and CD 19; ICOS and PD1 ; CTLA4 and PD1; LAG3 and PD1; TIM3 and PD1; PDL1 and PD1; LAG3 and PDL1; CTLA4 and PDL1; TIM3 and PDL1; TNF and HSA; IL6R and HSA; IL17A / F and HSA; RANKL and HSA; IL4 and IL 13; IL17 and IL 13; ICOSL and BAFF; IL-17A and BAFF; TNF and NGF; DLL4 and EGFR; ANG2 and EGFR; c-MET and EGFR; LGR5 and EGFR; HER3 and HER2; HER2 and HER2 (e.g., targeting distinct epitopes of HER2); DR5 and FAP; CD79B and CD32B; KLB and FGFR1; Pcrv and Psi; and A 42 and AP40 among other combinations.

[0063] Host cells

[0064] Standard recombinant methods can be used for the component parts may be amplified from another source and inserted into expression vectors, and then transfected into the cells. As should be apparent, the DNA segments encoding the polypeptides may be operably linked to control sequences in the expression vector(s) that ensure the expression of the polypeptides. Expression control sequences include, but arc not limited to, promoters (e.g., naturally-associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences, etc. The expression control sequences can be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells (e.g., COS or CHO cells). Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the antibodies.

[0065] Suitable expression vectors arc typically replicable in the host organisms cither as cpisomcs or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markersAtty. Dkt: OMN1-005WO (e.g., ampicillin-resistance, hygromycin-resistance, tetracycline resistance, kanamycin resistance or neomycin resistance) to permit detection of those cells transformed with the desired DNA sequences. In some embodiments, the first library will encode a selectable marker for a first selectable marker and the first library will encode a selectable marker for a second selectable marker.

[0066] Escherichia coli is an example of a prokaryotic host cell that can be used for cloning a subject antibody-encoding polynucleotide. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replicalion). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.

[0067] Other microbes, such as yeast, are also useful for expression. Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells, with suitable vectors having expression control sequences (e.g., promoters), an origin of replication, termination sequences and the like as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

[0068] In addition to microorganisms, mammalian cells (e.g., mammalian cells grown in in vitro cell culture) can also be used to express and produce the polypeptides of the present invention (e.g., polynucleotides encoding immunoglobulins or fragments thereof). See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B-cells or hybridomas.

[0069] Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (Queen ct al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Examples of suitable expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Co et al., J. Immunol. 148:1149 (1992).

[0070] Once synthesized (either chemically or recombinantly), the whole antibodies, their dimers, individual light and heavy chains, or other forms of a subject antibody (e.g., scFv, etc.) can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns,Atty. Dkt: OMN1-005WO column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, and the like (see generally Scopes, Protein Purification (Springer-Verlag, N.Y., (1982)). A subject antibody can be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99%, or more, pure, e.g., free from contaminants such as cell debris, macromolecules other than a subject antibody, etc.

[0071] The present disclosure also provides isolated genetically modified host cells (e.g., in vitro cells) into which the first and second libraries have been introduced. Suitable host cells include eukaryotic host cells, such as a mammalian cell, an insect host cell, a yeast cell; and prokaryotic cells, such as a bacterial cell. Introduction of a subject nucleic acid into the host cell can be effected, for example by calcium phosphate precipitation, DEAE dextran mediated transfection, liposome-mediated transfection, electroporation, or other known method. Chemical methods such as lipofection (lipid carriers) and calcium phosphate; and biological methods using modified viruses (viral vectors) for delivery, plus bacterial processes like transformation (natural uptake) and transduction can be used. The vector can be a plasmid, a viral vector, or any other type of nucleic acid that is suitable for introduction into a cell.

[0072] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like. Other cells include cells that natural produce antibodies, e.g., DT-40 cells and the like.

[0073] Depending on the libraries, the nucleic acid may be maintained in the cell episomally (i.e., not incorporated into the genome) or it may be incorporated into the nuclear genome of the cell.

[0074] Screening methods

[0075] The present library may be screened for bispecific antibodies that have desirable features by any of a variety of screening methods, several of which generally involve testing the antibodies for binding and affinity (using SPR or flow cytometry for flow cytometry), activity assays (e.g., testing for T-cell activation, cytokine release (e.g., via ELISA), or cytotoxicity, for example), functional assays (e.g., assessing mechanism of action, like T-cell or NK-cell engagement or cndocytosis), and in vivo evaluation (using, e.g., organoids or animal models for efficacy). Platforms for screening for bispecificAtty. Dkt: OMN1-005WO antibodies are known (see, e.g., Kim Protein Eng Des Sei. 2017, Fawcett MAbs. 2024 16: 2311992, and Slavney et al, Frontiers in Immunology 2024: 1469329, among many others). The screening may be done on cells that secrete the bispecific antibodies, or cells that present the bispecific antibodies on the cell surface (i.e., where the bispecific antibodies displayed on or are tethered to cell surface). Either way, nucleic acids encoding the heavy chain V region and antigen binding region of a single chain anlibody that has desirable characteristics can be readily amplified from a cell that produces the antibody and, if necessary, cloned into one or more other vectors and expressed along with the common light chain V region and, optionally, other pails of the bi-specific anlibody, in another cell. As would be apparent, once a candidate bi-specific antibody has been identified, it could be modified (e.g., humanized) and potentially reconfigured into a different format for bi-specific antibodies, among other things.

