Tl1a-related antibody compositions and methods of use

Abstract

The disclosure herein relates to the development and production of novel antibodies and antigen-binding fragments thereof that bind to TL1A and are useful in the treatment, prevention, and diagnosis of diseases, disorders, or inflammation, including, for example, autoimmune diseases, including rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, systemic lupus erythematosus, asthma, ulcerative colitis, Crohn's disease, psoriasis, primary biliary cirrhosis, primary biliary cholangitis, ankylosing spondylitis, and fibrosis, including intestinal fibrosis, pulmonary fibrosis, and liver fibrosis. Some of the elements of the final antibody structure are designed de novo by a computer system and its data training set, without reference to a particular reference molecule.

Description

[0001] Cross-references to related applications

[0002] This application is a PCT application that claims the benefit and priority of U.S. Provisional Application No. 63 / 542186, filed October 3, 2023; U.S. Provisional Application No. 63 / 554832, filed February 16, 2024; U.S. Provisional Application No. 63 / 675403, filed July 25, 2024; and U.S. Provisional Application No. 63 / 682593, filed August 13, 2024, the disclosures of which are hereby incorporated herein by reference in their entirety.

[0003] Merging of sequence lists

[0004] This application contains an electronically submitted sequence list XML, which is hereby incorporated in its entirety by reference. The copy of the XML was created on September 30, 2024, named 59413_SeqListing.xml, and is 132,207 bytes in size. Background Technology

[0005] TL1A, also known as TNFSF15, is a member of the tumor necrosis factor receptor family. It is expressed in various immune cells, such as monocytes, macrophages, dendritic cells, T cells, and non-immune cells, such as synovial fibroblasts and endothelial cells. TL1A competitively binds to death receptor 3 (DR3) or decoy receptor 3 (“DcR3”), providing stimulatory signals to downstream signaling pathways and then regulating the proliferation, activation, apoptosis, and chemokine production of cytokines in effector cells. Recent findings have shown aberrant expression of TL1A in autoimmune diseases, including rheumatoid arthritis, inflammatory bowel disease, psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, and ankylosing spondylitis. In vivo and in vitro studies have further demonstrated that TL1A is involved in the development and pathogenesis of these diseases. These accumulated data raise the possibility that the TL1A pathway could represent a significant therapeutic target for chronic immune diseases. Summary of the Invention

[0006] As described herein, this disclosure provides TL1A antibodies and related host cells, expression vectors, compositions, treatment methods, and methods for producing the foregoing.

[0007] In one embodiment, an antibody or antigen-binding fragment thereof is provided, the antibody or antigen-binding fragment thereof comprising at least one of the following: variable heavy chain complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, wherein: CDR-H1 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1, CDR-H2 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1, and CDR-H3 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1; and variable light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein: CDR-L1 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1, CDR-L2 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1, and CDR-L3 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1.

[0008] In another embodiment, an antibody or antigen-binding fragment thereof is provided, the antibody or antigen-binding fragment comprising at least one of the following: a variable heavy chain, wherein the variable heavy chain comprises a polypeptide sequence having at least 90% sequence identity with an amino acid sequence selected from any of the sequences provided in Sequence Listing 1 (including any sequence from SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136, or 138); and a variable light chain, wherein the variable light ... A polypeptide sequence having at least 90% sequence identity of the amino acid sequence of any of the sequences 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93 or 97.

[0009] In another embodiment, this disclosure provides an antibody or antigen-binding fragment thereof comprising a variable heavy chain complementarity-determining region (CDR-H1), CDR-H2, and CDR-H3, wherein CDR-H1 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including sequences from SEQ ID NO: 4, 14, 23, 28, 35, 39, 43, 46, 48, 64, 78, 84, 88, 94, 98, 109, 112, 115, 118, 120, 122, 125, 127, 130, or 137), and CDR-H2 ...). The CDR-H3 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any sequence from SEQ ID NO: 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128, or 135); and variable light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 comprises any of the sequences provided in Sequence Listing 1 (including any sequence from SEQ ID NO: 6, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, or 133); and variable light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 comprises any of the sequences selected from any of the sequences provided in Sequence Listing 1 (including any sequence from SEQ ID NO: 6, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, or 133). CDR-L2 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any of the sequences GAS, ATS, SAS, YAS, or SEQ ID NO: 62, 70, 72, 77, or 87), and CDR-L3 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any of the sequences SEQ ID NO: 3 or 63).

[0010] In yet another embodiment, this disclosure provides an antibody or antigen-binding fragment thereof comprising a variable heavy chain, wherein the variable heavy chain comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any of SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136, or 138); and a variable light chain, wherein the variable light chain comprises any of the sequences provided in Sequence Listing 1 (including any of SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136, or 138). A polypeptide sequence of any one of the sequences 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93 or 97. In another embodiment, an antibody or antigen-binding fragment thereof is provided, the antibody or antigen-binding fragment comprising a variable heavy chain complementarity-determining region (CDR-H1), CDR-H2, and CDR-H3, wherein CDR-H1 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any of SEQ ID NO: 4, 14, 23, 28, 35, 39, 43, 46, 48, 64, 78, 84, 88, 94, 98, 109, 112, 115, 118, 120, 122, 125, 127, 130, or 137), CDR-H2 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, or 133), and CDR-H3 comprises any of the sequences provided in Sequence Listing 1 (including any of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, or 133), and CDR-H3 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, or 133). NO: Any sequence of 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128 or 135) of a polypeptide sequence.In one embodiment, this disclosure also provides an antibody or antigen-binding fragment thereof comprising a variable light chain complementarity-determining region (CDR-L1), CDR-L2, and CDR-L3, wherein CDR-L1 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any of SEQ ID NO: 1, 12, 19, 27, 41, 53, 61, or 69), CDR-L2 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any sequence containing GAS, ATS, SAS, or YAS, or any of SEQ ID NO: 62, 70, 72, 77, or 87), and CDR-L3 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any of SEQ ID NO: 3 or 63). In another embodiment, an antibody or antigen-binding fragment thereof is provided, the antibody or antigen-binding fragment comprising a variable heavy chain complementarity-determining region (CDR-H1), CDR-H2, and CDR-H3, wherein CDR-H1 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any of SEQ ID NO: 4, 14, 23, 28, 35, 39, 43, 46, 48, 64, 78, 84, 88, 94, 98, 109, 112, 115, 118, 120, 122, 125, 127, 130, or 137), CDR-H2 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, or 133), and CDR-H3 comprises any of the sequences provided in Sequence Listing 1 (including any of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, or 133), and CDR-H3 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, or 133). The sequence comprises a polypeptide sequence of any of the sequences provided in Sequence Listing 1 (including any of the sequences of SEQ ID NO: 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128, or 135); and variable light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any sequence containing GAS, ATS, SAS, YAS, or any of the sequences of SEQ ID NO: 62, 70, 72, 77, or 87); and CDR-L3 comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any of the sequences of SEQ ID NO: 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128, or 135). A polypeptide sequence of either NO: 3 or 63.

[0011] In one embodiment, this disclosure provides an antibody or antigen-binding fragment thereof comprising a variable heavy chain, wherein the variable heavy chain comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any of the sequences SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136, or 138). In one embodiment, this disclosure provides an antibody or an antigen-binding fragment thereof comprising a variable light chain, wherein the variable light chain comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any of the sequences SEQ ID NO: 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93, or 97). In another embodiment, this disclosure provides an antibody or antigen-binding fragment thereof comprising a variable heavy chain, wherein the variable heavy chain comprises a polypeptide sequence selected from any of the sequences provided in Sequence Listing 1 (including any of SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136, or 138); and an antibody or antigen-binding fragment thereof comprising a variable light chain, wherein the variable light chain comprises ... selected from any of the sequences provided in Sequence Listing 1 (including SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, A polypeptide sequence of any one of the sequences 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93 or 97.

[0012] In one embodiment, this disclosure provides an antibody or an antigen-binding fragment thereof comprising (i) variable heavy chain complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, wherein (a) CDR-H1 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 4), (b) CDR-H2 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 5), and (c) CDR-H3 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 6); (ii) variable light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein: (a) CDR-L1 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 1); (b) CDR-L2 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (GAS); and (c) CDR-L3 comprises the amino acid sequence of the lead antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 1). 3); or (iii) the variable heavy chain complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NO: 9), and (ii) the variable light chain complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NO: 7). In one embodiment, this disclosure provides the aforementioned antibody capable of binding to human TL1A.

[0013] In yet another embodiment, this disclosure provides an antibody or an antigen-binding fragment thereof comprising: (a) a variable heavy chain comprising a polypeptide sequence having at least 90% sequence identity with the amino acid sequence (SEQ ID NO: 9) of the lead antibody 1 as shown in Sequence Listing 1; (b) a variable light chain comprising a polypeptide sequence having at least 90% sequence identity with the amino acid sequence (SEQ ID NO: 7) of the lead antibody 1 as shown in Sequence Listing 1; or (c) the variable heavy chain of (a) and the variable light chain of (b).

[0014] In one embodiment, this disclosure provides a pharmaceutical composition or drug comprising the aforementioned antibody or its antigen-binding fragment, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0015] A method for preventing disease, condition, or inflammation in a subject of need, said disease, condition, or inflammation including, for example, autoimmune diseases including rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, systemic lupus erythematosus, asthma, ulcerative colitis, Crohn's disease, psoriasis, primary biliary cirrhosis, primary biliary cholangitis, ankylosing spondylitis, and fibrosis, including intestinal fibrosis, pulmonary fibrosis, and liver fibrosis, said method comprising administering to the subject a therapeutically effective amount of the aforementioned antibody or antigen-binding fragment or the aforementioned pharmaceutical composition.

[0016] In one embodiment, this disclosure provides a method for treating a disease, condition, or inflammation in a subject of need, said disease, condition, or inflammation including, for example, autoimmune diseases including rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, systemic lupus erythematosus, asthma, ulcerative colitis, Crohn's disease, psoriasis, primary biliary cirrhosis, primary biliary cholangitis, ankylosing spondylitis, and fibrosis, including intestinal fibrosis, pulmonary fibrosis, and liver fibrosis, said method comprising administering to the subject (a) the aforementioned antibody or its antigen-binding fragment, or the aforementioned pharmaceutical composition.

[0017] In one embodiment, this disclosure provides a hybridoma that generates the aforementioned antibody or its antigen-binding fragment. In one embodiment, this disclosure provides a fusion protein comprising the aforementioned antibody or its antigen-binding fragment.

[0018] In one embodiment, this disclosure further provides an isolated nucleic acid encoding one or more or all of the aforementioned antibody's CDRs. In yet another embodiment, an expression vector is provided comprising the aforementioned isolated nucleic acid molecule. In still another embodiment, a host cell is provided comprising the aforementioned expression vector or the aforementioned isolated nucleic acid molecule.

[0019] In yet another embodiment, this disclosure provides a method for inhibiting the binding of TL1A to host cells expressing DR3, the method comprising contacting the host cells with the aforementioned antibody or its antigen-binding fragment.

[0020] In one embodiment, this disclosure also provides a method for generating an antibody or an antigen-binding fragment thereof, the method comprising: culturing a host cell according to any one of claims 56 to 59 in a culture medium under conditions that allow expression of a polypeptide encoded by the isolated nucleic acid and assembly of the antibody or an antigen-binding fragment thereof; and purifying the antibody or an antigen-binding fragment thereof from the cultured cells or cell culture medium.

[0021] In one embodiment, a method is provided for preparing an antibody or antigen-binding fragment thereof that is capable of binding to TL1A with higher affinity than a reference antibody. The method comprises: identifying a reference antibody or fragment thereof comprising one or more CDR sequences associated with TL1A binding activity; optionally identifying a frame sequence; modifying one or more amino acids in one or more of the CDR sequences to produce a variant antibody, wherein one or more of the amino acid modifications are predicted to cause the variant antibody to have a higher affinity for TL1A than the reference antibody, and wherein the prediction is generated by a computational system. In one embodiment, the computing system includes: one or more processors; and one or more non-transitory computer-readable media storing thereon: a machine learning model trained using training data, wherein the training data includes one or more training antibody sequence variants, each training antibody sequence variant having a corresponding measured binding property, the corresponding measured binding property representing the ability of each training antibody sequence variant to bind to a corresponding corresponding binding partner, and wherein the machine learning model is configured to output predicted antibody binding properties of the input antibody sequence variants; and instructions, which, when executed by the one or more processors, cause the computing system to: process the one or more antibody sequence variants with the machine learning model to generate one or more predicted binding properties, each predicted binding property corresponding to a corresponding antibody sequence variant among the one or more antibody sequence variants; analyze the one or more predicted binding properties to identify one or more antibody sequence variants of interest from the one or more antibody sequence variants, each of the one or more antibody sequence variants of interest having a corresponding one or more desired properties; and provide the one or more antibody sequence variants of interest as output. In another embodiment, the computing system includes: one or more processors; and one or more non-transitory computer-readable media storing thereon: a machine learning antibody prediction model trained to predict structural information of an antibody based on an input; and instructions that, when executed by the one or more processors, cause the computing system to: (1) receive a target input, the target input including one or more of the following: a primary sequence of a target binding partner, three-dimensional coordinates of a target binding partner, a primary sequence of a target binding partner epitope, or three-dimensional coordinates of a primary sequence of a target binding partner epitope, or a fragment or portion thereof; and (2) predict the structural information of a target antibody by processing the target input with the machine learning antibody prediction model.In another embodiment, the computing system includes: one or more processors; and one or more non-transitory computer-readable media storing thereon: a machine learning affinity prediction artificial neural network, the machine learning affinity prediction artificial neural network including: (i) one or more antibody prediction layers trained to predict antibody structural information from target input; (ii) one or more docking layers trained to generate docking complexes from two or more input three-dimensional antibodies; and (iii) one or more affinity prediction layers trained to predict affinity based on input docking complexes; wherein the one or more antibody prediction layers, the one or more docking layers, and the one or more affinity prediction layers are connected; and instructions, which, when executed by the one or more processors, cause the computing system to: receive a target input comprising one or more of a target binding partner sequence, a target binding partner, or a target epitope; and process the target input using the affinity prediction artificial neural network to generate a docking complex corresponding to the target input and a corresponding structural affinity value. In yet another embodiment, the computing system performs antigen-sensing antibody folding and includes: one or more processors; and one or more memories storing thereon: a machine learning model trained to predict output antibody digital representations corresponding to output antibodies using one or more training inputs, the one or more training inputs including one or more training antibody digital representations, each corresponding to a corresponding one of a plurality of training antibodies; and a set of computer-executable instructions that, when executed by the one or more processors, cause the computing system to: receive a plurality of input antibody digital representations, each corresponding to a corresponding one of a plurality of input antibodies; and process one or more of the plurality of input antibody digital representations using the machine learning model to produce one or more predicted output antibody digital representations.

[0022] In one embodiment, this disclosure also provides an antibody or antigen-binding fragment thereof that is capable of binding to TL1A with higher affinity than a reference antibody, the antibody or antigen-binding fragment comprising one or more of the following: at least one HCDR sequence, wherein the at least one HCDR sequence comprises one or more amino acid modifications compared to the reference antibody, and wherein the one or more amino acid modifications are predicted to give the antibody a higher affinity for TL1A than the reference antibody for TL1A, and wherein the prediction is generated by a computational system; and / or at least one LCDR sequence, wherein the at least one LCDR sequence comprises one or more amino acid modifications compared to the reference antibody, and wherein the one or more amino acid modifications are predicted to give the antibody a higher affinity for TL1A than the reference antibody for TL1A, and wherein the prediction is generated by a computational system; wherein at least one LCDR is unmodified compared to the reference antibody. In one embodiment, the antibody or antigen-binding fragment thereof comprises amino acid modifications of HCDR1, HCDR2, and HCDR3 compared to the reference antibody. In another embodiment, the antibody or antigen-binding fragment thereof comprises amino acid modifications of LCDR1 and LCDR2 compared to the reference antibody. In yet another embodiment, the antibody or its antigen-binding fragment comprises a frame region different from the frame region of the reference antibody. In yet another embodiment, the antibody or its antigen-binding fragment is generated by the foregoing method.

[0023] In other embodiments, the antibodies or fragments thereof provided herein (including, for example, ABS-101-A, ABS-101-B, or ABS-101-C) have superior properties relative to reference antibodies, including binding affinity, stability at high concentrations, and half-life characteristics.

[0024] By incorporating via reference

[0025] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same degree, as if specifically and individually indicated that each individual publication, patent or patent application is incorporated by reference. Attached Figure Description

[0026] Figure 1 A-1G demonstrates dose-dependent antibody inhibition of IFN-γ in whole blood by Heron1, Heron2B, or a novel antibody (mAb 4).

[0027] Figure 2 A-2B shows AI model predictions of ACE scores based on a high-quality hold-out dataset and subsequent ACE score predictions for novel AI-predicted HCDR sequences.

[0028] Figure 3 A-3B depicts HCDR sequences predicted by novel AI, with SPR binding affinity relative to the edit distance from Heron2B.

[0029] Figure 4 A summary of 20 high-priority, AI-optimized lead compounds was described.

[0030] Figure 5 A summary of quality and exploitability metrics for 20 high-priority AI-optimized lead compounds is presented.

[0031] Figure 6 A-6B shows the binding evaluation of 20 high-priority AI-optimized leads in mAb form with TL1A.

[0032] Figure 7 A-7B shows the cross-reactivity assessments of 20 high-priority, AI-optimized leads in mAb form.

[0033] Figure 8 A-8B shows that, via SPR, at pH 6.0, T relative to Heron 1 / 2 The elongation mutation increases FcRn affinity by approximately 10-fold.

[0034] Figure 9 The correlation between TL1A binding and surface target expression function assays and SPR assays was shown.

[0035] Figure 10A-10G The results of the NFKb affinity determination are shown.

[0036] Figures 11A-11E show the results of the apoptosis assay.

[0037] Figure 12 A-12B illustrates the generation of high-affinity variants of LCDR1 and LCDR2 through de novo LCDR modeling.

[0038] Figure 13 This demonstrates how to achieve high affinity and high diversity by combining AI-optimized HCDR with de novo AI-generated LCDR.

[0039] Figure 14 The study demonstrated that all 20 high-priority, AI-optimized lead compounds exhibited sub-nM affinity for human TL1A.

[0040] Figures 15A-15B An AI de novo design of the initial conjugate with the epitope of interest is shown.

[0041] Figure 16A-16B illustrates AI-guided lead optimization for improving therapeutic agents.

[0042] Figure 17 The lead candidate is shown to be combined with both the monomer and the trimer TL1A.

[0043] Figure 18 A-18B illustrates a lead candidate that blocks the binding of TL1A to DR3 and DcR3.

[0044] Figure 19 A-19B demonstrates that an AI-optimized lead candidate inhibits TL1A:DR3-driven apoptosis.

[0045] Figure 20 A-20B demonstrates an AI-optimized lead candidate that suppresses TL1A:DR3-driven NF-kB activation.

[0046] Figure 21 A-21B demonstrates an AI-optimized lead candidate that inhibits TL1A:DR3-driven INFγ release.

[0047] Figure 22 A variant of the lead is shown that is designed to prevent activation of the effector function.

[0048] Figure 23 The epitopes selected during AI design are shown to manage immunogenicity.

[0049] Figure 24 The low immunogenicity of the lead candidate enables sub-Q formulations and longer dosing intervals.

[0050] Figures 25A-25B The lead candidate is shown to perform well in the computer-simulated exploitability spectrum.

[0051] Figure 26 The study demonstrated that half-life extension mutations can improve antibody pharmacokinetics.

[0052] Figure 27 The results of the formulation and stability evaluation of ABS-101 are shown.

[0053] Figures 28A-28B The extended half-life profile of ABS-101-A is shown. Detailed Implementation

[0054] This disclosure provides antibodies and / or antigen-binding fragments thereof that specifically bind to TL1A (e.g., human TL1A) and antagonize TL1A function (e.g., TL1A-mediated inflammation suppression). Pharmaceutical compositions comprising these antibodies and / or their antigen-binding fragments are also provided, nucleic acids encoding these antibodies and / or their antigen-binding fragments, expression vectors and host cells for preparing these antibodies and / or their antigen-binding fragments, and methods for treating subjects using these antibodies and / or their antigen-binding fragments. Specifically, the antibodies and / or their antigen-binding fragments disclosed herein can be used to inhibit the binding of TL1A to death receptor 3 (DR3) and / or block DR3 and decoy receptor 3 (DcR3) on host cells. The antibody and / or its antigen-binding fragment may be used to treat and / or prevent diseases, conditions, or inflammation in the subject, including, for example, autoimmune diseases including rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, systemic lupus erythematosus, asthma, ulcerative colitis, Crohn's disease, psoriasis, primary biliary cirrhosis, primary biliary cholangitis, ankylosing spondylitis, and fibrosis, including intestinal fibrosis, pulmonary fibrosis, and liver fibrosis. All instances of "isolated antibody" described herein are also considered to be, but not necessarily, isolated antibodies. All instances of "isolated polynucleotide" described herein are also considered to be, but not necessarily, isolated polynucleotides. All instances of "antibody" described herein are also considered to be, but not necessarily, isolated antibodies. All instances of "polynucleotide" described herein are also considered to be, but not necessarily, isolated polynucleotides. In other respects, this disclosure provides a method for preparing an antibody or antigen-binding fragment thereof that is capable of binding to TL1A with a higher affinity than a reference antibody. The method comprises: identifying a reference antibody or fragment thereof containing one or more CDR sequences associated with TL1A binding activity; optionally identifying a frame sequence; modifying one or more amino acids in one or more of the CDR sequences to produce a variant antibody, wherein one or more of the amino acid modifications are predicted to cause the variant antibody to have a higher affinity for TL1A than the reference antibody, and wherein the prediction is generated by a computational system.

[0055] As used herein, the term "antibody" refers to a naturally occurring or partially or fully synthetic immunoglobulin (Ig). The term also covers any polypeptide or protein having a binding domain that is an antigen-binding domain or homologous to an antigen-binding domain. The term further includes "antigen-binding fragment" (as described herein) and other interchangeable terms.

[0056] Antibodies include, but are not limited to, any specific binding member, immunoglobulin class and / or isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM), and their biologically relevant fragments or specific binding members. Therefore, antibodies include, for example, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, recombinant antibodies, chemically engineered antibodies, deimmunized antibodies, affinity-matured antibodies, multispecific antibodies (e.g., bispecific antibodies and multireactive antibodies), heteroconjugated antibodies, antibody fragments, and combinations thereof (e.g., deimmunized monoclonal antibodies, deimmunized humanized antibodies, etc.).

[0057] As used herein, the term "TL1A-associated antibody" refers to an antibody that is specific to a TL1A-associated antigen or epitope.

[0058] Natural antibodies and natural immunoglobulins are typically heterotetraglycoproteins of approximately 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is usually linked to the heavy chain by a single covalent disulfide bond, although the number of disulfide bonds varies between heavy chains of different immunoglobulin isoforms. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (“V”) at one end. H "), followed by multiple constant structural domains ("C") H Each light chain has a variable structural domain ("V") at one end. L "), and has a constant structural domain (C) at its other end. L The constant domains of the light chain align with the first constant domain of the heavy chain, and the variable domains of the light chain align with the variable domains of the heavy chain. Specific amino acid residues are considered to form the interface between the variable domains of the light and heavy chains.

[0059] The antibodies or antigen-binding fragments thereof disclosed herein may include deletions at one end of the light chain. The antibodies or antigen-binding fragments thereof disclosed herein may include deletions of 3 or more amino acids at one end of the light chain. The antibodies or antigen-binding fragments thereof disclosed herein may include deletions of 7 or fewer amino acids at one end of the light chain. The antibodies or antigen-binding fragments thereof disclosed herein may include deletions of 3, 4, 5, 6, or 7 amino acids at one end of the light chain.

[0060] The antibodies or antigen-binding fragments thereof disclosed herein may include insertions in the light chain. The antibodies or antigen-binding fragments thereof disclosed herein may include insertions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids in the light chain. The antibodies or antigen-binding fragments thereof disclosed herein may include insertions of 3 amino acids in the light chain.

[0061] The “variable region” of an antibody refers to either or both of the variable region of the antibody light chain or the variable region of the antibody heavy chain. The variable regions of the heavy and light chains each consist of four frame regions (FRs) connected by three complementarity-determining regions (CDRs) (also known as hypervariable regions). The CDRs in each chain are held together tightly by the FRs and together with the CDRs from the other chain, promote the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, National Institutes of Health, Bethesda Md., MD) and (2) methods based on crystallographic studies of antigen-antibody complexes (Allazikani et al., 1997, J. Moles. Biol., 273:927-48). A CDR can refer to a CDR defined by any one method or by a combination of two methods.

[0062] The "constant region" of an antibody refers to either or both of the constant region of the antibody light chain and the constant region of the antibody heavy chain. The constant region does not change in terms of antigen specificity.

[0063] As used herein, the term "heavy chain region" includes the amino acid sequence of a constant domain derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain region comprises at least one of the following: C H 1. Structural domain, hinge (e.g., upper hinge area, middle hinge area, and / or lower hinge area) structural domain, C H 2. Structural domain, C H 3. A domain or a variant or fragment thereof. In one embodiment, the antibody or its antigen-binding fragment may include the Fc region of the immunoglobulin heavy chain (e.g., hinge portion, C...). H 2. Structural Domains and C H 3. Structural domains). In another embodiment, the antibody or its antigen-binding fragment lacks at least one region of a constant structural domain (e.g., C). H 2. All or part of the structural domains. In some embodiments, at least one and preferably all of the constant structural domains are derived from the human immunoglobulin heavy chain. For example, in a preferred embodiment, the heavy chain region comprises a fully human hinge domain. In other preferred embodiments, the heavy chain region comprises a fully human Fc region (e.g., a hinge, C, derived from human immunoglobulin). H 2. Domain sequence and C H3. Domain Sequence). In some embodiments, the constituent constant domains of the heavy chain region are derived from different immunoglobulin molecules. For example, the heavy chain region of a polypeptide may include a domain derived from an IgG1 molecule and a hinge region derived from an IgG3 or IgG4 molecule. In other embodiments, the constant domain is a chimeric domain comprising regions of different immunoglobulin molecules. For example, the hinge may comprise a first region from an IgG1 molecule and a second region from an IgG3 or IgG4 molecule. Those skilled in the art will understand that the constant domain of the heavy chain region can be modified such that the constant domain differs in amino acid sequence from naturally occurring (wild-type) immunoglobulin molecules. That is, the polypeptides disclosed herein may comprise one or more heavy chain constant domains (C) of the heavy chain constant domain. H 1. Hinges, C H 2 or C H 3) and / or light chain constant structural domains (C L Changes or modifications to ( ). Exemplary modifications include the addition, deletion, or substitution of one or more amino acids in one or more domains.

[0064] As used herein, the term "hinge region" includes the C-linked region of the heavy chain molecule. H 1. Structural Domains and C H 2. The hinge region. This hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper hinge domain, the middle hinge domain, and the lower hinge domain (Roux et al., Journal of Immunology, 1998, 161:4083).

[0065] As used herein, the term "Fv" refers to the smallest antibody fragment containing complete antigen recognition and antigen binding sites. This fragment consists of a dimer of a tightly non-covalently associated heavy chain variable region domain and a light chain variable region domain.

[0066] Regarding the "heavy chain variable region" or "V" of antibodies H "" refers to a segment of a heavy chain containing three CDRs between inserted side-connected extensions (called frame regions), which are typically more conservative than the CDRs and form a scaffold to support the CDRs.

[0067] Six hypervariable rings (three rings each in the H chain and L chain) provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific to the antigen) can recognize and bind antigens, albeit with a lower affinity than the intact binding site.

[0068] "Frame" or "FR" residues are those variable domain residues other than hypervariable region residues.

[0069] It should be understood in this art that an antibody is a glycoprotein or its antigen-binding moiety having at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. The heavy chains consist of heavy chain variable regions (V... H ) and heavy chain constant region (C H 1. C H 2 and C H 3) Composition. Light chains consist of light chain variable regions (V... L ) and light chain constant region (C L The antibody is composed of a heavy chain and a light chain. Both heavy and light chains have variable regions containing a framework region (FR or FWR) and a hypervariable region (HVR). The HVR consists of amino acid residues responsible for antigen binding. The hypervariable region typically contains amino acid residues from the complementarity-determining region (CDR), which has the highest sequence variability and / or is involved in antigen recognition. (Except for V...) H The CDR1 in the CDR typically contains amino acid residues that form the hypervariable ring. The CDR also contains "specificity-determining residues," or "SDRs," which are residues that contact the antigen. SDRs are contained within the CDR region, referred to as the abbreviation CDR or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are located at amino acid residues 31-34 of L1, amino acid residues 50-55 of L2, amino acid residues 89-96 of L3, amino acid residues 31-35B of H1, amino acid residues 50-58 of H2, and amino acid residues 95-102 of H3. (See, for example, Fransson, Frontiers in Biosciences 13:1619-1633 (2008)).

[0070] Unless otherwise stated, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered herein according to Kabat et al., ibid. The variable region is a domain of the antibody heavy or light chain involved in antibody-antigen binding. (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). Single V H or V L The structural domain is sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to specific antigens can use V... H or V L The domain binds to the antigen to screen for complementary V antibodies. L or VH Antibodies are isolated from libraries containing domains. (See, for example, Portolano et al., *Journal of Immunology* 150:880-87 (1993); Clarkson et al., *Nature* 352:624-28 (1991)). The four FWR regions are generally more conserved, while the CDR regions (CDR1, CDR2, and CDR3) represent hypervariable regions and are arranged from the NH2 end to the COOH end as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen, while, depending on the isotype, the constant regions can mediate the binding of immunoglobulins to host tissues or factors. Antibodies also include chimeric antibodies, humanized antibodies and recombinant antibodies, human antibodies derived from transgenic nonhuman animals, and antibodies selected from libraries using enrichment techniques available to the art.

[0071] The term "antibody heavy chain" refers to the larger of two types of polypeptide chains in an antibody molecule that exist in their natural conformation, and it usually determines the category to which the antibody belongs.

[0072] The term "antibody light chain" refers to the smaller of two types of polypeptide chains that exist in the antibody molecule in their naturally occurring conformation. κ ("kappa") and λ ("lambda") light chains refer to the two main isotypes of antibody light chains.

[0073] If an antibody or its antigen-binding fragment binds to a target antigen with a greater affinity and / or affinity than it binds to an epitope on an unrelated polypeptide, then the antibody or its antigen-binding fragment is “specifically bound” or “preferentially bound” to the target antigen. The specificity of an antibody or its antigen-binding fragment or portion thereof can be determined based on affinity and / or affinity. Methods for determining such specific binding are well known in the art. According to some embodiments of this disclosure, an antibody or its antigen-binding fragment may bind to human TL1A but not to TL1A from other subjects. In still other embodiments, an antibody or its antigen-binding fragment may bind to both human TL1A and TL1A from other subjects. In some embodiments, the antibodies or antigen-binding fragments disclosed herein specifically bind to the target antigens disclosed herein.

[0074] The equilibrium constant (K) for the dissociation of antigen and antigen-binding protein D Affinity, expressed as K, is a measure of the strength of binding between an antigenic determinant and an antigen-binding site on an antigen-binding protein. D The smaller the value, the stronger the binding strength between the antigenic determinant and the antigen-binding molecule. Alternatively, affinity can also be expressed as the affinity constant (K). A ), which is 1 / K DAs is clear to those skilled in the art, affinity can be determined in a manner known per se, depending on the specific antigen of interest. Therefore, when an antibody or its antigen-binding fragment has an affinity at least 50 times, such as at least 100 times, and preferably at least 1000 times, and at most 10,000 times or more (appropriately expressed as, for example, K) that is the affinity of the amino acid sequence or polypeptide to another target or polypeptide, affinity is considered. D When an antibody or its antigen-binding fragment binds to a first antigen, it is considered "specific" to the first target or antigen compared to a second target or antigen, as defined herein. Preferably, when an antibody or its antigen-binding fragment is "specific" to a target or antigen compared to another target or antigen, it can bind to the target or antigen but not to the other target or antigen. However, as will be understood by those skilled in the art, in some embodiments, when multiple different ligands share or partially share binding sites on a target, the antibody or its antigen-binding fragment can specifically bind to a target (such as TL1A) and have, for example, inhibitory / preventive effects against diseases, conditions, or inflammation, including, for example, autoimmune diseases including rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, systemic lupus erythematosus, asthma, ulcerative colitis, Crohn's disease, psoriasis, primary biliary cirrhosis, primary biliary cholangitis, ankylosing spondylitis, and fibrosis, including intestinal fibrosis, pulmonary fibrosis, and liver fibrosis.

[0075] K D It can be measured by any suitable determination. For example, K D It can be measured by radiolabeled antigen binding assay (RIA) (see, for example, Chen et al., Journal of Molecular Biology 293:865-881 (1999); Presta et al., Cancer Research 57:4593-4599 (1997)). For example, K D Surface plasmon resonance (SPR) measurements can be performed using (e.g., using BIACORE). ® -2000 or BIACORE ® -3000) is used for measurement. For example, K D Competitive ELISA can be used for measurement.

[0076] Affinity is a measure of the strength of binding between an antigen-binding molecule and its associated antigen. Affinity relates to two factors: the affinity between the antigenic determinant and its antigen-binding site on the antigen-binding molecule, and the number of associated binding sites present on the antigen-binding molecule. Typically, antigen-binding proteins will bind with respect to a dissociation constant (K0). D 10 -5 Up to 10 -12 M or smaller, and preferably 10-7 Up to 10 -12 M or smaller, and more preferably 10 -8 Up to 10 -12 M)(i.e., association constant (K) A ) is 10 5 Or 10 12 M -1 Or larger, and preferably 10 7 Up to 10 12 M -1 Or larger and more preferably 10 8 Up to 10 12 M -1 ) binds to its homologous or specific antigens. Any antigen greater than 10 -4 M of K D Value (or anything below 10) 4 M -1 K A The K-value is generally considered to indicate non-specific binding. K-values ​​are considered to indicate meaningful (e.g., specific) biological interactions. D Usually at 10 -10 M (0.1 nM) to 10 -5 Within the range of M (10000 nM). The stronger the interaction, the greater its K. D The lower the affinity, the better. Preferably, the binding site on the anti-LAP antibody or its antigen-binding fragment described herein will bind with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. The specific binding of the antigen-binding protein to the antigen or antigenic determinant can be determined by any suitable method known per se in the art or by different variants thereof, including, for example, Scatchard analysis and / or competitive binding assays (such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assay); and other techniques mentioned herein.

[0077] As used in this article, the term "k" on "" refers to the rate constant of association between the antibody or its antigen-binding fragment and the antigen.

[0078] As used in this article, the term "k" off "" refers to the rate constant of dissociation of an antibody or its antigen-binding fragment from the antibody / antigen complex.

[0079] It should be understood that this disclosure is not limited to specific formulations or process parameters, as these can certainly vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Furthermore, it should be understood that in practice, many methods and materials similar to or equivalent to those described herein can be used.

[0080] According to this application, conventional molecular biology, microbiology, and recombinant DNA techniques can be employed in the art. The definitions contained herein are supplementary to those in the art and, for the purposes of this application, should not be extrapolated to any related or unrelated circumstances, such as any jointly owned patents or applications. Therefore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0081] In this application, unless otherwise specified, the use of the singular includes the plural. It must be noted that, as used herein, the singular forms “a / an” and “the” include the plural referent unless the context clearly specifies otherwise. Furthermore, the use of the term “including” and other forms such as “include,” “includes,” and “included” is not restrictive.

[0082] As used herein, the terms “and / or” and “any combination thereof” and their grammatical equivalents are used interchangeably. These terms can convey a specific consideration of any combination. For illustrative purposes only, the phrases “A, B and / or C” or “A, B, C or any combination thereof” can mean “A alone; B alone; C alone; A and B; B and C; A and C; and A, B and C”.

[0083] The term "or" can be used together or separately, unless the context specifically requests separate use.

[0084] The terms "about" or "approximately" mean within an acceptable margin of error for a particular value, as determined by a person skilled in the art, which will depend in part on how the value was measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may mean within one or more standard deviations. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term may mean within orders of magnitude of the value, preferably within 5 times and more preferably within 2 times. When a particular value is described in this application and claims, unless otherwise indicated, it should be assumed that the term "about" means within an acceptable margin of error for the particular value.

[0085] As used in this specification and claims, the terms “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional unlisted elements or method steps. It is contemplated that any embodiments discussed in this specification can be implemented with respect to any method or composition of this disclosure, and vice versa. Furthermore, the compositions of this disclosure can be used to implement the methods of this disclosure.

[0086] As used herein, the term "consistently of" refers to those elements required for a given embodiment. This term allows for the presence of elements that do not materially affect the basic, novel, or functional characteristics of the embodiments described in this disclosure.

[0087] As used herein, the term "composed of" means the compositions, methods and their corresponding components as described herein, which do not include any elements not described in this description of the embodiments.

[0088] The references to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" in the specification mean that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments of this disclosure.

[0089] TL1A and anti-TL1A antibodies

[0090] This article provides antibodies and their antigen-binding fragments that bind to TL1A and can be used to treat and prevent diseases, conditions, or inflammations, including, for example, autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, systemic lupus erythematosus, asthma, ulcerative colitis, Crohn's disease, psoriasis, primary biliary cirrhosis, primary biliary cholangitis, ankylosing spondylitis, and fibrosis, including intestinal fibrosis, pulmonary fibrosis, and liver fibrosis.

[0091] Tumor necrosis factor (TNF)-like cytokine 1A (TL1A) is a member of the TNF superfamily and was first identified in 2002 (Xu WD et al., *Fronts Immunol.*, July 14, 2022; 13:913-28). The tnfsf15 gene encoding TL1A is located on human chromosome 9q32 and mouse chromosome 4. TL1A is a type 2 transmembrane protein that self-assembles into a stable trimer through interaction with the TNF homologous domain (THD). It is primarily expressed in a membrane-bound form and forms a stable trimer. Soluble TL1A (sTL1A) is produced via alternative splicing or TNF-α convertase (TACE) cleavage (Migone TS et al., Immunity (2002) 16(3):479–92; Zhan C et al., Biochemistry (2009) 48(32):7636–45). TL1A is constitutively expressed in endothelial cells and is upregulated in response to tumor necrosis factor-α (TNF-α) stimulation. When dendritic cells (DCs) and macrophages are triggered by the Fc region (FcγR) of toll-like receptor 4 (TLR4), TLR11, or IgG, the expression of TL1A in these cells increases (Schreiber TH et al., Immunol Res (2013) 57(1-3):3–11; Fang L et al., J Exp Med (2008) 205(5):1037–48; and Prehn JL et al., J Immunol (2007) 178(7):4033–8). Mitogen-activated protein kinase (MAPK), nuclear factor κ light chain enhancer (NF-κB) of activated B cells, and caspase-8 signaling pathways regulate immune responses ranging from apoptosis to autoimmunity (Wen L et al., J Biol Chem (2003) 278(40):39251–8; and Micheau O et al., Cell (2003) 114(2):181–90). TL1A binds to its receptor, death receptor 3 (DR3), and subsequently activates downstream signaling, then participates in innate and adaptive immune homeostasis. The binding of APC-derived TL1A to lymphocyte DR3 provides a co-stimulatory signal to activated lymphocytes. DR3 signaling affects the proliferative activity and cytokine production of effector lymphocytes, but also significantly influences the regulation of T cell development and repressive function. DR3 has also been found to be highly expressed by innate lymphoid cells (ILCS) that respond to TL1A stimulation.Recent studies using transgenic and knockout mice, along with neutralizing and agonistic antibodies against these two proteins, have clearly demonstrated that TL1A / DR3 is a key mediator of several chronic immune diseases, including inflammatory bowel disease (IBD). TL1A and DR3 are abundant in inflamed intestinal regions of patients with IBD and mice with experimental ileitis or colitis, and actively participate in mucosal homeostasis and immune pathways underlying intestinal inflammation.

[0092] Soluble TL1A (sTL1A) has been shown to be detectable in the serum and body fluids of patients with T-cell-mediated inflammatory autoimmune diseases such as rheumatoid arthritis (RA), psoriatic arthritis (PsA), and ankylosing spondylitis (AS). TL1A also plays an important role in the pathogenesis of these diseases (Aiba Y et al., MediatorsInflamm (2013) 2013:258164). In recent years, TL1A has attracted much attention as an important mediator of inflammation because anti-TL1A antibody therapy may be a promising treatment for inflammatory conditions (Danese S et al., Clin Gastroenterology and Hepatology (2021) 19(11):2324–32.e6).

[0093] Kokkotis and Bamias (Kokkotis, G and Bamias, G., ExpertRev. Clin. Immunol., 2022, 18(6): 551-55) also described the relationship between TL1A and inflammatory bowel disease (IBD). TL1A was first reported in 2002 (Migone et al., Immunology (2002) 16 pp. 479-92). It was found to be a member of the TNF protein superfamily (TNFSF) and encoded by the Tnfsf15 gene located on human chromosome 9q32 and mouse chromosome 4. In 2005, Yamazaki et al. first reported that specific genetic variants of Tnfsf15 and tnfsf15_28 were closely associated with susceptibility to IBD in Japanese patients, while the genes were monomorphic in a Caucasian population from the UK (Yamazaki et al., “Single nucleotide polymorphisms in TNFSF15 confer susceptibility to Crohn's disease”, Human Molecular Genetics 14 (3400-506) (2005)). Further studies in these two populations revealed five distinct SNPs (tnfsf15_26, 31, 35, 36, and 41), which were polymorphic in both groups, forming three different haplotypes that influence IBD susceptibility. In both populations, haplotype A was identified as a high-risk biomarker for IBD susceptibility, while haplotype B was found to be a low-risk genetic factor. Although haplotype C was frequently detected, it was not significantly associated with IBD risk in either population. Two years later, Picornell et al. investigated the aforementioned three haplotypes in Jewish and non-Jewish IBD patients and a control group in Los Angeles, USA. In the non-Jewish population, similar to previous studies, haplotype B was less frequent in both CD and UC patients compared to controls, highlighting a potential protective role (Picornell Y. et al., “TNFSF15 is an ethnic-specific IBD gene”. Inflammatory Bowel Dis. (2007) 13:1333–8.20). On the other hand, haplotype A was not observed to be associated with IBD in either population, suggesting that TNFSF15 polymorphism is ethnic-specific. Independent studies from Asia and Europe further support these findings.

[0094] Pfizer has developed a fully human anti-TL1A antibody (PF-06480605) that binds to the trimer form of TL1A (Banfield, Christopher et al., British Journal of Clinical Pharmacology 86.4 (2020): 812-24; Danese S et al., Clinical Gastroenterology and Hepatology, 2021. 19: 2324-2332.e6). Prometheus Bioscience developed a humanized anti-TL1A mouse IgG1 mAb (PRA023) that binds to both the trimer and monomeric forms of TL1A (Sands, B. et al., Journal of Crohn's and Colitis 17. Supplement_1 (2023): i56-i59; Feagan, BG et al., Journal of Crohn's and Colitis 2023 17: i162–i164). Glenmark Pharmaceuticals developed a humanized anti-TL1A mAb (WO 2014 / 106602; US 9,290,576). Pelican Therapeutics developed a human TL1A-Ig fusion protein agonist of TNFRSF25 / DR3 (PTX-45).