[0076] In some embodiments, the first and second libraries can be combined into single molecules, e.g., by PCR or random ligation, such that each molecule contains a member of the first library and a member of the second library, before transfection into the cells. In these embodiments, these cells only need to receive a single molecule nucleic acid (as opposed to two or three). In some embodiments, the molecule could be potentially targeted to a single locus, e.g., via transposase, nuclease or recombinase-mediated integration, which potentially makes the screening extremely efficient. Alternatively, the different libraries can be transfected into the cells without being combined into single molecules (separately or pre-mixed). The term “introducing a member of the first library and a member of the second library into the same cell” and “introducing pairwise combinations of members of the first and second libraries into the same cells” covers all of these embodiments (e.g., pre-combining the libraries into single nucleic acids before transfection, separately transfecting the libraries, or pre-mixing the libraries and then transfecting them).

[0077] In any embodiment, the screening may be done using a platform in which the nucleic acid encoding the bispecific antibody and the function of the antibody are linked such that the nucleic acid encoding an antibody that has desirable characteristics can be readily amplified and / or cloned after the antibody has has been identified. Such platforms include cell and phage display systems, as well as microfluidic-, droplet-, bead and microdrop-based systems. Other platforms are known and can be used.

[0078] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition ofAtty. Dkt: OMN1-OQ5WO matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

Atty. Dkt: OMNI-OQ5WO CLAIMSWhat is claimed is:

1. A two-library system for producing bispecific antibodies, comprising:(a) a first library of nucleic acids, wherein members of the first library encode a light chain fusion polypepride comprising:(i) a common light chain comprising a light chain C region and a light chain V region; and(ii) an antigen binding region of a single chain antibody that binds to a first antigen; wherein:the sequence of the antigen binding region of the single chain antibody varies in the first library; andthe sequence of the common light chain does not vary in the first library; and(b) a second library of nucleic acids, wherein the members of the second library encode an antibody heavy chain comprising a heavy chain V region and a heavy chain C region,wherein:the sequence of the heavy chain V region varies in the second library; and the antibody heavy chain binds pairs with the common light chain of (a) to bind to the second antigen.

2. The system of claim 1 , wherein expression of a member of the first library and a member of the second library in a cell results in a bispecific antibody comprising:a first binding domain that binds to the first antigen; anda second binding domain that binds to the second antigen.

3. The system of claim 2, wherein:the first binding domain comprises the antigen binding region of a single chain antibody; and the second binding domain comprises a light chain V region from the common light chain and the heavy chain V region.

4. The system of claim 2 or 3, wherein the bispecific antibody comprises two arms, wherein each arm comprises a first binding domain and a second binding domain.Atty. Dkt: OMN1-OQ5WO5. The system of any prior claim, wherein in (a) the common light chain is N-terminal to the antigen binding region of the single chain antibody.

6. The system of any prior claim, wherein in (a) the common light chain is C-terminal to the antigen binding region of the single chain antibody.

7. The system of any prior claim, wherein the single chain antibody is a VHH antibody, a picobody, or a scFv.

8. The system of any prior claim, wherein the first library comprises at least 10 members and, independently, the second library comprises at least 10 members.

9. The system of any prior claim, wherein the members of the first library are housed in one or more containers that are separate to containers that house the members of the second library.

10. The system of any prior claim, wherein the members of the first library are in separate containers and the members of the second library are in separate containers.

11. The system of any prior claim, wherein the light chain V region of (a), the antigen binding region of the single chain antibody of (a) and / or the heavy chain V region of (b) are humanized or were made by diversifying human sequences.

12. The system of any prior claim, does not comprise a third library of nucleic acids or a separate vector comprising a common light chain.

13. A method for producing a bispecific antibody, comprising:(a) obtaining a two-library system of any prior claim;(b) introducing a member of the first library and a member of the second library into the same cell; and(c) incubating the cell to provide for expression of the bispecific antibody.Atty. Dkt: OMN1-005WO 14. The method of claim 13, wherein the bispecific antibody of (c) is displayed on the tethered to cell surface.

15. The method of claim 13, wherein the bispecific antibody of (c) is secreted from the cell.

16. The method of any of claims 13-15, further comprising:(d) testing the bispecific antibody for binding to the first antigen and the second antigen.

17. The method of claim 16, wherein testing comprises detecting binding of the bispecific antigen to the first antigen and the second antigen at the same time.

18. The method of any of claims 13-17, wherein:step (b) comprises introducing pairwise combinations of members of the first and second libraries into the same cells; andstep (c) comprises incubating the cells to provide for expression of multiple bispecific antibodies.

19. The method of claim 18, further comprising:(d) independently testing the multiple bispecific antibodies for binding to the first antigen and the second antigen.

20. The method of claim 19, further comprising:(e) identilying at least one bispecific antibody based on at least one binding characteristic.

21. The method of any of claims 13-20, wherein the method is done without introducing any further nucleic acids into the cells.

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