[0095] For example, in US 9,068,003, 8,642,741, 9,556,277, 10,822,422, 2021 / 037193, 10,316,083, 11,474,112, 9,683,998, 10,138,296, 10,968,279, 10,322,174, 10,689,439, 11,136,386, 2020 / 0362025, 11,292,848, 2022 / 029024, 2021 / 0395824, WO 2022 / 103961, WO2022 / 119842, WO2022 / 178158, WO Additional antibodies and related methods are described in 2022 / 178159, WO 2022 / 232253 and WO 2023 / 009545.

[0096] In some respects, this disclosure provides nucleic acid and polypeptide sequences of TL1A-related antibodies.

[0097] As used herein, the terms “protein,” “peptide,” and “polypeptide” are used interchangeably to refer to a series of amino acid residues linked together by peptide bonds between the α-amino and carboxyl groups of adjacent residues. The terms “protein,” “peptide,” and “polypeptide” refer to polymers of amino acids, including modified amino acids (e.g., phosphorylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. “Protein” and “polypeptide” are generally used to refer to relatively large polypeptides, while the term “peptide” is generally used to refer to smaller polypeptides; however, the use of these terms overlaps in the art. When referring to gene products and fragments thereof, the terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein. As used herein, the term “fusion protein” refers to a polypeptide comprising the amino acid sequence of an antibody or a fragment thereof and the amino acid sequence of a heterologous polypeptide (i.e., an unrelated polypeptide).

[0098] As used herein, an “isolated” nucleic acid molecule or “isolated” nucleic acid sequence is a nucleic acid molecule that meets either of the following criteria: (1) it is identified and isolated from at least one contaminating nucleic acid molecule that is normally associated with it in its natural source; or (2) it is considered isolated if the sequence of the nucleic acid of interest can be determined by cloning, amplification, labeling, or otherwise distinguishing it from background nucleic acids. Isolated nucleic acid molecules differ from those found in nature or in their environment. Therefore, isolated nucleic acid molecules are distinct from those present in natural cells. However, isolated nucleic acid molecules include those found in cells that typically express antibodies, where, for example, the nucleic acid molecule is located at a chromosomal location different from that in natural cells.

[0099] As used herein, the terms “synthetic polynucleotide,” “synthetic gene,” or “synthetic polypeptide” mean a corresponding polynucleotide sequence or a portion thereof, or an amino acid sequence or portion thereof, derived from a sequence that has been designed, de novo synthesized, or modified compared to an equivalent naturally occurring sequence. Synthetic polynucleotides (antibody or antigen-binding fragments) or synthetic genes can be prepared by methods known in the art, including but not limited to the chemical synthesis of nucleic acid or amino acid sequences. Synthetic genes typically differ from naturally occurring genes at the amino acid or polynucleotide level (or both) and are typically located within an environment controlling the synthetic expression sequence. Synthetic gene polynucleotide sequences may not necessarily encode proteins with different amino acids compared to natural genes; for example, they may also encompass synthetic polynucleotide sequences incorporating different codons but encoding the same amino acid (i.e., nucleotide changes representing silent mutations at the amino acid level).

[0100] The percentage of sequence identity (%) relative to a reference polypeptide sequence is the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after sequence alignment and cleavage (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. Alignment for determining the percentage of amino acid sequence identity can be performed in several ways within the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or MegAlign (DNASTAR). ® Software. Those skilled in the art can determine the appropriate parameters used for sequence alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. However, for the purposes of this document, the amino acid sequence identity % values ​​were generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office at 20559 Washington, D.C., registered under U.S. Copyright Registry No.: TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and remained unchanged.

[0101] When using ALIGN-2 for amino acid sequence alignment, the amino acid sequence identity % of a given amino acid sequence A with (and, or relative to) a given amino acid sequence B (which can be alternatively expressed as a given amino acid sequence A having or containing a certain amino acid sequence identity % with (and, or relative to) a given amino acid sequence B) can be calculated as follows: 100 multiplied by the fraction X / Y, where X is the number of amino acid residues that the sequence alignment program ALIGN-2 assesses as a complete match when aligning A and B, and where Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the amino acid sequence identity % of A with B will not be equal to the amino acid sequence identity % of B with A. Unless otherwise specifically stated, all amino acid sequence identity % values ​​used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraphs.

[0102] In some embodiments, this disclosure further provides variable heavy chains (V) of antibodies according to a number of embodiments of this disclosure. H ) and variable light chains (V L The complete nucleic acid and polypeptide sequences of V are also provided.H and V L The nucleic acid and polypeptide sequences of the three complementarity-determining regions (CDRs).

[0103] One aspect of this disclosure relates to nucleic acid molecular sequences encoding antibody peptides or antigen-binding fragments thereof as described herein. In some embodiments, the nucleic acid sequence encoding a heavy chain peptide is selected from any of the sequences provided in Sequence Listing 1 herein. In some embodiments, the nucleic acid sequence encoding a light chain peptide is selected from any of the sequences provided in Sequence Listing 1 herein. In some embodiments, the nucleic acid sequence encodes a CDR1, CDR2, or CDR3 peptide with a variable heavy chain, such that:

[0104] (a) The nucleic acid sequence encoding the CDR1 polypeptide of the variable heavy chain is selected from any of the sequences provided in Sequence Listing 1 herein, including any of the sequences SEQ ID NO: 4, 14, 23, 28, 35, 39, 43, 46, 48, 64, 78, 84, 88, 94, 98, 109, 112, 115, 118, 120, 122, 125, 127, 130, or 137.

[0105] (b) The nucleic acid sequence encoding the CDR2 polypeptide of the variable heavy chain is selected from any of the sequences provided in Sequence Listing 1 herein, including any of the sequences of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131 or 133, or

[0106] (c) The nucleic acid sequence encoding the CDR3 polypeptide of the variable heavy chain is selected from any of the sequences provided in Sequence Listing 1 of this document, including any of the sequences of SEQ ID NO: 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128 or 135.

[0107] In some embodiments, the nucleic acid sequence encodes a CDR1, CDR2, or CDR3 polypeptide of a variable light chain, such that:

[0108] (a) The nucleic acid sequence encoding the CDR1 region of the variable light chain polypeptide is selected from any of the sequences provided in Sequence Listing 1 herein, including any of SEQ ID NO: 1, 12, 19, 27, 41, 53, 61 or 69.

[0109] (b) The nucleic acid sequence encoding the CDR2 region of the variable light chain polypeptide is selected from any of the sequences provided in Sequence Listing 1 herein, including any sequence containing GAS, ATS, SAS, or YAS, or any sequence of SEQ ID NO: 62, 70, 72, 77, or 87, or

[0110] (c) The nucleic acid sequence encoding the CDR3 region of the variable light chain polypeptide is selected from any of the sequences provided in Sequence Listing 1 of this document, including any of the sequences of SEQ ID NO: 3 or 63.

[0111] In some embodiments, the nucleic acid sequence encodes HCDR1 (SEQ ID NO: 4), HCDR2 (SEQ ID NO: 5), HCDR3 (SEQ ID NO: 20), LCDR1 (SEQ ID NO: 19), LCDR2 (GAS), LCDR3 (SEQ ID NO: 3), VL (SEQ ID NO: 21), CL (SEQ ID NO: 8), VH (SEQ ID NO: 22), CH (SEQ ID NO: 10) and / or the Fc region (SEQ ID NO: 139) of ABS-101-A, and HCDR1 (SEQ ID NO: 28), HCDR2 (SEQ ID NO: 29), HCDR3 (SEQ ID NO: 30), LCDR1 (SEQ ID NO: 27), LCDR2 (GAS), LCDR3 (SEQ ID NO: 3), VL (SEQ ID NO: 31), CL (SEQ ID NO: 8), VH (SEQ ID NO: 32), CH (SEQ ID NO: 10) and / or the Fc region (SEQ ID NO: 139) of ABS-101-B. 10) and / or Fc region (SEQ ID NO: 139) or HCDR1 (SEQ ID NO: 4), HCDR2 (SEQ ID NO: 5), HCDR3 (SEQ ID NO: 20), LCDR1 (SEQ ID NO: 41), LCDR2 (SAS), LCDR3 (SEQ ID NO: 3), VL (SEQ ID NO: 42), CL (SEQ ID NO: 8), VH (SEQ ID NO: 22), CH (SEQ ID NO: 10) and / or Fc region (SEQ ID NO: 139) of ABS-101-C.

[0112] Another aspect of this disclosure relates to nucleic acid molecules comprising a nucleic acid sequence encoding an antibody polypeptide or an antigen-binding fragment thereof as described herein. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence encoding a heavy chain polypeptide of an antibody. In some embodiments, the nucleic acid sequence encoding the heavy chain polypeptide is selected from the sequences provided in Sequence Listing 1 herein. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence encoding a light chain polypeptide of an antibody. In some embodiments, the nucleic acid sequence encoding the light chain polypeptide is selected from the sequences provided in Sequence Listing 1 herein.

[0113] In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence encoding a CDR1 polypeptide of a variable heavy chain. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding a CDR2 polypeptide of a variable heavy chain. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding a CDR3 polypeptide of a variable heavy chain. In some embodiments, the nucleic acid sequence encoding the CDR1 polypeptide of a variable heavy chain (CDR-H1) comprises a sequence selected from the sequences provided in Sequence Listing 1 herein (including any of SEQ ID NO: 4, 14, 23, 28, 35, 39, 43, 46, 48, 64, 78, 84, 88, 94, 98, 109, 112, 115, 118, 120, 122, 125, 127, 130, or 137). In some embodiments, the nucleic acid sequence encoding the CDR2 polypeptide (CDR-H2) with a variable heavy chain comprises a sequence selected from the sequences provided in Sequence Listing 1 herein (including any of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, or 133). In some embodiments, the nucleic acid sequence encoding the CDR3 polypeptide (CDR-H3) with a variable heavy chain comprises a sequence selected from the sequences provided in Sequence Listing 1 herein (including any of SEQ ID NO: 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128, or 135). In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence encoding the CDR1 polypeptide with a variable light chain. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding the CDR2 polypeptide with a variable light chain. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence encoding a CDR3 polypeptide of a variable light chain. In some embodiments, the nucleic acid sequence encoding the CDR1 region (CDR-L1) of the variable light chain polypeptide comprises a sequence selected from the sequences provided in Sequence Listing 1 herein (including any sequence of SEQ ID NO: 1, 12, 19, 27, 41, 53, 61, or 69). In some embodiments, the nucleic acid sequence encoding the CDR2 region (CDR-L2) of the variable light chain polypeptide comprises a sequence selected from the sequences provided in Sequence Listing 1 herein (including sequences containing GAS, ATS, SAS, or YAS, or sequences of SEQ ID NO: 62, 70, 72, 77, or 87). In some embodiments, the nucleic acid sequence encoding the CDR3 region (CDR-L3) of the variable light chain polypeptide comprises a sequence selected from the sequences provided in Sequence Listing 1 herein (including any sequence of SEQ ID NO: 3 or 63).

[0114] Nucleic acids according to at least some embodiments of this disclosure can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes), cDNA encoding the light and heavy chains of the antibody prepared by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding the antibody can be recovered from the library. Once DNA fragments encoding VH and VL regions are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operatively linked to another DNA fragment encoding another protein (such as an antibody constant region or a flexible linker). As used in this context, the term "operatively linked" is intended to mean joining two DNA fragments such that the amino acid sequences encoded by said two DNA fragments remain within the frame. By linking the DNA encoding VH with the DNA encoding the heavy chain constant region (C... H 1. C H 2 and C H 3) Another DNA molecule can be operatively linked to convert the isolated DNA encoding the VH region into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, E.A. et al. (1991), *Protein Sequences of Immunological Significance*, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. Heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, but are most preferably IgG1 or IgG4 constant regions. For Fab fragment heavy chain genes, the DNA encoding VH can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0115] By operatively linking the DNA encoding the VL region to another DNA molecule encoding the light chain constant region CL, the isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat, EA et al. (1991), *Protein Sequences of Immunological Significance*, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a κ or λ constant region, but is most preferably a κ constant region. To generate the scFv gene, DNA fragments encoding VH and VL are operatively linked to another fragment encoding a flexible linker (e.g., the amino acid sequence (Gly-4-Ser)3), such that the VH and VL sequences can be expressed as a continuous single-stranded protein, wherein the VL and VH regions are joined by a flexible linker (see, for example, Bird et al. (1988) Science 242:423-26; Huston et al. (1988) Proceedings of the National Academy of Sciences 85:5879-5883; and McCafferty et al. (1990) Nature 348:552-554).

[0116] It is also envisioned that nucleic acids may contain nucleotide sequences substantially different from those described but still encoding at least as described herein and / or as known in the art, antibody or antigen-binding fragment thereof, due to the degeneracy of the genetic code. Of course, the genetic code is well known in the art. Therefore, it will be conventional for those skilled in the art to generate such degenerate nucleic acid variants encoding specific antibodies of this disclosure. See, for example, Ausubel et al., ibid., and this disclosure includes such nucleic acid variants.

[0117] In some embodiments, the nucleic acid is a nucleic acid encoding any of the amino acid sequences of the antibodies listed in Sequence Listing 1 herein. In some embodiments, the nucleic acid sequence is a nucleic acid sequence that is at least 80% identical (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the amino acid sequences encoding any of the antibodies in Sequence Listing 1 herein. In some embodiments, the nucleic acid is a nucleic acid that hybridizes with any or more of the nucleic acid sequences provided herein. In some embodiments, hybridization is performed under moderate conditions. In some embodiments, hybridization is performed under highly stringent conditions, such as: a first wash at about 65°C with at least about 6X SSC and 1% SDS, followed by a wash at about 42°C with 0.1X SSC containing about 20% (v / v) formamide for 10 minutes, and then a wash at 65°C with 0.2X SSC and 0.1% SDS.

[0118] Nucleic acids can be constructed using conventional recombinant DNA techniques in the art. In some embodiments, the nucleic acids disclosed herein are placed in an expression vector suitable for expression in selected host cells. Vectors containing nucleic acids encoding antibody or antigen-binding fragments of this document are provided. Vectors containing nucleic acids encoding heavy and / or light chains are also provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In one embodiment, the nucleic acids encoding the light chain and the nucleic acids encoding the heavy chain are isolated separately using the procedures outlined above. In one embodiment, the isolated nucleic acids encoding the light chain and the isolated nucleic acids encoding the heavy chain may be inserted into separate expression plasmids or together into the same plasmid, provided that each plasmid is under suitable promoter and translational control. In some embodiments, a suitable promoter is an inducible promoter. In some embodiments, a suitable promoter is a constitutive promoter. In some embodiments, the heavy and light chains are expressed as part of a single polypeptide, for example, when the antibody is scFv.

[0119] In some embodiments, a first vector contains a nucleic acid encoding a heavy chain, and a second vector contains a nucleic acid encoding a light chain. In some embodiments, the first and second vectors are transfected into host cells in similar amounts (e.g., similar molar amounts or similar mass amounts). In some embodiments, the first and second vectors are transfected into host cells in a molar ratio or mass ratio between 5:1 and 1:5. In some embodiments, a mass ratio between 1:1 and 1:5 is used for the vector encoding the heavy chain and the vector encoding the light chain. In some embodiments, a mass ratio of 1:2 is used for the vector encoding the heavy chain and the vector encoding the light chain. In some embodiments, a vector optimized for expressing a polypeptide in CHO cells or CHO-derived cells or NSO cells is selected. Exemplary such vectors are described, for example, in Running Deer et al., *Biotechnol. Prog.* 20:880-889 (2004).

[0120] Contemplated alternative vectors for antibody expression and purification are described in U.S. Patent Nos. 9,617,335, 11,371,048, U.S. Publication No. 2018 / 0282405, 11,584,785, and PCT / US22 / 82294.

[0121] Exemplary TL1A-related antibodies or antigen-binding fragments thereof

[0122] This disclosure provides TL1A-associated antibodies or antigen-binding fragments thereof. In some embodiments, the antibody or antigen-binding fragment thereof blocks or otherwise inhibits the binding of TL1A to death receptor 3 (DR3; TNFRSF25) and / or blocks DR3 and DcR3. In some embodiments, the TL1A-related antibody or its antigen-binding fragment comprises one or more of the following regions of ABS-101-A as shown in Sequence Listing 1: HCDR1 (SEQ ID NO: 4), HCDR2 (SEQ ID NO: 5), HCDR3 (SEQ ID NO: 20), LCDR1 (SEQ ID NO: 19), LCDR2 (GAS), LCDR3 (SEQ ID NO: 3), VL (SEQ ID NO: 21), CL (SEQ ID NO: 8), VH (SEQ ID NO: 22), CH (SEQ ID NO: 10) and / or Fc region (SEQ ID NO: 139): HCDR1, HCDR2, HCDR3, LCDR1 (SEQ ID NO: 27), LCDR2 (GAS), LCDR3 (SEQ ID NO: 3), VL (SEQ ID NO: 31), CL (SEQ ID NO: 8), VH (SEQ ID NO: 32), CH (SEQ ID NO: 10), and / or Fc region (SEQ ID NO: 139): HCDR1, HCDR2, HCDR3, LCDR1 (SEQ ID NO: 27), LCDR2 (GAS), LCDR3 (SEQ ID NO: 3), VL (SEQ ID NO: 31), CL (SEQ ID NO: 8), VH (SEQ ID NO: 32), CH (SEQ ID NO: 139 ... HCDR2, HCDR3, LCDR1 (SEQ ID NO: 27), LCDR2 (GAS), LCDR3 (SEQ ID NO: 3), VL (SEQ 10) and / or one or more of the Fc region (SEQ ID NO: 139) ABS-101-B, or one or more of HCDR1 (SEQ ID NO: 4), HCDR2 (SEQ ID NO: 5), HCDR3 (SEQ ID NO: 20), LCDR1 (SEQ ID NO: 41), LCDR2 (SAS), LCDR3 (SEQ ID NO: 3), VL (SEQ ID NO: 42), CL (SEQ ID NO: 8), VH (SEQ ID NO: 32), CH (SEQ ID NO: 10) and / or Fc region (SEQ ID NO: 139) ABS-101-C.

[0123] On one hand, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain (V) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the sequences provided in SEQ ID NO: 1 or in the tables herein (including any of the sequences SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136, or 138). H In some embodiments, V having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to a reference sequence. H The sequence contains substitutions (e.g., conserved substitutions), insertions, or deletions, but the antibody or its antigen-binding fragment containing the sequence retains the same ability to bind to TL1A as the parent. In some embodiments, any of the sequences provided in Sequence Listing 1 herein (including any of SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136, or 138) has a total of 1 to 10 amino acids substituted, inserted, and / or deleted in its amino acid sequence. In some embodiments, the substitution, insertion, or deletion occurs in regions outside the CDR (e.g., in the FR). Optionally, the antibody or its antigen-binding fragment comprises any of the sequences provided in Sequence Listing 1 herein (including any of the sequences SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136, or 138). H A sequence, including one or more post-translational modifications of the sequence.

[0124] In some embodiments, V HThe sequence comprises one, two, or three CDRs selected from the following: (a) CDR-H1, which comprises the amino acid sequence of any of the sequences provided in Sequence Listing 1 of this document (including any of the sequences of SEQ ID NO: 4, 14, 23, 28, 35, 39, 43, 46, 48, 64, 78, 84, 88, 94, 98, 109, 112, 115, 118, 120, 122, 125, 127, 130, or 137); (b) CDR-H2, which comprises the amino acid sequence of any of the sequences provided in Sequence Listing 1 of this document (including any of the sequences of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, or 133); and (c) CDR-H3, which comprises any of the sequences provided in Sequence Listing 1 of this document (including any of the sequences of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, or 133). IDNO: any sequence of amino acids of 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128 or 135.

[0125] In some embodiments, V H The sequence comprises one, two, or three CDRs selected from the following: (a) CDR-H1, which comprises the amino acid sequence of any of the sequences provided in Sequence Listing 1 herein (including any of the sequences SEQ ID NO: 4, 14, 23, 28, 35, 39, 43, 46, 48, 64, 78, 84, 88, 94, 98, 109, 112, 115, 118, 120, 122, 125, 127, 130, or 137); and (b) CDR-H2 ...). (c) an amino acid sequence of any of the sequences 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, or 133; and (d) a CDR-H3 comprising the amino acid sequence of any of the sequences provided in Sequence Listing 1 herein, namely 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128, or 135, wherein the selected CDR-H1, CDR-H2, and CDR-H3 are paired according to Sequence Listing 1.

[0126] On one hand, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment comprises a light chain variable domain (V) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the sequences provided in Sequence Listing 1 herein (including any of SEQ ID NO: 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93, or 97). L In some embodiments, V having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to a reference sequence. L The sequence contains substitutions (e.g., conserved substitutions), insertions, or deletions, but the antibody or its antigen-binding fragment containing the sequence retains the same ability to bind to TL1A as the parent. In some embodiments, any of the sequences provided in Sequence Listing 1 herein (including any of SEQ ID NO: 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93, or 97) has a total of 1 to 10 amino acids substituted, inserted, and / or deleted in its amino acid sequence. In some embodiments, the substitution, insertion, or deletion occurs in regions outside the CDR (e.g., in the FR). Optionally, the antibody or its antigen-binding fragment comprises any of the sequences provided in Sequence Listing 1 herein (including any of the sequences of SEQ ID NO: 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93 or 97) with V L A sequence, including one or more post-translational modifications of the sequence.

[0127] In some embodiments, V LThe sequence comprises one, two, or three CDRs selected from the following: (a) CDR-L1, which comprises the amino acid sequence of any of the sequences provided in Sequence Listing 1 of this document (including any of the sequences of SEQ ID NO: 1, 12, 19, 27, 41, 53, 61, or 69); (b) CDR-L2, which comprises the amino acid sequence of any of the sequences provided in Sequence Listing 1 of this document (including any sequence containing GAS, ATS, SAS, or YAS, or any of the sequences of SEQ ID NO: 62, 70, 72, 77, or 87); and (c) CDR-L3, which comprises the amino acid sequence of any of the sequences provided in Sequence Listing 1 of this document (including any of the sequences of SEQ ID NO: 3 or 63).

[0128] In some embodiments, V L The sequence comprises one, two, or three CDRs selected from the following: (a) CDR-L1, which comprises the amino acid sequence of any of the sequences provided in Sequence Listing 1 of this document (including any of the sequences of SEQ ID NO: 1, 12, 19, 27, 41, 53, 61, or 69); (b) CDR-L2, which comprises the amino acid sequence of any of the sequences provided in Sequence Listing 1 of this document (including any sequence containing GAS, ATS, SAS, or YAS, or any of the sequences of SEQ ID NO: 62, 70, 72, 77, or 87); and (c) CDR-L3, which comprises the amino acid sequence of any of the sequences provided in Sequence Listing 1 of this document (including any of the sequences of SEQ ID NO: 3 or 63), wherein the selected CDR-H1, CDR-H2, and CDR-H3 are paired according to Sequence Listing 1.

[0129] On the one hand, an antibody or an antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises: (a) V H The V H The amino acid sequence comprising any of the sequences provided in Sequence Listing 1 of this article (including any of the sequences SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136 or 138), and (b) V L The V LThe amino acid sequence comprising any of the sequences provided in Sequence Listing 1 of this document (including any of the sequences of SEQ ID NO: 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93 or 97), and optionally including post-translational modifications of these sequences.

[0130] On the one hand, an antibody or an antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises: (a) V H The V H The amino acid sequence comprising any of the sequences provided in Sequence Listing 1 of this article (including any of the sequences SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136 or 138), and (b) V L The V L An amino acid sequence comprising any of the sequences provided in Sequence Listing 1 of this document (including any of SEQ ID NO: 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93, or 97), wherein the selected V H and V L Pair according to sequence list 1.

[0131] On one hand, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises: (a) CDR-H1, wherein the CDR-H1 is selected from any of the sequences provided in Sequence Listing 1 herein, including any of the sequences SEQ ID NO: 4, 14, 23, 28, 35, 39, 43, 46, 48, 64, 78, 84, 88, 94, 98, 109, 112, 115, 118, 120, 122, 125, 127, 130 or 137; and (b) CDR-L1, wherein the CDR-L1 is selected from any of the sequences provided in Sequence Listing 1 herein, including any of the sequences SEQ ID NO: 1, 12, 19, 27, 41, 53, 61 or 69, wherein the selected CDR-H1 and CDR-L1 are paired according to Sequence Listing 1.

[0132] On one hand, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises: (a) CDR-H2, wherein the CDR-H2 is selected from any of the sequences provided in Sequence Listing 1 herein, including any of the sequences of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131 or 133, and (b) CDR-L2, wherein the CDR-L2 is selected from any of the sequences provided in Sequence Listing 1 herein, including sequences comprising GAS, ATS, SAS or YAS, or sequences of SEQ ID NO: 62, 70, 72, 77 or 87, wherein the selected CDR-H2 and CDR-L2 are paired according to Sequence Listing 1.

[0133] On one hand, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises: (a) CDR-H3, wherein the CDR-H3 is selected from any of the sequences provided in Sequence Listing 1 herein, including any of the sequences of SEQ ID NO: 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128 or 135, and (b) CDR-L3, wherein the CDR-L3 is selected from any of the sequences provided in Sequence Listing 1 herein, including any of the sequences of SEQ ID NO: 3 or 63, wherein the selected CDR-H3 and CDR-L3 are paired according to Sequence Listing 1.

[0134] On one hand, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises: (a) CDR-H1, wherein CDR-H1 is selected from any of the sequences provided in Sequence Listing 1 herein, including any of SEQ ID NO: 4, 14, 23, 28, 35, 39, 43, 46, 48, 64, 78, 84, 88, 94, 98, 109, 112, 115, 118, 120, 122, 125, 127, 130 or 137; CDR-H2, wherein CDR-H2 is selected from any of the sequences provided in Sequence Listing 1 herein, including any of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131 or 133; and CDR-H3, wherein CDR-H3 is selected from any of the sequences provided in Sequence Listing 1 herein, including any of SEQ ID NO: 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128, or 135; and (b) CDR-L1, wherein CDR-L1 is selected from any of the sequences provided in Sequence Listing 1 herein, including any of the sequences SEQ ID NO: 1, 12, 19, 27, 41, 53, 61, or 69; CDR-L2, wherein CDR-L2 is selected from any of the sequences provided in Sequence Listing 1 herein, including sequences containing GAS, ATS, SAS, or YAS, or sequences SEQ ID NO: 62, 70, 72, 77, or 87; and CDR-L3, wherein CDR-L3 is selected from any of the sequences provided in Sequence Listing 1 herein, including sequences containing SEQ ID NO: Either sequence 3 or 63, wherein the selected CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are paired according to sequence list 1.

[0135] Lead antibody and high-priority antibody

[0136] The present disclosure describes antibodies numbered 1-20 and other variant antibodies numbered 21-100 as described herein, in conjunction with Sequence Listing 1. In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof comprising one or more variable regions selected from the group consisting of: (a) a V region comprising the amino acid sequence of antibody 1 as shown in Sequence Listing 1. H (b) V containing the amino acid sequence of antibody 1 as shown in sequence listing 1. L , and (c) their combination.

[0137] On one hand, this disclosure provides an antibody or an antigen-binding fragment thereof comprising at least one, two, three, four, five, or six CDRs selected from the following: (a) CDR-H1, wherein CDR-H1 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 4); (b) CDR-H2, wherein CDR-H2 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 5); (c) CDR-H3, wherein CDR-H3 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 6); (d) CDR-L1, wherein CDR-L1 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 1); (e) CDR-L2, wherein CDR-L2 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (including GAS); and (f) CDR-L3, wherein CDR-L3 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 3).

[0138] On one hand, this disclosure provides an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising at least one, at least two, or all three selected from the following V H CDR sequences: (a) CDR-H1, which comprises the amino acid sequence of lead antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 4); (b) CDR-H2, which comprises the amino acid sequence of lead antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 5); and (c) CDR-H3, which comprises the amino acid sequence of lead antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 6); and (d) V L The V L It contains the amino acid sequence of the lead antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 7).

[0139] On one hand, this disclosure provides an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising at least one, at least two, or all three selected from the following V L CDR sequences: (a) CDR-L1, which comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 1); (b) CDR-L2, which comprises the amino acid sequence (GAS) of antibody 1 as shown in Sequence Listing 1; and (c) CDR-L3, which comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 3); and V HThe V H It contains the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 9).

[0140] On one hand, this disclosure provides an antibody or an antigen-binding fragment thereof comprising CDR:CDR-H3, wherein CDR-H3 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 6); and CDR-L3, wherein CDR-L3 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 3).

[0141] On the one hand, the disclosure herein provides an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising at least one, at least two, or all three selected from the following V L CDR sequences: (a) CDR-L1, wherein CDR-L1 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 1); (b) CDR-L2, wherein CDR-L2 comprises the amino acid sequence (GAS) of antibody 1 as shown in Sequence Listing 1; and (c) CDR-L3, wherein CDR-L3 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 3). In one aspect, this disclosure provides an antibody or an antigen-binding fragment thereof comprising at least one, at least two, or all three V molecules selected from the following: H CDR sequences: (a) CDR-H1, which comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 4); (b) CDR-H2, which comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 5); and (c) CDR-H3, which comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 6).

[0142] On one hand, this disclosure provides an antibody or an antigen-binding fragment thereof comprising a CDR: (a) CDR-H1, wherein the CDR-H1 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 4); (b) CDR-H2, wherein the CDR-H2 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 5); (c) CDR-H3, wherein the CDR-H3 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 6); (d) CDR-L1, wherein the CDR-L1 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 1); (e) CDR-L2, wherein the CDR-L2 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (GAS); and (f) CDR-L3, wherein the CDR-L3 comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 3).

[0143] On the one hand, the antibody or its antigen-binding fragment contains a V that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (including SEQ ID NO: 9). H Sequence. In some embodiments, V has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to a reference sequence. H The sequence contains substitutions (e.g., conserved substitutions), insertions, or deletions, but the antibody or its antigen-binding fragment containing said sequence retains its ability to bind to the antigen. In some embodiments, a total of 1 to 10 amino acids in the amino acid sequence of antibody 1 (including SEQ ID NO: 9) as shown in Sequence Listing 1 have been substituted, inserted, and / or deleted. In some embodiments, the substitution, insertion, or deletion occurs in regions outside the CDR (e.g., in the FR). Optionally, the antibody or its antigen-binding fragment contains the V of the amino acid sequence of antibody 1 (including SEQ ID NO: 9) as shown in Sequence Listing 1. HThe sequence, including post-translational modifications of the sequence. In one specific embodiment, VH comprises one, two, or three CDRs selected from the following: (a) CDR-H1, which comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 4); (b) CDR-H2, which comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 5); and (c) CDR-H3, which comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (SEQ ID NO: 6).

[0144] On one hand, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain (V) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of the lead antibody 1 as shown in Sequence Listing 1 (including SEQ ID NO: 7). L In some embodiments, V having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to a reference sequence. L The sequence contains substitutions (e.g., conserved substitutions), insertions, or deletions, but the antibody or its antigen-binding fragment containing said sequence retains its ability to bind to the antigen. In some embodiments, a total of 1 to 10 amino acids in any of the amino acid sequences of antibody 1 as shown in Sequence Listing 1 (including SEQ ID NO: 7) have been substituted, inserted, and / or deleted. In some embodiments, the substitution, insertion, or deletion occurs in regions outside the CDR (e.g., in the FR). Optionally, the antibody or its antigen-binding fragment contains the V sequence of antibody 1 as shown in Sequence Listing 1. L The sequence (including SEQ ID NO: 7), including post-translational modifications of the sequence. In one specific embodiment, V L The CDR comprises one, two, or three selected from the following: (a) CDR-L1, which comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (including SEQ ID NO: 1); (b) CDR-L2, which comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (including the sequence containing GAS); and CDR-L3, which comprises the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (including SEQ ID NO: 3).

[0145] On one hand, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises V as in any of the embodiments provided above. HAnd V as in any of the embodiments provided above L In some embodiments, the antibody comprises V H The V H The antibody contains the amino acid sequence of antibody 1 as shown in Sequence Listing 1 (including SEQ ID NO: 9); and V in antibody 1 as shown in Sequence Listing 1. L Sequences (including SEQ ID NO: 7), including post-translational modifications of these sequences.

[0146] In other embodiments, the above aspects were repeated and contemplated for the lead antibody numbers 3, 4, 5, 6, 7, 8, 9, 15 and 20 as described herein and provided in Sequence Listing 1.

[0147] The antibodies or antigen-binding fragments thereof disclosed herein may comprise a CDR3 region of at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. The antibodies or antigen-binding fragments thereof disclosed herein may comprise a CDR3 region of at least about 18 amino acids in length.

[0148] In some embodiments, the antibodies provided herein have a concentration of about 1 μM, 100 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM or less (e.g., 10 μM). −8 M or smaller, such as 10 −8 M to 10 −13 M, for example, 10 −9 M to 10 −13 The dissociation constant (K) of M) D Another aspect of this disclosure provides an antibody or antigen-binding fragment thereof with increased affinity for its target, such as an affinity-matured antibody. An affinity-matured antibody is an antibody that has one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody without such alterations, such alterations resulting in improved affinity of the antibody for the antigen. These antibodies can be expressed at approximately 5 × 10⁻⁶. −9 M, 2×10 −9 M, 1×10 −9 M, 5×10 −10 M, 2×10 −9 M, 1×10 −10 M, 5×10 −11 M, 1×10 −11 M, 5×10 −12 M, 1×10−12 M or smaller K D Binding to antigens. In some embodiments, this disclosure provides an antibody or antigen-binding fragment thereof that has an increased affinity of at least 1.5, 2, 2.5, 3, 4, 5, 10, 20, or more times compared to germline antibodies containing heavy chain sequences and light chain sequences or both. In other embodiments, antibodies are provided that compete with antibodies as described herein for binding to the same epitope. In some embodiments, antibodies or antigen-binding fragments thereof that bind to the same epitope and / or compete with antibodies for binding to the same epitope exhibit effector functional activities, such as Fc-mediated cytotoxicity, including ADCC activity.

[0149] Methods for generating anti-TL1A antibodies

[0150] Antibody synthesis and purification

[0151] Starting with computer-simulated nucleic acid sequences, antibody peptides can be synthesized and purified using conventional procedures. In one embodiment, an artificial genetic construct encoding an antibody or an antibody fragment thereof is synthesized (see, for example, Khorana, HG et al., Journal of Molecular Biology 72(2):209-217 (1972); Itakura, K. et al., Science 198(4321):1056-1063 (1977); and Edge, MD et al., Nature 292(5825):756-762 (1981)). The DNA template of the synthetic gene construct can then be cloned into a suitable expression vector and operatively linked with a regulatory control sequence, transformed into a suitable host for amplification, and the resulting amplified expression vector can be purified and transfected into a suitable host for transient expression of the final polypeptide encoding an antibody or an antibody fragment thereof (see, for example, Vazquez-Lombardi, R. et al., Nat. Protoc. 13(1):99-117 (2018)).

[0152] Using the information provided herein, such as the nucleic acid and amino acid sequences of antibodies, nucleic acids encoding antibodies or antigen-binding fragments thereof can be obtained. Such nucleic acids can be obtained, for example, using conventional methods disclosed in the art. The nucleic acids disclosed herein can be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning, or synthesized, or any combination thereof. DNA can be triple-stranded, double-stranded, single-stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA can be a coding strand (also referred to as a sense strand), or it can be an antisense strand (also referred to as an antisense strand).

[0153] As used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a nucleotide polymer polynucleotide of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase. Nucleic acids may contain modified nucleotides, such as methylated nucleotides and their analogs. Modifications to the nucleotide structure may be conferred before or after polymer assembly, if present. Nucleotide sequences may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with labeled components. Other types of modifications include, for example, "caps," substitutions for one or more naturally occurring nucleotides with analogues; internucleotide modifications, such as those with uncharged bonds (e.g., methylphosphonates, triphosphates, phosphoramides, carbamates, etc.) and those with charged bonds (e.g., thiophosphates, dithiophosphates, etc.); internucleotide modifications containing side groups, such as those containing proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); internucleotide modifications containing intercalating agents (e.g., acridine, psoralen, etc.); internucleotide modifications containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.); internucleotide modifications containing alkylating agents; internucleotide modifications containing modified bonds (e.g., α-anomeric nucleic acids, etc.); and unmodified forms of polynucleotides. Furthermore, any hydroxyl group typically present in sugars can be replaced, for example, by a phosphonate group or a phosphate group, protected by a standard protecting group, activated to prepare additional linkages with other nucleotides, or conjugated to a solid support. The 5′ and 3′ terminal OH groups can be phosphorylated or substituted via amine or organic end-capping with 1 to 20 carbon atoms. Other hydroxyl groups can also be derived as standard protecting groups. Polynucleotides can also contain similar forms of ribose or deoxyribose known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars (such as arabinose, xylose), piperanose, furanose, sedoheptulose, acyclic analogs, and non-basic nucleoside analogs (such as methylriboside). One or more phosphate diesters can be replaced with alternative linking groups.These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S (“thioide”), P(S)S (“dithioide”), (O)NR2 (“amid”), P(O)R, P(O)OR', CO, or CH2 (“methylacetal”), wherein each R or R' is independently H or optionally contains an ether (-O-) bond, an aryl, alkenyl, cycloalkyl, cycloalkenyl, or alkyl substituted or unsubstituted alkyl group (1-20 Cs). Not all links in a polynucleotide need to be identical. The foregoing description applies to all polynucleotides mentioned herein, including isolated nucleic acids, RNA, and DNA.

[0154] In the context of this disclosure, the following abbreviations for common nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine. In some embodiments, the nucleic acid molecule comprises isolated nucleic acids.

[0155] Nucleic acids can be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. When purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) using standard techniques, said standard techniques include, but are not limited to, alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. (F. Ausubel et al., eds. (1987) *Current Protocols in Molecular Biology*, Greene Publishing and Wiley Interscience, New York). According to at least some embodiments of this disclosure, nucleic acids can be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0156] Artificial gene synthesis

[0157] Various standard recombinant DNA techniques can be used to manipulate structural domains or functional segments within antibody nucleic acid sequences. Once the encoding V is obtained... H and V L These DNA fragments can be further manipulated, for example, converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the gene encoding V... L or V HA DNA fragment is operatively linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operatively linked" is intended to mean joining two DNA fragments such that the amino acid sequences encoded by the two DNA fragments remain within the frame. This is achieved by encoding V... H DNA and coding heavy chain constant regions (C H 1. C H 2 and C H 3) Another DNA molecule can be operatively linked to encode V H The isolated DNA from the region is converted into the full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, EA et al. (1991), *Protein Sequences of Immunological Significance*, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. Heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, but are most preferably IgG1 or IgG4 constant regions. For the Fab fragment heavy chain gene, encoding V... H DNA can interact with only the C-chain heavy chain. H Another DNA molecule in the constant region can be operatively linked.

[0158] By encoding V L DNA has a constant region C encoding the light chain. L Another DNA molecule can be operatively linked to encode V. L The isolated DNA from the regions is converted into full-length light chain genes (and Fab light chain genes). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat, EA et al. (1991), *Protein Sequences of Immunological Significance*, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments covering these regions can be obtained by standard PCR amplification. Light chain constant regions can be κ or λ constant regions, but κ constant regions are most preferred.

[0159] In order to produce the scFv gene, the gene encoding V will be... H and V L The DNA fragment is operatively linked to another fragment encoding a flexible linker (e.g., encoding the amino acid sequence (Gly-Gly-Gly-Gly-Ser)3), enabling V to... H Sequence and V L The sequence can be expressed as a continuous single-chain protein, where V L District and V HThe area is joined by a flexible joint (see, for example, Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proceedings of the National Academy of Sciences 85:5879-5883; McCafferty et al. (1990) Nature 348:552-554).

[0160] Host cell selection and transformation

[0161] On the one hand, this article provides a host cell containing the isolated nucleic acid described above or a vector containing the isolated nucleic acid. The vector may be a cloning vector or an expression vector. Suitable host cells for cloning or expressing the DNA in the vector described above are prokaryotic cells, yeast cells, or higher eukaryotic cells. Suitable prokaryotic cells for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, including Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens), and Shigella, and Bacilli, including Bacilli subtilis and Bacilli licheniformis (e.g., DD, published April 12, 1989). Bacillus licheniformis 41 P, Pseudomonas species such as P. aeruginosa, and Streptomyces are disclosed in 266,710. A preferred Escherichia coli cloning host is Escherichia coli 294 (ATCC 31,446), but other strains such as Escherichia coli B, Escherichia coli Xl 776 (ATCC 31,537), and Escherichia coli W3110 (ATCC 27,325) are suitable. These examples are illustrative and not limiting. Further embodiments are described below.

[0162] purification

[0163] On the one hand, this document discloses purified antibodies or antigen-binding fragments as provided herein. Once expressed, the complete antibodies, their dimers, single light and heavy chains, or other immunoglobulin forms disclosed herein can be recovered and purified using known techniques, such as immunoadsorption or immunoaffinity chromatography, chromatography such as HPLC (high performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination of these methods. See typically Scopes, *Protein Purification* (Springer-Verlag, NY, 1982).

[0164] Specifically, for pharmaceutical use, substantially pure immunoglobulins with at least about 90% to 95% homogeneity are advantageous, as are those with 98% to 99% or higher homogeneity. When using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium (including from microbial cultures). If antibodies are produced intracellularly, then as a first step, particulate debris from host cells or lysed fragments can be removed, for example, by centrifugation or ultrafiltration. Better et al. in *Science* 240: 1041–1043 (1988); *ICSU Short Reports* 10: 105 (1990); and *Proceedings of the National Academy of Sciences* 90: 457–461 (1993) describe procedures for isolating antibodies secreted into the periplasmic space of *E. coli*. (See also Carter et al., Bio / Technology 10: 163-167 (1992)).

[0165] Antibody compositions prepared from microbial or mammalian cells can be purified using, for example, hydroxyapatite chromatography, cation or anion exchange chromatography, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and any isotype of the immunoglobulin Fe domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., *Journal of Immunological Methods* 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., *Journal of the European Society for Molecular Biology* 5: 1567-1575 (1986)). Agarose is the most common matrix for affinity ligand attachment, but other matrices are also available. Compared to the flow rates and processing times achievable with agarose, mechanically stable matrices such as controlled-porosity glass or poly(divinyl)styrene can achieve faster flow rates and shorter processing times. In antibodies containing C...H In the case of a 3-domain antibody, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) can be used for purification. Depending on the antibody to be recovered, other techniques for protein purification are also available, such as fractionation on an ion-exchange column, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation. Once humanized or recombinant human antibodies are partially purified or purified to homogeneity as needed, they can then be used for therapeutic purposes or for developing and performing assays, immunofluorescence staining, etc. See generally Volumes I and II, *Immunol. Meth* (edited by Lefkovits and Pernis, Acad. Press, NY, 1979 and 1981).

[0166] Antibody composition and structural conformation

[0167] In the various embodiments of this disclosure, the resulting antibody peptides may present a range of compositions or structural conformations. These include, herein, bispecific antibodies, multispecific antibodies, multivalent antibodies, chimeric antibodies, human antibodies, humanized antibodies, monoclonal antibodies, deimmunizing antibodies, or combinations thereof.

[0168] The various variants described herein are known in the art and are common knowledge to those skilled in the art in producing anti-TL1A antibodies and / or antigen-binding fragments thereof comprising one or more of these variants.

[0169] Bispecific antibodies and multispecific antibodies

[0170] In some embodiments, it may be desirable to generate multispecific (e.g., bispecific) monoclonal antibodies, including monoclonal antibodies, human antibodies, humanized antibodies, or variant antibodies that have binding specificity to at least two different epitopes. In some embodiments, the antibodies disclosed herein are multispecific. Exemplary bispecific antibodies may bind to two different epitopes of an antigen (e.g., a SARS-CoV-2-associated antigen). Alternatively, the antigen-binding region may be combined with a region that binds to triggering molecules on leukocytes to focus cellular defense mechanisms on cells expressing the antigen, such as T-cell receptor molecules (e.g., CD2 or CD3) or IgG Fe receptors (FcγRs) such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). Bispecific antibodies may be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0171] According to another method for preparing bispecific antibodies, the interface between antibody molecule pairs can be engineered to maximize the percentage of heterodimers recovered from recombinant cell cultures. Preferred interfaces include the C0 of the antibody constant domain. H At least a portion of the 3-domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensating "cavity" of the same or comparable size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism for improving the yield of heterodimers compared to undesirable end products such as isodimers.

[0172] Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, in a heteroconjugation, one antibody may be conjugated to avidin, and the other to biotin. Heteroconjugated antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents and numerous cross-linking techniques have been considered.

[0173] Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical bonds. Brennan et al., *Science* 229: 81 (1985) described a procedure in which intact antibody proteins are hydrolyzed to produce F(ab')2 fragments. These fragments are reduced in the presence of a dithiol complexing agent, sodium arsenite, to stabilize the ortho-dithiol and prevent the formation of intermolecular disulfides. The resulting Fab' fragment is then converted to a thionitrobenzene (TNB) derivative. One of the Fab'-TNB derivatives is then converted back to Fab' thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The resulting bispecific antibody can be used as a reagent for the selective immobilization of enzymes. In yet another embodiment, Fab'-SH fragments recovered directly from *E. coli* can be chemically conjugated in vitro to form bispecific antibodies. (Shalaby et al., Journal of Experimental Medicine 175:217-225 (1992)).

[0174] Exemplary techniques for preparing multispecific antibodies include recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities, engineered electrostatic redirection effects for preparing antibody Fc-heterodimeric molecules, crosslinking two or more antibodies or fragments, generating bispecific antibodies using leucine zippers, preparing bispecific antibody fragments using "dual antibody" techniques, using single-chain Fv (sFv) dimers, preparing trispecific antibodies, and "knob-in-hole" engineering (see, for example, Milstein and Cuello, Nature 305: 537 (1983); Traunecker et al., Journal of the European Society for Molecular Biology 10: 3655 (1991); U.S. Patents 4,676,980 and 5,731,168; Brennan et al., Science, 229: 81 (1985); Kostelny et al., Journal of Immunology 148(5):1547-1553). (1992); Hollinger et al., Proceedings of the National Academy of Sciences 90:6444-48 (1993); Gruber et al., Journal of Immunology 152:5368 (1994); and Tutt et al., Journal of Immunology 147:60 (1991). Engineered antibodies with three or more functional antigen-binding sites have also been envisioned.

[0175] chimeric antibodies

[0176] In some embodiments, the antibodies provided herein are chimeric. A chimeric antibody is an antibody in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species. In one instance, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In another instance, a chimeric antibody is a “class-switching” antibody, in which the class or subclass has been changed from that of the parent antibody. A chimeric antibody includes its antigen-binding fragment. For more information, see, for example, Jones et al., Nature 321: 522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988); Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992); and Morrison et al., Proceedings of the National Academy of Sciences 81: 6851-6855 (1984).

[0177] Human antibodies

[0178] In some embodiments, the antibodies described herein are human antibodies. Human antibodies can be generated using various techniques known in the art (see, for example, van Dijk and van de Winkel, *Curr. Opin. Pharmacol.* 5: 368-74 (2001); and Lonberg, *Curr. Opin. Immunol.* 20:450-459 (2008)). A human antibody is a human antibody having an amino acid sequence corresponding to that of an antibody of non-human origin produced by humans or human cells or derived from a human antibody reservoir or other human antibody coding sequence. This definition of a human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be prepared by administering an immunogen (e.g., TL1A) to a transgenic animal that has been modified to produce a complete human antibody in response to antigen stimulation or a complete antibody with a human variable region. (See, for example, Lonberg, Nature Biotechnology 23:1117-1125 (2005)). The human variable region of a complete antibody produced from such animals can be further modified, for example, by combining it with different human constant regions.

[0179] In one embodiment, the antibody described herein is a fully human antibody.

[0180] Human antibodies can also be prepared using hybridoma-based methods. For example, human antibodies can be generated from human myeloma and mouse-human heterologous myeloma cell lines using human B-cell hybridoma techniques and other methods (see, for example, Kozbor, *Journal of Immunology* 133: 3001 (1984); Brodeur et al., *Monoclonal Antibody Production Techniques and Applications*, pp. 51-63 (1987); Boerner et al., *Journal of Immunology* 147: 86 (1991); Li et al., *Proceedings of the National Academy of Sciences* 103:3557-3562 (2006); Ni, *Xiandai Mianyixue*, 26(4):265-268 (2006); Vollmers and Brandlein, *Histology and Histopathology*). 20(3):927-937 (2005); and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology 27(3):185-91 (2005). Human antibodies can also be generated by isolating variable domain sequences of Fv clones selected from human phage display libraries. Such variable domain sequences can then be combined with desired human constant domains.

[0181] Recombinant human antibodies

[0182] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated, or isolated in a recombinant manner, such as (a) antibodies isolated from transgenic or transchromosomally modified animals (e.g., mice) or hybridomas prepared therefrom that are transgenic with respect to the human immunoglobulin gene; (b) antibodies isolated from host cells transformed to express human antibodies, such as from transfected tumors; (c) antibodies isolated from recombinant combined human antibody libraries; and (d) antibodies prepared, expressed, generated, or isolated by any other means involving splicing the human immunoglobulin gene sequence into other DNA sequences. Such recombinant human antibodies have variable regions, in which the frame region and CDR region are derived from the immunoglobulin sequences disclosed herein. However, in some embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when using animals transgenic with respect to the human Ig sequence, in vivo somatic cell mutagenesis), and therefore the V of the recombinant antibody... H District and V LThe amino acid sequence of the region is derived from human immunoglobulin V. H Sequence and V L Sequences that are related to the sequence but may not be naturally present in human antibody germline libraries in vivo.

[0183] Humanized antibodies

[0184] In some embodiments, the antibodies provided herein are humanized antibodies. In one embodiment, a humanized antibody is an antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, the humanized antibody will comprise at least one, and typically substantially all, of the two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of the non-human antibody, and all or substantially all of the FRs correspond to those of the human antibody. Optionally, the humanized antibody may comprise at least a portion of the antibody constant region derived from the human antibody. See, for example, Almagro and Fransson, *Fronts in Bioscience* 13:1619-1633 (2008); Riechmann et al., *Nature* 332:323-329 (1988); Queen et al., *Proceedings of the National Academy of Sciences* 86:10029-10033 (1989); Kashmiri et al., *Methods* 36:25-34 (2005); Padlan, *Molecular Immunol* 28:489-498 (1991); Dall'Acqua et al., *Methods* 36:43-60 (2005); Osbourn et al., *Methods* 36:61-68 (2005); and Klimka et al., *British Journal of Cancer*. 83:252-260 (2000).

[0185] Non-human antibodies can be humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Humanized antibodies may contain one or more variable domains comprising one or more CDRs or portions thereof derived from a non-human antibody. Humanized antibodies may contain one or more variable domains comprising one or more FRs or portions thereof derived from a human antibody sequence. Humanized antibodies may optionally contain at least a portion of a human constant region. In some embodiments, one or more FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0186] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using the “best fit” method; frame regions derived from sequences of human antibodies derived from specific subgroups of light chain variable regions or heavy chain variable regions; human mature (somatic mutant) frame regions or human germline frame regions; and frame regions derived from screening FR libraries (see, for example, Sims et al., Journal of Immunology 151:2296 (1993); Carter et al., Proceedings of the National Academy of Sciences 89:4285 (1992); Presta et al., Journal of Immunology 151:2623 (1993); Baca et al., Journal of Biochemistry 272:10678-10684 (1997); and Rosok et al., Journal of Biochemistry 271:22611-22618 (1996)).

[0187] Monoclonal antibodies

[0188] Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, meaning that individual antibodies comprising said population are identical, except for the possible presence of naturally occurring mutations in small quantities. In some embodiments, the antibodies of this disclosure are monoclonal. In a preferred embodiment, the monoclonal antibody can be prepared using a recombinant DNA method, or in alternative embodiments, it can be prepared using a hybridoma method first described by Kohler et al., Nature, 256:495 (1975).

[0189] Deimmunizing antibodies

[0190] The immunogenicity of the antibodies or antigen-binding fragments described herein may be optionally assessed, and deimmunization (i.e., reducing the immunoreactivity of the antibody by altering one or more T-cell epitopes) may be performed as needed. As used herein, “deimmunized antibody” means that one or more T-cell epitopes in the antibody sequence have been modified such that, upon administration of the antibody to a subject, a reduced T-cell response is observed compared to an antibody that has not been deimmunized but retains its binding activity. Analysis of the immunogenicity and T-cell epitopes present in the antibodies and antigen-binding fragments described herein can be performed using software and specific databases known in the art. Exemplary software and databases include iTope™, developed by Antitope of Cambridge, England. iTope™ is a computer simulation technology for analyzing the binding of peptides to human MHC class II alleles. iTope™ software predicts the binding of peptides to human MHC class II alleles and thereby provides an initial screening of the location of such “potential T-cell epitopes.” iTope™ software predicts favorable interactions between the amino acid side chains of the peptide and specific binding pockets within the binding grooves of 34 human MHC class II alleles. The location of key binding residues was determined through computer simulation to generate a 9-meric peptide that overlaps by one amino acid across the variable region sequence of the test antibody. Each 9-meric peptide can be tested against each of the 34 MHC class II allotypes and scored based on its potential “match” and interaction with the MHC class II binding groove. Peptides that produce a high average binding score (>0.55 in the iTope™ scoring function) against >50% of MHC class II alleles are considered potential T-cell epitopes. Within these regions, the core 9-amino acid sequence binding to the peptide within the MHC class II groove is analyzed to identify MHC class II pocket residues (P1, P4, P6, P7, and P9) and potential T-cell receptor (TCR) contact residues (P1, P2, P3, P5, and P8). After identifying any T-cell epitope, amino acid residue changes, substitutions, additions, and / or deletions can be introduced to remove the identified T-cell epitope. Such changes can be made to maintain antibody structure and function while still removing the identified epitope. Exemplary changes may include, but are not limited to, conservative amino acid changes.

[0191] Engineered antibodies and modified antibodies

[0192] Using the V disclosed herein, which has a fragment derived from an antibody or its antigen-binding fragment. H Sequence and / or V LAntibodies can be engineered using one or more antibodies from the sequence as starting materials to further prepare antibodies according to at least some embodiments of this disclosure, wherein the modified antibodies may have properties altered relative to the starting antibody. This document provides the V of the antibodies disclosed herein. H Chain and V L The complete amino acid and nucleic acid sequences of the chain region are provided in this article. The V of the antibody described herein is also provided. H and V L The amino acid and nucleic acid sequence of the CDR3 region. This can be achieved by modifying one or both variable regions (i.e., V...). H and / or V L Antibodies can be engineered by modifying one or more residues within a region (e.g., within one or more CDR regions and / or one or more frame regions). Alternatively or concurrently, antibodies can be engineered by modifying residues within a constant region, for example, to alter the effector function of the antibody.

[0193] One type of variable region engineering that can be performed is CDR transplantation. Antibodies primarily interact with target antigens through amino acid residues located in the six complementarity-determining regions (CDRs) of the heavy and light chains. For this reason, the amino acid sequences within the CDRs are more diverse among individual antibodies than those outside the CDRs. Because the CDR sequence dominates most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific antibodies by constructing expression vectors comprising a CDR sequence from a specific antibody (e.g., the antibody disclosed herein) that has been grafted onto a frame sequence from a different antibody with different properties (see, for example, Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. et al. (1989) Proceedings of the National Academy of Sciences 86:10029-10033; U.S. Patent No. 5,225,539 to Winter and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370 to Queen et al.).

[0194] Suitable framework sequences can be obtained from public DNA databases that include germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in: the “VBase” human germline sequence database (available on the Internet), and Kabat, EA et al. (1991), “Protein Sequences of Immunological Significance,” 5th edition, U.S. Department of Health and Human Services, NIH Publication No.: 91-3242; Tomlinson, IM et al. (1992), “Human Germline V…”. H The sequence library revealed approximately fifty groups of V with different hypercyclic rings. H Section (The Repertoire of Human Germline V) H Sequences Reveals about Fifty Groups of V H "Segments with Different Hypervariable Loops" (Journal of Molecular Biology 227:776-798); and Cox, JPL et al. (1994) "Segments with Different Hypervariable Loops" H The section's directory reveals a strong bias in its use (A Directory of Human Germ-line V) H (Segments Reveals a Strong Bias in their Usage) "Eur. J. Immunol. 24:827-836; the contents of each of the references are explicitly incorporated herein by reference.

[0195] Another type of variable region modification is to make V H and / or V L Amino acid residue mutations within the CDR 1, CDR 2, and / or CDR 3 regions can be performed to improve one or more binding properties (e.g., affinity) of the antibody of interest. Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and their effect on antibody binding or other functional properties of interest can be evaluated in appropriate in vitro or in vivo assays. Preferably, conserved modifications (as discussed above) are introduced. Mutations can be amino acid substitutions, additions, or deletions, but substitution is preferred. Furthermore, typically no more than one, two, three, four, or five residues within the CDR regions are altered.

[0196] Engineered antibodies according to at least some embodiments of this disclosure include antibodies against V. H and / or V LAntibodies are created by modifying framework residues within the antibody, for example, to improve antibody properties. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "reverse mutation" one or more framework residues into the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutations may contain framework residues different from the germline sequence from which the antibody originated. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody originated.

[0197] In addition to modifications performed within the frame or CDR region, or alternatively within the frame or CDR region, antibodies according to at least some embodiments of this disclosure may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity. Furthermore, antibodies according to at least some embodiments of this disclosure may be chemically modified (e.g., one or more chemical moieties may be linked to the antibody) or modified to alter their glycosylation to again alter one or more functional properties of the antibody. Such embodiments have been described above. The residues in the Fc region are numbered according to the EU index of Kabat.

[0198] In one embodiment, for C H The hinge region of 1 is modified to alter the number of cysteine ​​residues in the hinge region, for example, by increasing or decreasing them. This method is further described in U.S. Patent No. 5,677,425 to Bodmer et al. Regarding C... H The number of cysteine ​​residues in the hinge region of the antibody can be altered to, for example, promote the assembly of the light and heavy chains or to increase or decrease antibody stability. In another embodiment, the Fc hinge region of the antibody is mutated to reduce the antibody's biological half-life. More specifically, one or more amino acid mutations are introduced into the C13C ... H 2-C H In the 3-domain interface region, the antibody exhibits impaired SpA binding relative to the natural Fc-hinge domain staphylococcal protein A (SpA). This method is further described in detail in U.S. Patent No. 6,165,745 to Ward et al.

[0199] In another embodiment, the antibody is modified to increase its biological half-life. Various methods are possible. For example, to increase the biological half-life, it can be modified at C... H 1 or C L The antibody is modified within the region to contain C from the Fc region of IgG. H The two loops of the 2-domain salvage receptor binding epitopes are as described in U.S. Patent Nos. 5,869,046 and 6,121,022 to Presta et al.

[0200] On the other hand, this article provides variants of antibodies or their antigen-binding fragments.

[0201] Substitution variants, insertion variants, and deletion variants

[0202] In some embodiments, amino acid sequence variants of the antibodies provided herein are considered. Variants typically differ from the polypeptides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or synthetically generated, for example by modifying one or more polypeptide sequences of the above-described polypeptide sequences of this disclosure, and evaluating one or more biological activities of the polypeptide as described herein, and / or using any of a variety of techniques well known in the art. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to achieve a final construct, provided that the final construct possesses the desired properties, such as antigen binding.

[0203] In some embodiments, antibody variants or antigen-binding fragments thereof with one or more amino acid substitutions are provided. Sites of interest for mutagenesis via substitution include CDR and FR. Amino acid substitutions can be introduced into the antibody of interest, and the desired activity of the product can be screened, such as retained / improved antigen binding, reduced immunogenicity, and / or improved ADCC or CDC function.

[0204]

[0205] Hydrophobic amino acids include: leucine, Met, Ala, Val, Leu, and Ile. Neutral hydrophilic amino acids include: Cys, Ser, Thr, Asn, and Gln. Acidic amino acids include Asp and Glu. Basic amino acids include: His, Lys, and Arg. Amino acids with residues that affect chain orientation include: Gly and Pro. Aromatic amino acids include: Trp, Tyr, and Phe.

[0206] In some embodiments, substitution, insertion, or deletion can occur within one or more CDRs, wherein the substitution, insertion, or deletion does not significantly reduce antibody-antigen binding. For example, conserved substitutions that substantially do not reduce binding affinity can be made within a CDR. Such changes can occur outside of CDR “hotspots” or SDRs. In variant V H Sequence and V L In some embodiments of the sequence, each CDR remains unchanged or contains no more than one, two, or three amino acid substitutions.

[0207] Modifications (e.g., substitutions) can be made in the CDR to improve antibody affinity, for example. Such modifications can be made in CDR-encoding codons, which have a high mutation rate during somatic maturation (see, for example, Chowdhury, *Methods in Molecular Biology* 207:179-196 (2008)), and the binding affinity of the resulting variants can be tested. Affinity maturation (e.g., using error-prone PCR, strand shuffling, CDR randomization, or oligonucleotide directed mutagenesis) can be used to improve antibody affinity (see, for example, Hoogenboom et al., *Methods in Molecular Biology* 178:1-37 (2001)). The CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling (see, for example, Cunningham and Wells, *Science* 244:1081-1085 (1989)). Specifically, CDR-H3 and CDR-L3 are often targeted. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be used to identify the contact sites between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine if they contain the desired properties.

[0208] Amino acid sequence insertions and deletions include amino and / or carboxyl terminus fusions of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions and deletions of single or multiple amino acid residues. Examples of terminus insertions include antibodies having an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusions with peptides that increase the serum half-life of the antibody, for example, at the N-terminus or C-terminus. The term "tagged epitope" refers to an antibody fused with an epitope tag. The epitope-tagged peptide has sufficient residues to provide an epitope against which an antibody can be prepared, but the epitope-tagged peptide is short enough that it does not interfere with the activity of the antibody. The epitope tag is preferably unique enough that the antibody against it does not substantially cross-react with other epitopes. Suitable tagged peptides typically have at least 6 amino acid residues and generally have about 8 to 50 amino acid residues (preferably about 9 to 30 residues). Examples include influenza HA-tagged peptides and their antibody 12CA5 (Field et al., *Molecular and Cellular Biology* 8:2159-2165 (1988)); c-myc tags and their 8F9, 3C7, 6E10, G4, B7, and 9E10 antibodies (Evan et al., *Molecular and Cellular Biology* 5(12):3610-16 (1985)); and the herpes simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., *Protein Engineering* 3(6):547-553 (1990)). Other exemplary tags are polyhistidine sequences, typically about six histidine residues, which allow the separation of such labeled compounds using nickel chelation. This disclosure also covers other tags and labels well known and conventionally used in the art, such as FLAG. ® Tag (Eastman Kodak, Rochester, NY).

[0209] Other insertion variants of antibody molecules include the fusion of the N-terminus or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody. Examples of intra-sequence insertion variants of antibody molecules include the insertion of 3 amino acids in the light chain. Examples of terminal deletions include antibodies with 7 or fewer amino acids missing from the end of the light chain.

[0210] Glycosylation variants

[0211] In some embodiments, antibodies are modified to increase or decrease their glycosylation (e.g., by altering the amino acid sequence to create or remove one or more glycosylation sites). The carbohydrates linked to the antibody's Fc region can be modified. Natural antibodies derived from mammalian cells typically contain C-type glycosylation sites linked to the Fc region via N-bonds. H 2. Asn297-linked branched dianthin oligosaccharides (see, for example, Wright et al., Trends in Biotechnology (TIBTECH) 15:26-32 (1997)). Oligosaccharides can be various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, and fucose linked to GlcNAc in the “stem” of the dianthin oligosaccharide structure. For example, oligosaccharides in antibodies can be modified to produce antibody variants with certain improved properties. Antibody glycosylation variants can possess improved ADCC and / or CDC functions.

[0212] In some embodiments, antibody variants are provided having a carbohydrate structure lacking fucose (directly or indirectly) linked to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the glycan chain at Asn297 relative to the sum of all glycan structures linked to Asn297 (see, for example, WO 08 / 077546). Asn297 refers to the asparagine residue located at approximately position 297 (Eu number of the Fc region residue) in the Fc region; however, due to minor sequence variations in the antibody, Asn297 can also be located approximately 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants can have improved ADCC function (see, for example, Okazaki et al. Journal of Molecular Biology 336:1239-1249 (2004); and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)). Cell lines (e.g., knockout cell lines) and their methods of use can be used to generate defucosylated antibodies, such as Lec13 CHO cells lacking protein fucosylation and α-1,6-fucosyltransferase gene (FUT8) knockout CHO cells (see, for example, Ripka et al., Archives of Biochemistry and Biophysics, 249:533-545 (1986); Yamane-Ohnuki et al., Biotechnology and Bioengineering, 87:614 (2004); Kanda, Y. et al., Biotechnology and Bioengineering, 94(4):680-688 (2006)). Other antibody glycosylation variants have also been envisioned.

[0213] In another embodiment, the glycosylation of the antibody is modified. For example, deglycosylated antibodies (i.e., antibodies lacking glycosylation) can be prepared. Glycosylation can be altered to, for example, increase the antibody's affinity for the antigen. Such carbohydrate modification can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be performed, resulting in the elimination of one or more variable region framework glycosylation sites, thereby eliminating the glycosylation at said sites. Such non-glycosylation can increase the antibody's affinity for the antigen. Such methods are described in further detail in U.S. Patents Nos. 5,714,350 and 6,350,861 to Co et al. Conservative substitution involves replacing one amino acid with another member of its class. Non-conservative substitution involves replacing one of these classes with a member of another class.

[0214] Therefore, the antibodies or antigen-binding fragments thereof in this disclosure can be produced by host cells having one or more of the following: exogenous and / or high endogenous glycosyltransferase activities. Genes with glycosyltransferase activity include β(1,4)-N-acetylglucosamine transferase III (GnTII), α-mannosidase II (ManII), β(1,4)-galactosyltransferase (GalT), β(1,2)-N-acetylglucosamine transferase I (GnTI), and β(1,2)-N-acetylglucosamine transferase II (GnTII). Glycosyltransferases can comprise fusions including Golgi positioning domains (see, for example, Lifely et al., Glycobiology 318:813-22 (1995); Schachter, Biochem. Cell Biol. 64:163-81 (1986)). In some embodiments, the antibody can be expressed in a host cell containing a disrupted or inactivated glycosyltransferase gene. Therefore, in some embodiments, this disclosure relates to a host cell comprising (a) an isolated nucleic acid containing a sequence encoding a polypeptide having glycosyltransferase activity; and (b) an isolated polynucleotide encoding an antibody of this disclosure or an antigen-binding fragment thereof. In one specific embodiment, the modified antibody produced by the host cell has an IgG constant region comprising an Fc region or a fragment thereof. In another embodiment, the antibody is a humanized antibody comprising an Fc region or a fragment thereof.

[0215] Antibodies with altered glycosylation produced by host cells according to this disclosure can exhibit increased Fc receptor binding affinity (e.g., increased binding to Fcγ-activating receptors such as FcγRIIIa receptors) and / or increased effector function. Increased effector function can be an increase in one or more of the following: increased antibody-dependent cytotoxicity, increased antibody-dependent phagocytosis (ADCP), increased cytokine secretion, increased antigen uptake mediated by immune complexes of antigen-presenting cells, increased Fc-mediated cytotoxicity, increased binding to NK cells, increased binding to macrophages, increased binding to polymorphonuclear cells (PMNs), increased binding to monocytes, increased cross-linking of target-bound antibodies, increased apoptosis induced by direct signal transduction, increased dendritic cell maturation, and increased T cell sensitization. Therefore, in one aspect, this disclosure provides glycoforms of antibodies with increased effector function compared to antibodies that have not been glycoengineered. (See, for example, Tang et al., *Journal of Immunology* 179: 2815-2823 (2007)).

[0216] This disclosure also relates to a method for generating an antibody or antigen-binding fragment thereof having modified oligosaccharides as described herein, the method comprising (a) culturing a host cell engineered to express at least one polypeptide encoding a polypeptide having glycosyltransferase activity under conditions allowing for the generation of antibodies according to this disclosure, wherein the polypeptide having glycosyltransferase activity is expressed in an amount sufficient to modify the oligosaccharide in the Fc region of the antibody generated by the host cell; and (b) isolating the antibody. In another embodiment, two polypeptides having glycosyltransferase activity are present. Antibodies or antigen-binding fragments thereof generated by the method of this disclosure may have increased Fc receptor binding affinity and / or increased effector function.

[0217] In some embodiments, the percentage of bipartite N-linked oligosaccharides in the Fc region of the antibody is at least about 10% to about 100% of the total oligosaccharides, specifically at least about 50%, more specifically at least about 60%, at least about 70%, at least about 80%, or at least about 90-95%. In yet another embodiment, the antibody produced by the method of this disclosure has an increased proportion of non-fucosylated oligosaccharides in the Fc region as a result of modification of its oligosaccharides by the method of this disclosure. In some embodiments, the percentage of non-fucosylated oligosaccharides is at least about 20% to about 100%, specifically at least about 50%, at least about 60% to about 70%, and more specifically at least about 75%. The non-fucosylated oligosaccharides may be heterozygous or complex. In yet another embodiment, the antibody or antigen-binding fragment thereof produced by the method of this disclosure has an increased proportion of bipartite oligosaccharides in the Fc region as a result of modification of its oligosaccharides by the method of this disclosure. In some embodiments, the percentage of the dimeric oligosaccharides is at least about 20% to about 100%, specifically at least about 50%, at least about 60% to about 70%, and more specifically at least about 75%.

[0218] In another embodiment, this disclosure relates to antibodies or antigen-binding fragments thereof engineered by the methods of this disclosure to have increased effector functionality and / or increased Fc receptor binding affinity. In some embodiments, the antibody is a complete antibody. In some embodiments, the antibody is an antibody fragment containing an Fc region, or a fusion protein comprising a region equivalent to the Fc region of an immunoglobulin.

[0219] On one hand, this disclosure provides a host cell expression system for generating antibodies or antigen-binding fragments thereof having modified glycosylation patterns as disclosed herein. Specifically, this disclosure provides a host cell system for generating glycoforms of the antibodies or antigen-binding fragments thereof disclosed herein, which have enhanced therapeutic value. Therefore, this disclosure provides a host cell expression system selected or engineered to express polypeptides with glycosyltransferase activity. Any type of cultured cell line (including the cell lines discussed above) can be used as a background to engineer the host cell lines of this disclosure. In some embodiments, CHO cells, BHK cells, NSO cells, SP2 / O cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, other mammalian cells, yeast cells, insect cells or plant cells are used as background cell lines to generate the engineered host cells of this disclosure.

[0220] Host cells containing the coding sequence of the antibody or its antigen-binding fragment of the present disclosure and expressing a biologically active gene product can be identified by at least four general methods: (a) DNA-DNA or DNA-RNA hybridization; (b) presence or absence of “marker” gene function; (c) assessment of transcriptional levels, such as by means of expression of the corresponding mRNA transcript in the host cells; and (d) detection of the gene product, such as by means of immunoassay or by means of its biological activity.

[0221] Cysteine-engineered antibody variants

[0222] In some embodiments, it may be desirable to generate cysteine-engineered antibodies or antigen-binding fragments thereof, such as "thioMAb," wherein one or more residues of the antibody are replaced with cysteine ​​residues. In some embodiments, the substituted residues appear at accessible sites on the antibody. Reactive thiol groups may be positioned at sites that conjugate with other parts, such as pharmaceutical parts or linker-pharmaceutical parts, to generate immunoconjugates. In some embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat number) of the light chain; A118 (EU number) of the heavy chain; and S400 (EU number) of the Fc region of the heavy chain. Cysteine-engineered antibodies can be generated as described.

[0223] Any cysteine ​​residues that do not participate in maintaining the proper conformation of monoclonal antibodies, human antibodies, humanized antibodies, or variant antibodies can be replaced with serine residues to improve the oxidative stability of the molecule and prevent undesirable cross-linking. Conversely, cysteine ​​bonds can be added to antibodies to improve their stability (specifically, when the antibody is an antibody fragment such as an Fv fragment).

[0224] Fc region variants

[0225] Mutations in residues within the Fc receptor binding site can lead to alterations in effector function, such as changes in ADCC, CDC activity, and / or half-life. As described in more detail above, mutations include, for example, the insertion, deletion, and / or substitution of one or more residues, including alanine substitution, conserved substitution, non-conserved substitution, and / or substitution at the same position with a corresponding amino acid residue from a different IgG subclass (e.g., replacing an IgG1 residue with a corresponding IgG2 residue at the same position).

[0226] The Fc region in this article is the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. Fc includes native sequence Fc regions and variant Fc regions. Fc region variants may contain human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) that include amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0227] Previous studies have mapped the binding site of FcγR on human and mouse IgG primarily to the lower hinge region consisting of IgG residues 233-239. Other studies have proposed additional broad regions, such as Gly316-Lys338 of human Fcγ receptor I, Lys274-Arg301 and Tyr407Arg416 of human Fcγ receptor III, or specific residues outside the lower hinge, such as Asn297 and Glu318 of mouse IgG2b interacting with mouse Fcγ receptor II. Reports of the 3.2-A crystal structure of the human IgG Fc fragment with human Fcγ receptor IIIA describe IgG1 residues such as Leu234-Ser239, Asp265-Glu269, Asn297-Thr299 and Ala327-Ile332 involved in binding to Fc receptor γIIIA. Based on the crystal structure, it has been proposed that, in addition to the lower hinge (Leu234-Gly237), IgG C... H Residues in the 2-domain loop FG (residues 326-330) and BC (residues 265-271) may play a role in binding to the Fcγ receptor IIA. See Shields et al., Journal of Biochemistry 276(9):6591-6604 (2001). Shields et al. reported that the IgG1 residues involved in binding to all Fc receptors are located near the hinge in the C domain. H Within the 2 domains, they are divided into two categories: 1) positions that can directly interact with all FcRs, including Leu234-Pro238, Ala327 and Pro329 (and possibly Asp265); 2) positions that affect the properties or positions of carbohydrates, including Asp265 and Asn297.

[0228] In some embodiments, this disclosure contemplates antibody variants having some, but not all, effector functions, making them desirable candidates for applications where in vivo antibody half-life is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the effect of one or more Fc amino acid modifications on CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding capacity.

[0229] Fc variants altered by binding to Fcγ receptors

[0230] In some cases, Fc variants exhibit altered affinity for one or more Fcγ receptors (FcγRs). For example, Fc variants may exhibit increased affinity for one or more Fcγ receptors (FcγRs), decreased affinity for one or more Fcγ receptors (FcγRs), or a combination thereof. In one instance, Fc variants exhibit increased ADCC activity. In yet another instance, the Fc region is modified to increase the ability to induce antibody-dependent cytotoxicity (ADCC). The binding sites of FcγRI (Fc gamma RI), FcγRII (Fc gamma RII), FcγRIII (Fc gamma RIIII), and FcRn on human IgG1 have been mapped, and variants with altered binding have been described. Non-limiting examples of such modifications are described, for example, in U.S. Patent No. 6,737,056; Presta's PCT Publication WO 00 / 42072; Shields, RL et al. (2001) Journal of Biochemistry 276:6591-6604; and U.S. Patent No. 7,332,581. In some embodiments, the constant region of the antibody disclosed herein is replaced by IGHG1.

[0231] Armour et al. (Molecular Immunology, 2003; 40(9):585-93) identified an IgG1 variant that reacted with the activating receptor FcγRIIa at most 1 / 10 the efficiency of wild-type IgG1 with the activating receptor FcγRIIa, but with only a four-fold reduction in binding to the inhibitory receptor FcγRIIb. Mutations were made in the region of amino acids 233-236 and / or at amino acid positions 327, 330, and 331. See also WO 99 / 58572.

[0232] Non-limiting examples of in vitro assays for evaluating the ADCC activity of molecules of interest are described, for example, in U.S. Patent Nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive assays (e.g., ACTI™ and CYTOTOX 96) may be used. ® Non-radioactive cytotoxicity assays). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo (e.g., in animal models) (see, for example, Clynes et al., Proceedings of the National Academy of Sciences 95:652-656 (1998)).

[0233] C1q binds to reduced Fc variants

[0234] In another case, the Fc variant exhibits reduced C1q binding. C1q binding assays can also be performed to confirm whether the antibody can or cannot bind to C1q and therefore contains or lacks CDC activity (Idusogie et al., *Journal of Immunology* 164: 4178-84 (2000)). To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., *Journal of Immunological Methods* 202:163 (1996); Cragg, MS et al., *Blood* 101:1045-52 (2003); and Cragg et al., *Blood* 103:2738-43 (2004)).

[0235] In another instance, one or more amino acids may be substituted with different amino acid residues, resulting in altered Clq binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC) of the antibody. This method is further described in detail in U.S. Patent No. 6,194,551 to Idusogie et al. In another instance, one or more amino acid residues are altered to thereby change the antibody's ability to fix complement. This method is further described in PCT Publication WO 94 / 2935 1 to Bodmer et al. In one case, the Fc variants provided herein may contain mutations at amino acid positions 329, 331, and / or 322 (using Kabat numbering) and exhibit reduced Clq binding and / or CDC activity. In some cases, the Clq binding and / or CDC activity of the antibody may be reduced by mutating amino acid residues 318, 320, and / or 322 (using Kabat numbering) of the heavy chain; substitution of residue 297 (Asn) may result in the removal of the antibody's cleavage activity.

[0236] The cytotropic activity of IgG1 is due to its heavy chain CH 2. Domain Characteristics. In one case, where the Fc variant is IgG, amino acid residues 234-237 remain identical to the wild type to maintain the molecule's cytotropic activity. In other cases, IgG2 antibodies containing the complete ELLGGP sequence (residues 233-238) may be more active than wild-type IgG1.

[0237] In some cases, the Clq binding and / or cleavage activity of IgG1 antibodies can be reduced by mutating the amino acid residue Pro331 to Ser. In other cases, the Clq binding and / or cleavage activity of IgG4 antibodies can be reduced by mutating the amino acid residue Pro of Ser331 (Xu et al., Journal of Biochemistry 1994; 269(5):3469-74).

[0238] Fc variants with interchain disulfide bonds or double Fc regions

[0239] In yet another embodiment, it may be desirable to modify the antibodies of this disclosure relative to the effector function in order to enhance the therapeutic efficacy of the antibodies. For example, one or more cysteine ​​residues may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The resulting homodimeric antibody may have improved internalization capacity, increased complement-mediated cell killing, and / or antibody-dependent cytotoxicity (ADCC). See Caron et al., Journal of Experimental Medicine 176:1191-95 (1992) and Shapes, B. Journal of Immunology 148:2918-22 (1992). Alternatively, the antibody may be engineered to have a double Fc region, and may thereby have enhanced complement cleavage and / or ADCC capacity. See Stevenson et al., Anti-Cancer Drug Design 3: 219-30 (1989).

[0240] Fc variants with FcRn binding and increased in vivo half-life

[0241] This paper also envisions Fc variants with altered binding affinity to the neonatal receptor (FcRn). Fc variants with improved affinity for FcRn are expected to have a longer serum half-life, and such variants could be used in treatments of subjects for whom a long half-life of the administered peptide is desired, for example, to treat chronic infections. Conversely, Fc variants with reduced FcRn binding affinity are expected to have a shorter half-life, and such variants could be administered to subjects for whom shorter circulation time is preferred, such as those undergoing in vivo diagnostic imaging or those using antibodies that would produce toxic side effects if circulated in the bloodstream for an extended period. FcRn binding and in vivo clearance / half-life determination can be performed using methods known in the art (see, for example, Petkova, SB et al., *International Journal of Immunology* 18(12):1759-1769 (2006)).

[0242] By citation to Schuurman et al., *Molecular Immunology* 2001; 38(1):1-8, which are incorporated herein by reference, a mutation of Cys226, one of the hinge cysteine ​​residues involved in heavy chain inter-linkage, to serine produces more stable heavy chain linkages. Mutating the IgG4 hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequence Cys-Pro-Pro-Cys also significantly stabilized the covalent interactions between heavy chains. By citation to Angal et al., *Molecular Immunology* 1993; 30(1):105-8, which are incorporated herein by reference, a mutation of serine at amino acid position 241 in IgG4 to proline (found at the same position in IgG1 and IgG2) produces homogeneous antibodies and prolongs serum half-life and improves tissue distribution compared to the original chimeric IgG4. Other such instances of Fc region variants have also been envisioned (see, for example, Duncan & Winter, Nature 322:738-40 (1988); Chan CA and Carter PJ (2010) Nature Review of Immunology 10:301-316); and Shields et al., Journal of Biochemistry 9(2): 6591-6604 (2001).

[0243] antigen-binding fragments

[0244] The terms "antibody fragment," "antigen-binding fragment," or "antibody-binding domain" refer to at least a portion of an antibody or its recombinant variant containing an antigen-binding domain (i.e., the antigenic determination variable region of the complete antibody) sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, single-chain (sc)Fv ("scFv") antibody fragments, linear antibodies, and single-domain antibodies such as sdAb (V L or V H Camelidae V H H domain and multispecific antibodies formed from antibody fragments.

[0245] Antibody fragments can be prepared using a variety of techniques, including but not limited to the proteolytic digestion of intact antibodies and the production of recombinant host cells (e.g., Escherichia coli or bacteriophages), as described herein.

[0246] Fv is the smallest antibody fragment containing both an intact antigen recognition and binding site. This fragment is a dimer consisting of a tightly non-covalently associated heavy chain variable region domain and a light chain variable region domain. Six hypervariable rings (three from the H chain and three from the L chain) are derived from the folds of these two domains, providing amino acid residues for antigen binding and conferring antigen-binding specificity to the antibody. However, even a single variable region (or half of an Fv containing only the three antigen-specific CDRs) can recognize and bind antigens, albeit with a lower affinity than the intact binding site.

[0247] The term "scFv" refers to a fusion protein comprising at least one antibody fragment including a variable region comprising a light chain and at least one antibody fragment comprising a variable region comprising a heavy chain, wherein the light chain variable region and the heavy chain variable region are continuously linked by a short, flexible polypeptide linker and are capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, scFv may have V in any order. L Variable region and V H Variable regions, such as those relative to the N-terminus and C-terminus of the polypeptide, may include V... L -Connector-V H It may contain V H -Connector-V L .

[0248] Double antibodies are produced by... V H domain and VConstructing scFv fragments with short linkers (approximately 5-10 residues) between the L domains enables interchain rather than intrachain pairing of the V domains, resulting in bivalent fragments for preparing small antibody fragments. Bispecific biantibodies are heterodimers of two crossed scFv fragments, where the V domains of the two antibodies... H Domain and V L The domains are presented on different polypeptide chains. (See, for example, Hollinger et al., Proceedings of the National Academy of Sciences 90:6444-6448 (1993)).

[0249] Domain-specific antibodies (dAbs) can be produced in fully human form and are the smallest known antibody-antigen binding fragments, ranging from approximately 11 kDa to approximately 15 kDa. dAbs are stable variable regions of the heavy and light chains of immunoglobulins (V0 and V1, respectively). H and V L They are highly expressed in microbial cell cultures, exhibiting favorable biophysical properties, including, but not limited to, solubility and temperature stability, and are well-suited for selection and affinity maturation via in vitro selection systems such as phage display. dAbs are biologically active monomers and, due to their small size and inherent stability, can be formed into larger molecules, thereby producing drugs with prolonged serum half-lives or other pharmacological activities.

[0250] Fv and scFv are the only species with complete binding sites lacking constant regions. Therefore, they are well-suited for reducing nonspecific binding during in vivo use. scFv fusion proteins can be constructed to produce fusions of effector proteins at the N-terminus or C-terminus of the scFv. Antibody fragments can also be "linear antibodies." Such linear antibody fragments can be monospecific or bispecific.

[0251] In an alternative embodiment of this disclosure, the antigen-binding fragment can be generated in a variety of forms, wherein the antigen-binding domain is expressed as part of a continuous polypeptide chain, including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv) derived from a human antibody (Harlow et al., 1999, in: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, in: Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proceedings of the National Academy of Sciences 85:5879-5883; Bird et al., 1988, Science 242:423-426). In such embodiments, the antigen-binding domain comprises:

[0252] (a) One or more of the following (e.g., one, two, or all three):

[0253] (i) Heavy chain complementarity determination region 1 (CDR-H1), wherein the CDR-H1 comprises a sequence selected from any of the sequences provided in Sequence Listing 1 herein (including any of the sequences SEQ ID NO: 4, 14, 23, 28, 35, 39, 43, 46, 48, 64, 78, 84, 88, 94, 98, 109, 112, 115, 118, 120, 122, 125, 127, 130, or 137),

[0254] (ii) Heavy chain complementarity determination region 2 (CDR-H2), wherein the CDR-H2 comprises a sequence selected from any of the sequences provided in Sequence Listing 1 herein (including any of the sequences SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131 or 133),

[0255] (iii) Heavy chain complementarity determination region 3 (CDR-H3), wherein the CDR-H3 comprises a sequence selected from any of the sequences provided in Sequence Listing 1 herein (including any of the sequences SEQ ID NO: 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128, or 135), and / or

[0256] (b) One or more of the following (e.g., one, two, or all three):

[0257] (i) Light chain complementarity determination region 1 (CDR-L1), wherein the CDR-L1 comprises a sequence selected from any of the sequences provided in Sequence Listing 1 herein (including any of the sequences SEQ ID NO: 1, 12, 19, 27, 41, 53, 61 or 69),

[0258] (ii) Light chain complementarity determination region 2 (CDR-L2), wherein the CDR-L2 comprises a sequence selected from any of the sequences provided in Sequence Listing 1 herein (including any sequence containing GAS, ATS, SAS, or YAS, or the sequence of SEQ ID NO: 62, 70, 72, 77, or 87).

[0259] (iii) Light chain complementarity determination region 3 (CDR-L3), wherein the CDR-L3 comprises a sequence selected from any of the sequences provided in Sequence Listing 1 herein (including any of the sequences of SEQ ID NO: 3 or 63).

[0260] In one embodiment, the antigen-binding domain includes the heavy chain variable region and / or the light chain variable region described herein. In some embodiments:

[0261] (a) The heavy chain variable region comprises a sequence selected from any of the sequences provided in Sequence Listing 1 herein (including any of the sequences SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136, or 138), and / or

[0262] (b) The light chain variable region contains a sequence selected from any of the sequences provided in the sequence listing 1 herein (including any of the sequences SEQ ID NO: 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93 or 97).

[0263] In one embodiment, the antigen-binding domain is an scFv comprising a heavy chain variable region and a light chain variable region (e.g., the heavy chain variable region and light chain variable region described herein) containing an amino acid sequence. In one embodiment, the antigen-binding domain (e.g., scFv) comprises:

[0264] (a) A heavy chain variable region, the heavy chain variable region comprising:

[0265] (i) an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region provided herein, or

[0266] (ii) sequences having 85-99% (e.g., 90-99% or 95-99%) identity with the amino acid sequences provided herein (including any of the sequences SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136 or 138), and / or

[0267] (b) A light chain variable region, said light chain variable region comprising:

[0268] (i) an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of the light chain variable region provided herein, or

[0269] (ii) A sequence having 85-99% (e.g., 90-99% or 95-99%) identity with the amino acid sequence provided herein (including any of the sequences SEQ ID NO: 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93 or 97).

[0270] In some embodiments, the above aspects envision antibodies numbered 1, 3, 4, 5, 6, 7, 8, 9, 15 and 20 as described herein and provided in Sequence Listing 1.

[0271] In some embodiments, the above aspects envision antibodies ABS-101-A, ABS-101-B, and ABS-101-C as described herein and provided in Sequence Listing 1.

[0272] Synthesis of antigen-binding fragments

[0273] Once the code V is obtained H and V L These DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the gene encoding V... L or V HA DNA fragment is operatively linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operatively linked" is intended to mean joining two DNA fragments such that the amino acid sequences encoded by the two DNA fragments remain within the frame. This is achieved by encoding V... H DNA and coding heavy chain constant regions (C H 1. C H 2 and C H 3) Another DNA molecule can be operatively linked to encode V H The isolated DNA from the region is converted into the full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, EA et al. (1991), *Protein Sequences of Immunological Significance*, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. Heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions; most preferably, IgG1 or IgG4 constant regions. For Fab fragment heavy chain genes, encoding V... H DNA can interact with only the C-chain heavy chain. H Another DNA molecule in the constant region can be operatively linked.

[0274] By encoding V L DNA has a constant region C encoding the light chain. L Another DNA molecule can be operatively linked to encode V. L The isolated DNA from the regions is converted into full-length light chain genes (and Fab light chain genes). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat, EA et al. (1991), *Protein Sequences of Immunological Significance*, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments covering these regions can be obtained by standard PCR amplification. Light chain constant regions can be κ or λ constant regions, but κ constant regions are most preferred.

[0275] In order to produce the scFv gene, the gene encoding V will be... H and V L The DNA fragment is operatively linked to another fragment encoding a flexible linker (e.g., encoding the amino acid sequence (Gly-Gly-Gly-Gly-Ser)3), enabling V to... H Sequence and V L The sequence can be expressed as a continuous single-chain protein, where V L District and V HThe area is joined by a flexible joint (see, for example, Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proceedings of the National Academy of Sciences 85:5879-5883; McCafferty et al. (1990) Nature 348:552-554).

[0276] Alternative pathways for antibody production

[0277] As an alternative to direct synthesis using recombinant DNA methods, the antibodies or antigen-binding fragments described in this disclosure can be generated via hybridomas. In the hybridoma approach, as described herein, mice or other suitable host animals (such as hamsters or macaques) are immunized to induce the production or ability to produce lymphocytes that will specifically bind to the proteins used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent (such as polyethylene glycol) to form hybridoma cells (Goding, *Monoclonal Antibodies: Principles and Practice*, pp. 59-103 (Academic Press, 1986)).

[0278] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells. Preferred myeloma cells are those that fuse efficiently, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to the culture medium. Human myeloma and mouse-human heterologous myeloma cell lines for producing human monoclonal antibodies have also been described (Kozbor, *Journal of Immunology*, 133: 3001 (1984); Brodeur et al., *Monoclonal Antibody Production Techniques and Applications*, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Exemplary mouse myeloma cell lines include mouse myeloma cell lines derived from MOP-21 and MC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, California, USA) and SP-2 or X63-Ag8-653 cells (available from the American Type Culture Collection, Rockville, Md., USA). The production of monoclonal antibodies against antigens in the culture medium for growing hybridoma cells is measured. Therefore, in one aspect, this disclosure provides a hybridoma that produces the antibodies or antigen-binding fragments described herein. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis (Munson et al., Analytical Biochemistry 107:220 (1980)).

[0279] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, the clones can be subcloned using a limited dilution procedure and grown using standard methods (Goding, *Monoclonal Antibodies: Principles and Practice*, pp. 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640. Additionally, in animals, hybridoma cells can be grown in vivo as ascites tumors. The monoclonal antibodies secreted by the subclones are appropriately separated from the culture medium, ascites, or serum using routine immunoglobulin purification procedures (e.g., protein A-agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography).

[0280] Screening methods for identifying target antigens

[0281] Combining affinity

[0282] Antibody binding affinity can be screened using methods known in the art. For example, gel shift assays, Western blotting, competitive assays with radiolabeled markers, co-fractionation by chromatography, co-precipitation, cross-linking, ELISA, etc., can be used, as described in, for example, *Laboratory Guide to Molecular Biology* (1999), John Wiley & Sons, NY, which is incorporated herein by reference in its entirety.

[0283] To preliminarily screen for antibodies that bind to the desired epitopes on the antigen, routine cross-blocking assays can be performed, as described in *Antibodies: A Laboratory Handbook*, Cold Spring Harbor Laboratory, edited by Harlow and David Lane (1988). Routine competitive binding assays can also be used, where the unknown antibody is characterized by its ability to inhibit the binding of the antigen to the antigen-specific antibody of this disclosure. The complete antigen, fragments thereof, or linear epitopes can be used. Epitope mapping is described in Champe et al., *Journal of Biochemistry* 270: 1388-94 (1995).

[0284] In one variant of the in vitro assay, this disclosure provides a method comprising the steps of: (a) contacting an immobilized antigen with a candidate antibody, and (b) detecting the binding of the candidate antibody to the antigen. In an alternative embodiment, the candidate antibody is immobilized and the binding of the antigen is detected. Immobilization is performed using any method well known in the art, including covalent bonding with a carrier, beads, or chromatographic resin, and non-covalent, high-affinity interactions such as antibody binding, or using streptavidin / biotin binding, wherein the immobilized compound includes a biotin moiety. Detection of binding can be performed by (a) using a radioactive label on an unimmobilized compound, (b) using a fluorescent label on an unimmobilized compound, (c) using an antibody with immunospecificity to the unimmobilized compound, (d) using a label on an unimmobilized compound that excites a fluorescent carrier to which the immobilized compound is attached, and other techniques well known in the art and of conventional practice.

[0285] In some embodiments, this document provides an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is capable of binding to human TL1A with an affinity at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times that of a reference antibody binding to human TL1A. In one embodiment, the antibody or antigen-binding fragment is capable of binding to human TL1A with an affinity at least 8.30 times that of a reference antibody binding to human TL1A. In other embodiments, this document provides an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is capable of binding to human FcRn with an affinity at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times that of a reference antibody binding to human FcRn. In one embodiment, the antibody or antigen-binding fragment is capable of binding to human FcRn with an affinity that is at least 6.6-9.1 times greater than the affinity of a reference antibody for binding to the human FcRn. In other embodiments, the antibody or antigen-binding fragment is expressed in concentrations of approximately 0.05 nM, 0.10 nM, 0.15 nM, 0.20 nM, 0.25 nM, 0.3 nM, 0.25 nM, 0.40 nM, 0.45 nM, 0.50 nM, 0.55 nM, 0.60 nM, 0.65 nM, 0.70 nM, 0.75 nM, 0.80 nM, 0.85 nM, 0.90 nM, 0.95 nM, 1.00 nM, 1.10 nM, 1.20 nM, 1.30 nM, 1.40 nM, 1.50 nM, 1.60 nM, 1.70 nM, 1.80 nM, 1.90 nM, 2.0 nM, 3.0 nM, 4.0 nM, 5.0 nM, 6.0 nM. Affinities of nM, 7.0 nM, 8.0 nM, 9.0 nM, or 10.0 nM bind to human TL1A.

[0286] Regulator activity

[0287] Another aspect of this disclosure relates to a method for identifying antibodies that modulate (i.e., reduce) the activity of a target antigen, the method comprising contacting the target antigen with the antibody and determining whether the antibody alters the activity of the antigen. The activity in the presence of the test antibody is compared to the activity in the absence of the test antibody. If the activity of the sample containing the test antibody is lower than the activity of the sample without the test antibody, the antibody will have inhibitory activity.

[0288] Antibodies that regulate (i.e., increase, decrease, or block) the activity or expression of a desired target can be identified by incubating a proposed modulator with cells expressing the desired target and determining the effect of the proposed modulator on the activity or expression of the target. The selectivity of antibodies that regulate the activity of a target peptide or polynucleotide can be assessed by comparing their effect on the target peptide or polynucleotide with their effect on other related compounds. Selective modulators can include, for example, antibodies that specifically bind to the target peptide or the nucleic acid encoding the target peptide, and other proteins, peptides, or organic molecules. Modulators of target activity are therapeutically useful in treating diseases and physiological conditions involving normal or abnormal activity of the target peptide. Targets can be, for example, TL1A.

[0289] In one embodiment of this disclosure, a method for screening antibodies that modulate the activity of a target antigen includes contacting the antibody with a target antigen peptide and determining whether a complex exists between the antibody and the target antigen. In such assays, ligands are typically labeled. After appropriate incubation, free ligands separate from those present in bound form, and the amount of free or unlabeled ligand is a measure of the ability of a particular antibody to bind to a target antigen.

[0290] High-throughput screening

[0291] As described herein, the AI-based de novo design, optimization / affinity maturation and / or folding methods envisioned in this disclosure employ a variety of experimental “wet lab” techniques, including, for example, ACE and qaACE as described herein.

[0292] This disclosure also envisions a high-throughput screening (HTS) assay for identifying antibodies that interact with or inhibit the biological activity of antigens (e.g., inhibit enzyme activity, binding activity, etc.). HTS assays allow for the efficient screening of large numbers of compounds. Cell-based HTS systems are considered to investigate the interaction between antibodies and their target antigens and their binding partners. HTS assays are designed to identify “hit” or “lead compound” compounds with desired properties, thereby allowing for the design of modifications to improve those properties. The chemical modification of the “hit” or “lead compound” is typically based on a identifiable structure / activity relationship between the “hit” and the target antigen.

[0293] HTS arrays can consist of one or more protein arrays (e.g., antibody arrays, antibody microarrays, protein microarrays). The array can contain one or more antibodies or antigen-binding fragments thereof immobilized on a solid support. Methods for generating and using such arrays are well known in the art (e.g., Buessow et al., Nucleic Acids Res. 1998; Lueking et al., Molecular and Cellular Proteomics. 2003; Angenendt et al., Molecular and Cellular Proteomics 2006). In some embodiments, a very small amount (e.g., 1 µg to 500 µg) of antibody or antigen-binding fragment thereof is immobilized. In some embodiments, a single sample will contain 1 μg to 100 μg, 1 μg to 50 μg, 1 μg to 20 μg, 3 μg to 100 μg, 3 μg to 50 μg, 3 μg to 20 μg, 5 μg to 100 μg, 5 μg to 50 μg, or 5 μg to 20 μg of antibody. On the other hand, at least one of a plurality of samples will contain 1 μg to 100 μg, 1 μg to 50 μg, 1 μg to 20 μg, 3 μg to 100 μg, 3 μg to 50 μg, 3 μg to 20 μg, 5 μg to 100 μg, 5 μg to 50 μg, or 5 μg to 20 μg of antibody. The solid-phase support refers to an insoluble functionalized material to which the antibody can be reversibly linked, directly or indirectly, thereby separating it from unwanted materials (e.g., excess reagents, contaminants, and solvents). Examples of solid supports include, for example, functionalized polymeric materials, such as agarose or its bead form Sepharose. ®The antibody array comprises: dextran, polystyrene, and polypropylene, or mixtures thereof; a dense disk containing a microfluidic channel structure; a protein array chip; a pipette tip; a membrane, such as a nitrocellulose or PVDF membrane; and microparticles, such as paramagnetic or nonparamagnetic beads. In some embodiments, the affinity medium is bound to a solid-phase support, and the antibody is indirectly linked to the solid-phase support via the affinity medium. On one hand, the solid-phase support comprises a protein A affinity medium or a protein G affinity medium. “Protein A affinity medium” and “protein G affinity medium” each refer to a solid phase on which a natural or synthetic protein is bound, the natural or synthetic protein comprising, respectively, the Fc-binding domain of protein A or protein G, or a mutant variant or fragment of the Fc-binding domain of protein A or protein G, which retains affinity for the Fc portion of the antibody. Antibody arrays can be fabricated by transferring antibodies in an organized, high-density form onto a solid surface, followed by chemical immobilization. Representative techniques for fabricating arrays include photolithography, inkjet and contact printing, liquid dispensing, and piezoelectric techniques. The pattern and size of the antibody array are determined for each specific application. Users can easily control the size of each antibody spot. Antibodies can be linked to a variety of surfaces through diffusion, adsorption / absorption, or covalent cross-linking and affinity. Antibodies can be directly spotted onto ordinary glass surfaces. To keep the antibodies in a humid environment during the printing process, a high percentage of glycerol (e.g., 30-40%) can be used in the sample buffer, and spotting can be performed in a humidity-controlled environment.

[0294] ADCC and CDC measurements

[0295] On the one hand, the antibodies or antigen-binding fragments thereof disclosed herein are envisioned as therapeutic antibodies for treating diseases, conditions, or inflammations, including, for example, autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, systemic lupus erythematosus, asthma, ulcerative colitis, Crohn's disease, psoriasis, primary biliary cirrhosis, primary biliary cholangitis, ankylosing spondylitis, and fibrosis, including intestinal fibrosis, pulmonary fibrosis, and liver fibrosis, as well as infections. Therefore, antibodies or antigen-binding fragments thereof can be further screened in antibody-dependent cell-mediated cytotoxicity (ADCC) assays and / or complement-dependent cytotoxicity (CDC) assays. "ADCC activity" refers to the ability of an antibody to elicit an ADCC response. ADCC is a cell-mediated reaction in which antigen-nonspecific cytotoxic cells expressing FcRs (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to the surface of target cells and subsequently cause lysis (i.e., "killing") of the target cells. The primary mediators are natural killer (NK) cells. NK cells express only FcγRIII, of which FcγRIIIA is the activating receptor and FcγRIIIB is the repressive receptor; monocytes express FcγRI, FcγRII, and FcγRIII (Ravetch et al. (1991), *Annu. Rev. Immunol.* 9:457-92). ADCC activity can be measured in vitro, for example... 51 Cr release assays were performed using peripheral blood mononuclear cells (PBMCs) and / or NK effector cells as described in Examples and Shields et al. (2001), *Journal of Biochemistry* 276:6591-6604, or directly using another suitable method known in the art. ADCC activity can be expressed as the antibody concentration at half the maximum lysis level of the target cells. Therefore, in some embodiments, the concentration of the antibody or its antigen-binding fragment disclosed herein is at most 1 / 2, 1 / 3, 1 / 5, 1 / 10, 1 / 20, 1 / 50, or 1 / 100 of the concentration of the wild-type control itself when the lysis level is the same as the half-maximum lysis level of the wild-type control.

[0296] Additionally, in some embodiments, the antibodies or antigen-binding fragments of this disclosure may exhibit higher maximum target cell lysis compared to wild-type controls. For example, the maximum target cell lysis of the antibodies or Fc fusion proteins of this disclosure may be 10%, 15%, 20%, 25%, or more of the maximum target cell lysis of wild-type controls. "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to cleave a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component (C1q) of the complement system to a molecule (e.g., an antibody) that is complexed with a homologous antigen. To assess complement activation, a CDC assay may be performed, for example as described in the Journal of Immunological Methods, Gazzano-Santoro et al., 202:163 (1996).

[0297] target antigen

[0298] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein bind to TL1A. In some embodiments, anti-TL1A antibodies or fragments thereof bind to the same or similar or overlapping or partial epitopes that bind to reference molecules as described herein.

[0299] In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein binds to an epitope on a target antigen (e.g., TL1A). In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein binds to multiple epitopes on TL1A (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more).

[0300] The binding affinity and dissociation rate of the antibodies or antigen-binding fragments disclosed herein to the epitopes on TL1A can be determined using methods known in the art. Binding affinity can be measured by ELISA, RIA, flow cytometry, or surface plasmon resonance such as BIACORE™. Dissociation rate can be measured by surface plasmon resonance. Preferably, binding affinity and dissociation rate are measured by surface plasmon resonance. More preferably, binding affinity and dissociation rate are measured using BIACORE™.

[0301] Epitope plotting

[0302] As used herein, the term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, called a complementary site. A single antigen may have more than one epitope. Therefore, different antibodies can bind to different regions on the antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated from adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include a sugar, phosphoryl, or sulfonyl moiety on the antigen.

[0303] Various techniques known to those skilled in the art can be used to determine whether an antibody's antigen-binding domain interacts with one or more amino acids within a peptide or protein. Exemplary techniques include, for example, conventional cross-blocking assays (such as those described in *Antibodies*, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)), alanine scanning mutation analysis, peptide blotting analysis (Reineke, 2004, *Methods in Molecular Biology* 248:443-463), and peptide cleavage analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of the antigen can be employed (Tomer, 2000, *Protein Science* 9:487-96). Another method that can be used to identify amino acids within a peptide that interact with an antibody's antigen-binding domain is hydrogen / deuterium exchange detected by mass spectrometry. Generally, hydrogen / deuterium exchange methods involve deuterating the protein of interest and then binding the antibody to the deuterated protein. Next, the protein / antibody complex is transferred to water to allow hydrogen-deuterium exchange at all residues except those protected by the antibody (which remain deuterated). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterated residues corresponding to the specific amino acids that interact with the antibody. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Analytical Chemistry 73:256A-265A. X-ray crystallography of the antigen / antibody complex can also be used for epitope mapping.

[0304] Epitopes on the target antigen that bind to the antibody or antigen-binding fragments disclosed herein may consist of a single, continuous sequence of three or more amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) of the target antigen. Alternatively, epitopes may consist of multiple non-continuous amino acids (or amino acid sequences) of the antigen (e.g., TL1A). Additional epitope mappings, including the envisioned epitopes, are provided in the examples herein.

[0305] Contemplated alternative host cells for antibody expression and purification are described in U.S. Patent Nos. 9,617,335, 11,371,048, 2018 / 0282405, 11,584,785, and PCT / US22 / 82294.

[0306] Antibodies produced from prokaryotic host cells

[0307] On the one hand, this article provides a host cell containing the isolated nucleic acid described above or a vector containing the isolated nucleic acid. The vector may be a cloning vector or an expression vector. Suitable host cells for cloning or expressing the DNA in the vector described above are prokaryotic cells, yeast cells, or higher eukaryotic cells. Suitable prokaryotic cells for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia coli (e.g., *Escherichia coli*), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., *Salmonella typhimurium*), Serratia (e.g., *Serratia demersum*), and Shigella, as well as Bacillus, such as *Bacillus subtilis* and *Bacillus licheniformis* (e.g., *Bacillus licheniformis* 41 P disclosed in DD 266,710, published April 12, 1989), and Pseudomonas, such as *Pseudomonas aeruginosa* and *Streptomyces*. A preferred host for Escherichia coli cloning is Escherichia coli 294 (ATCC 31, 446), but other strains such as Escherichia coli B, Escherichia coli Xl 776 (ATCC 31, 537), and Escherichia coli W3110 (ATCC 27, 325) are suitable. These examples are illustrative and not limiting.

[0308] Contemplated alternative host cells for antibody expression and purification are described in U.S. Patent Nos. 9,617,335, 11,371,048, 2018 / 0282405, 11,584,785, and PCT / US22 / 82294.

[0309] As described herein, in another embodiment, the SoluPro *E. coli* strain is contemplated (see, for example, WO / 2014 / 025663 and WO / 2017 / 106583). As described in International Publication WO2017 / 106583, which is incorporated herein by reference in its entirety, the production of antigen-binding proteins on a commercial scale and in soluble form is addressed by providing suitable host cells capable of growing at high cell density in fermentation cultures and producing soluble gene products in oxidized host cytoplasm through highly controlled induced gene expression. Prokaryotic cells possessing these qualities are produced by combining some or all of the following characteristics: (1) genetically modifying the host cell to have oxidized cytoplasm by increasing the expression or function of oxidized polypeptides in the cytoplasm and / or by decreasing the expression or function of reducing polypeptides in the cytoplasm. Specific examples of such genetic alterations are provided herein. Optionally, the host cell may also be genetically modified to express chaperone proteins and / or cofactors and / or glycosylated polypeptide gene products that contribute to the production of the desired gene product. (2) The host cell contains one or more expression constructs designed for the expression of one or more gene products of interest; in some embodiments, at least one expression construct contains an inducible promoter and a polynucleotide encoding a gene product expressed by the inducible promoter. (3) The host cell contains additional gene modifications designed to improve certain aspects of the expression of the gene product derived from the expression construct. In a particular embodiment, the host cell (A) has an altered gene function of at least one gene encoding a transporter protein of at least one inducible promoter, and as another example, wherein the gene encoding the transporter protein is selected from the group consisting of araE, araE, araG, araH, rhaT, xylF, xylG, and xylH, or particularly araE, or wherein the altered gene function is more specifically derived from the expression of araE of the constitutive promoter; and / or (B) has a reduced level of gene function of at least one gene encoding a protein that metabolizes the inducer protein of at least one inducible promoter, and as another example The gene encoding the protein that metabolizes the inducer of at least one inducible promoter is selected from the group consisting of: araA, araB, araD, prpB, prpD, rhaA, rhaB, rhaD, xylA, and xylB; and / or (C) has a reduced level of gene function of at least one gene encoding the protein involved in the biosynthesis of the inducer of at least one inducible promoter, in another embodiment, the gene being selected from the group consisting of: scpA / sbm, argK / ygfD, scpB / ygfG, scpC / ygfH, rmlA, rmlB, rmlC, and rmlD.

[0310] Prokaryotic cells with oxidized cytoplasm

[0311] Examples of host cells are provided, which allow for the efficient and low-cost expression of gene products comprising components of multimeric products. In addition to isolated cells in a culture, host cells may also include cells that are part of a multicellular organism or cells grown within a different organism or system of organisms. In some embodiments of this disclosure, the host cell is a microbial cell, such as yeast (Saccharomyces, Schizosaccharomyces, etc.) or a bacterial cell, either Gram-positive or Gram-negative bacteria, or Escherichia coli, or Escherichia coli strain B, or Escherichia coli (strain B) EB0001 cells (also known as Escherichia coli ASE (DGH) cells), or Escherichia coli (strain B) EB0002 cells. In growth experiments of Escherichia coli host cells with oxidized cytoplasm, specifically Escherichia coli B strains Shuffle® Express (NEB catalog number C3028H) and Shuffle® T7 Express (NEB catalog number C3029H) and Escherichia coli K strain Shuffle® T7 (NEB catalog number C3026H), these Escherichia coli B strains with oxidized cytoplasm were able to grow to much higher cell densities than their closest counterparts, Escherichia coli K strains (International Publication No. WO 2017 / 106583).

[0312] Certain alterations can be made to the gene function of host cells containing inducible expression constructs to promote efficient and homogeneous induction of host cell populations via inducers. In some embodiments, as measured by the method described in Example 9 of International Publication No. WO 2017 / 106583 by Khlebnikov et al., the combination of expression construct, host cell genotype, and induction conditions results in at least 75% (more preferably at least 85%, and most preferably at least 95%) of the cells in the culture expressing the gene product from each inducible promoter. For host cells other than *E. coli*, these alterations can involve the function of genes structurally similar to those in *E. coli*, or genes performing functions similar to those in *E. coli* within the host cell. Alterations to host cell gene function include eliminating or reducing gene function by completely deleting the gene's protein-coding sequence, deleting a sufficient portion of the gene, inserting a sequence into the gene, or otherwise altering the gene sequence to reduce the level of the functional gene product produced by said gene. Alterations in host cell gene function also include enhancing gene function by, for example, altering the natural promoter to produce a stronger promoter that directs higher levels of gene transcription, or introducing missense mutations into the protein-coding sequence that produce a more active gene product. Alterations in host cell gene function include any alteration of gene function, including, for example, altering the natural inducible promoter to produce a constitutively activated promoter. In addition to altering gene function related to the transport and metabolism of inducers (as described herein and associated with inducible promoters) and / or changing the expression of chaperone proteins, the host cell's reductive-oxidative environment may also be altered.

[0313] Host cell reducing oxidative environment

[0314] In bacterial cells such as *E. coli*, proteins requiring disulfide bonds are typically exported to the periplasm, where the formation and isomerization of disulfide bonds are catalyzed by the Dsb system, which includes DsbABCD and DsbG. Increased expression of combinations of cysteine ​​oxidases (DsbA), disulfide isomerases (DsbC), or Dsb proteins, typically transported entirely to the periplasm, has been used for the expression of disulfide-requiring heterologous proteins (Makino et al., *Microb Cell Fact*, May 14, 2011; 10: 32). Cytoplasmic forms of these Dsb proteins can also be expressed, such as cytoplasmic versions of DsbA and / or DsbC lacking a signal peptide and therefore not transported to the periplasm ('cDsbA' or 'cDsbC'). Cytoplasmic Dsb proteins, such as cDsbA and / or cDsbC, serve to make the host cell cytoplasm more oxidative and thus more conducive to disulfide bond formation in heterologous proteins produced in the cytoplasm. The host cytoplasm can also be made less reducing and therefore more oxidizing by directly altering the thioredoxin and glutathione / glutathionease system: mutant strains deficient in glutathione reductase (gor) or glutathione synthase (gshB), along with thioredoxin reductase (trxB), make the cytoplasm oxidizing. These strains cannot reduce ribonucleotides and therefore cannot grow in the absence of exogenous reducing agents such as dithiothreitol (DTT). Repressive mutations in the gene ahpC that encode the peroxidase AhpC (such as ahpC* or ahpCA, Lobstein et al., Microbial Cell Factory 2012 May 8; 11: 56) convert it to a dithioreductase that produces reduced glutathione, thus allowing electrons to be directed to the ribonucleotide reductase enzyme and enabling gor and trxB deficient cells or gshB and trxB deficient cells to grow in the absence of DTT. Different mutant forms of AhpC can allow strains deficient in γ-glutamylcysteine ​​synthase (gshA) activity and trxB-deficient strains to grow in the absence of DTT; these include AhpC V164G, AhpC S71F, AhpC E173 / S71F, AhpC E171Ter, and AhpC dupl62-169 (Faulkner et al., Proceedings of the National Academy of Sciences, May 6, 2008; 105(18):6735-6740, e.g., May 2, 2008). In such strains with oxidative cytoplasm, exposed protein cysteine ​​is readily oxidized by thioredoxin catalysis, reversing its physiological function and leading to disulfide bond formation.Other proteins that can help reduce oxidative stress in host cells with oxidized cytoplasm include HPI (hydroperoxidase I) catalase-peroxidase encoded by *E. coli* katG and HPII (hydroperoxidase II) catalase-peroxidase encoded by *E. coli* katE, which disproportionately convert peroxides to water and O2 (Farr and Kogoma, *Microbiol Rev*, Dec 1991; 55(4): 561-85; Review). Increasing the levels of KatG and / or KatE proteins in host cells through induced co-expression or by increasing constitutive expression levels is one aspect of some embodiments of this disclosure.

[0315] Another possible alteration of the host cell is the expression of Ervlp, a thiol oxidase derived from the inner membrane space of yeast mitochondria in the host cytoplasm. This thiol oxidase has been shown to increase the production of various complex disulfide-bonded proteins of eukaryotic origin in the cytoplasm of *E. coli*, even in the absence of mutations in gor or trxB (Nguyen et al., *Microbial Cell Factory*, Jan 7, 2011; 10: 1).

[0316] The host cell containing the expression construct preferably also expresses cDsbA and / or cDsbC and / or Ervlp; lacks trxB gene function; also lacks gor, gshB or gshA gene function; optionally has increased katG and / or katE gene function levels; and expresses an appropriate AhpC mutant form so that the host cell can grow in the absence of DTT.

[0317] Cofactor cellular transport

[0318] When using the expression system of this disclosure to produce enzymes that require cofactors, it is helpful to use host cells capable of synthesizing cofactors from available precursors or taking up cofactors from the environment. Common cofactors include ATP, coenzyme A, flavin adenine dinucleotide (FAD), NAD+ / NADH, and heme. Polynucleotides encoding cofactor transport peptides and / or cofactor synthesis peptides can be introduced into host cells, and such peptides can be constitutively expressed or inducibly co-expressed with the active gene product to be produced by the methods of this disclosure.

[0319] protease

[0320] The ability of a host cell to degrade expressed protein products may be altered due to the lack or reduction of activity of one or more proteases. Exemplary proteases include, but are not limited to, Clp, ClpP, OmpT, Lon, FtsH, ClpX, ClpY, ClpA, ClpQ, ClpAP, ClpXP, ClpAXP, ClpYQ, ClpY, and proteases encoded by yaeL, sppA, tldD, sprT, yhbU, ptrA, frvX, hyaD, hybD, hycH, envC, ddpX, degP, degQ, degS, hslV, hslU, pepB, pepP, sohB, yggG, pepE, pepN, pepQ, abgA, pepT, iadA, pepA, pepD, ptrB, ycaL, ycbZ, yegQ, ygeY, ypdF, hycI, sgcX, and htpX (Gottesman, *Annu Rev Genet*). 30: 465-506, 1996).

[0321] Glycosylation of polypeptide gene products

[0322] Host cells can alter their ability to glycosylate peptides. For example, eukaryotic host cells may have eliminated or attenuated the gene function of glycosyltransferase and / or oligosyltransferase genes, thereby impairing normal eukaryotic glycosylation of peptides into glycoproteins. Prokaryotic host cells that do not normally glycosylate peptides (such as Escherichia coli) can be altered to express a set of eukaryotic and prokaryotic genes that provide glycosylation function (DeLisa et al., WO 2009 / 089154A2).

[0323] Other technologies related to host cell engineering include Rosano et al. (Fronts in Microbiology, 5, 2014), Jensen et al. (Sci Rep, 5: 17874, 2015), Gu et al. (Biotech, 9(3): 77, 2019), Meyer et al. (NatChem Biol, 15: 169-204, 2019), and Euler et al. (PLoSONE, 11: e0146408, 2016).

[0324] chaperone protein

[0325] In some embodiments, the active gene product may be co-expressed with another gene product, such as a chaperone protein, which promotes the formation of the active form of the gene product of interest within the host cell. A chaperone protein is a protein that assists in the non-covalent folding or unfolding and / or assembly or breakdown of other gene products, but is not present in the resulting monomeric or multimeric gene product structure when performing its normal biological function (having completed the folding and / or assembly process). Chaperone proteins can be expressed from inducible or constitutive promoters in the expression construct, or from the host cell chromosome. Exemplary chaperone proteins present in *E. coli* host cells include folding factors DnaK / DnaJ / GrpE, DsbC / DsbG, GroEL / GroES, IbpA / IbpB, Skp, Tig (triggering factor), and FkpA, which have been used to prevent the aggregation of cytoplasmic or periplasmic proteins. DnaK / DnaJ / GrpE, GroEL / GroES, and ClpB can work synergistically to help protein folding, and various combinations of these chaperone proteins have been shown to promote the expression of gene products that fold correctly. When eukaryotic proteins are expressed in prokaryotic host cells, eukaryotic chaperone proteins from the same or related eukaryotic species, such as protein disulfide isomerases (PDIs), can be co-expressed (e.g., inducibly co-expressed) with the gene product of interest.

[0326] In one embodiment, the modified cell line is provided with the accessory protein PnlP-1 as described in WO2023 / 122448.

[0327] In one embodiment, the materials and assays described herein do not require chaperone proteins.

[0328] Chromosomal protease deficiency and metabolic engineering

[0329] During the development of SoluPro (see, for example, WO 2017 / 106583), single-gene knockout studies were conducted to generate hosts capable of producing higher titers of the proteins of interest. clpA and ptsP were identified throughout the various cell line development programs. Subsequently, single gene deletions were combined into a single host to achieve even higher titers.

[0330] clpA is a natural host protein that guides the ClpAP protease to degrade unfolded or abnormal proteins. SoluPro (see, for example, WO 2017 / 106583) is constructed from parental strain B, specifically BL21, which lacks the Lon protease, which degrades many foreign proteins. This follows the general approach of knocking out proteases to increase the titer of heterologous proteins. Clp proteins, as chaperones, are highly conserved, present in all organisms, and contain ATP and polypeptide binding sites. ClpA (the ATPase component of the ATP-dependent ClpAP protease) also functions as a molecular chaperone. ClpA is a member of the class I Hsp100 family, which forms a homohexameric ring structure in the presence of ATP to perform its chaperone activity. ClpA catalyzes the ATP-dependent unfolding of substrate proteins and mediates their translocation into the proteolytic cavity of ClpP (Reid et al., Proceedings of the National Academy of Sciences, 98(7):3768-72, 2001). ClpA hexamers interact with ClpP via a considered IGL / F motif nested in the helical-loop-helical region near the C-terminus of ClpP's nucleotide-binding domain 2 (NBD2) (Kim et al., *Nature Structural Biology*, 8: 230-33, 2001). In the absence of the proteolytic component ClpP, ClpA catalyzes protein remodeling reactions, such as remodeling the inactive dimer of RepA into two active monomers (Walker et al., *Journal of the European Society for Molecular Biology*, 1: 945-51, 1994).

[0331] ptsP is the natural host protein encoding enzyme I (EI) of the nitrogen-associated phosphotransferase system (PTS). This gene is involved in the transport and phosphorylation of glucose and other sugars as part of the initial steps of the phosphorylation cascade. Literature suggests that the potential reason for increased titers is that ptsP knockout can achieve faster acetate assimilation through both upregulation of actP (acetate permease) and increased cAMP / CRP levels, thereby increasing the transcription of acetyl-CoA synthase Acs. In other words, this phenotype of increased acetate tolerance is particularly associated with high-cell-density fermentation, where acetate accumulation can be mitigated by ptsP deficiency to more efficiently utilize an alternative carbon source besides glucose.

[0332] Incorporating regulatory factors to reduce plasmid size

[0333] Typically, plasmids encode four distinct components: the origin of replication, antibiotic resistance markers, the gene of interest, and regulatory proteins that control the expression of the gene of interest. Several regulatory proteins are usually involved in controlling gene expression. These regulatory proteins, or genetic sensors, and all their associated genetic elements significantly increase plasmid size; each sensor typically exceeds 1000 base pairs. This increase in size increases the workload carried by the plasmid in maintaining the cell and increases the chance of recombination or mutation in ways that could inactivate the protein of interest or some other essential plasmid-encoding elements. In summary, these larger plasmid sizes lead to the formation of inactivating plasmids and unintended selection during protein production, reducing the yield of the target protein.

[0334] Reduce prophage reactivation potential

[0335] Prephages are typically present in at least 50% of the bacterial genome. Specifically, in the SoluPro strain, the presence of a prephage region was detected and it was reactivated during the mitomycin C test (RCB) to induce the production of live phages. Subsequent sequencing of the phages revealed regions of interest, the aim of which is to selectively knock out certain regions of the genome to prevent the production of fully functional phages capable of lysing infection and lysogens.

[0336] Inhibit post-translational gluconyization of heterologous proteins

[0337] The SoluPro strain (see, for example, WO 2017 / 106583) produces heterologously expressed proteins that undergo glucosylation, an undesirable post-translational modification. It has been shown that the formation of glucosylation / phosphoglucosylation may be caused by the accumulation of 6-phosphogluconolactone due to the absence of phosphogluconolactone (PGL). This is inherent to the *E. coli* B strain lineage, as the mutation can be traced back to a UV-induced deletion of the galM-ybhJ locus found in the parental strain *E. coli* B707. PGL catalyzes the hydrolysis of 6-phosphogluconolactone to 6-phosphogluconate. Non-specific protein glucosylation can be inhibited by overexpression of heterologous PGL in the SoluPro host.

[0338] Cofactor cellular transport

[0339] Common cofactors include ATP, coenzyme A, flavin adenine dinucleotide (FAD), NAD+ / NADH, and heme. Polynucleotides encoding cofactor transport peptides and / or cofactor synthesis peptides can be introduced into host cells, and these peptides can be constitutively expressed or inducibly co-expressed with gene products produced by the methods disclosed herein.

[0340] Glycosylation of polypeptide gene products

[0341] Host cells can alter their ability to glycosylate peptides. For example, eukaryotic host cells may have eliminated or attenuated gene function in glycosyltransferase and / or oligosyltransferase genes, thereby impairing normal eukaryotic glycosylation of peptides into glycoproteins. Prokaryotic host cells that do not normally glycosylate peptides (such as *E. coli*) can be altered to express a set of eukaryotic and prokaryotic genes that provide glycosylation function (DeLisa et al., WO 2009 / 089154A2, July 16, 2009).

[0342] Assays: Activity-specific cell enrichment (ACE) assay and HiPrBind assay

[0343] As described in WO 2021 / 146626, the activity-specific cell enrichment (ACE) assay for identifying host cells expressing an active gene product of interest, rather than an inactive substance, is incorporated herein by reference in the relevant section. By way of example, an active gene product can be distinguished from an inactive substance by its ability to specifically bind to a binding partner molecule or by its ability to participate in a chemical or enzymatic reaction. The presence of appropriately formed disulfide bonds in a polypeptide gene product indicates its correct folding and presumes activity. In cell enrichment methods, the active gene product of interest is detected by utilizing an appropriate labeled complex that specifically binds to it, such as a labeled antigen if the gene product of interest is an antibody or Fab; or a labeled ligand if the gene product of interest is a receptor or receptor fragment, wherein the ligand specifically binds to the active conformation of the receptor; or a labeled substrate or labeled substrate analog if the gene product of interest is an enzyme, as an example. For any gene product of interest, if there is an available antibody or antibody fragment that specifically binds to the active gene product but not to the inactive gene product, the antibody or antibody fragment can be used to label the active gene product of interest when linked to a detectable portion.

[0344] The HiPrBind assay provides an efficient method for multiple interrogations of an active gene product, such as by providing at least two different interrogations of the characteristic features of the active gene product, or by simultaneously interrogating at least two characteristic features of the active gene product. The HiPrBind assay is described in WO 2021 / 163349, which is incorporated herein by reference in its relevant sections. This assay improves upon the basic principle of the yeast dual heterozygosity assay by bringing the multicomponent detection mechanism closer together, thereby placing the multicomponent detection mechanism in an environment where the detection mechanism can be active in generating a detectable signal. In a multicomponent (e.g., a two-component) detection system, one component stably associates with a first analyte-associated portion (i.e., the active gene product-associated portion), and the second component of the detection system stably associates with a different second analyte-associated portion. A detectable signal is generated when the two components of the detection system approach each other through the analyte-associated portions bound to the analyte. Because each of the analyte-associated portions is specific to the active gene product as an analyte, a signal is generated only when characteristic features of the active gene product are detected using two different mechanisms, or when two different characteristic features of the active gene product are detected simultaneously. The HiPrBind assay is versatile in detecting a variety of characteristic properties, such as in a simple example where a gene product is active in a homodimeric form, where each monomer requires a disulfide bond to fold properly. An active gene product associated moiety can be a binder that specifically binds to a properly folded and therefore active monomer, and a second active gene product associated moiety can be a different second binder that specifically binds to the dimer form of the gene product. Therefore, the HiPrBind assay in this embodiment simultaneously detects gene products that are correctly folded and in dimer form.

[0345] Available host cell strains with altered gene function. In order to create preferred host cell strains for the expression systems and methods of this disclosure, it is useful to start with strains that already contain the desired genetic alterations (Table A; International Publication No. WO 2017 / 106583).

[0346] Table A. Exemplary host cell strains

[0347]

[0348] Besides prokaryotic cells, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae or common baker's yeast are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are commonly used and available in this paper, such as *Schizosaccharomyces pombe*; *Kluyveromyces* hosts, such as *Kluyveromyces lactis*, *Kluyveromyces fragilis* (ATCC 12,424), *Kluyveromyces bulgaricus* (ATCC 16,045), *Kluyveromyces wickeramii* (ATCC 24,178), *Kluyveromyces waltii* (ATCC 56,500), *Kluyveromyces drosophilarum* (ATCC 36,906), *Kluyveromyces thermotolerans*, and *Kluyveromyces marxianus*; *Yarrowia* (EP 402,226); and *Pichiapastors* (EP 402,226). 183,070); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium and Aspergillus host such as Aspergillus nidulans and Aspergillus niger.

[0349] Suitable host cells for expressing glycosylated antibodies originate from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Various baculovirus strains and variants, along with corresponding permitted insect host cells derived from hosts such as the grassland armyworm (Spodoptera frugiperda) (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm). Several viral strains for transfection are publicly available, such as the L-1 variant of the alfalfa silver-striped armyworm (Autographa californica) NPV and the Bm-5 variant of the silkworm NP7 strain, and such viruses can be used herein according to this disclosure, particularly for transfecting grassland armyworm cells.

[0350] Plant cell cultures of cotton, corn, potatoes, soybeans, peanuts, tomatoes, tobacco, duckweed, and other plant cells can also be used as hosts. However, vertebrate cells are of greatest interest, and the propagation of vertebrate cells in cultures (tissue cultures) has become a routine procedure. Examples of useful mammalian host cell lines include Chinese hamster ovary cells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary / -DHFR (CHO, Urlaub et al., Proceedings of the National Academy of Sciences 77: 4216 (1980)); monkey kidney CV1 lines transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lines (subcloned to 293 or 293 cells grown in suspension culture [Graham et al., Journal of General Virology 36: 59 (1977)]); juvenile hamster kidney cells (BHK, ATCCCCL 10); mouse Celtoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); and monkey kidney cells (CV1 ATCC CCL). 70); African green monkey kidney cells (VER0-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals of the New York Academy of Sciences 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2).

[0351] The host cells are transformed or transfected with the expression or cloning vectors described above for antibody production, and the host cells are cultured in a conventional nutrient medium modified to induce promoters, select transformants, or amplify genes encoding desired sequences. Furthermore, novel vectors containing multiple copies of transcription units isolated by selective markers and transfected cell lines are particularly suitable and preferred for expressing the antibodies described herein.

[0352] For transfection of the expression vector and production of the chimeric, humanized, or complex human antibodies described herein, the recipient cell line can be myeloma cells. Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by the transfected immunoglobulin nucleic acid sequence and possess mechanisms for immunoglobulin glycosylation. For example, in some embodiments, the recipient cells are myeloma cells SP2 / 0 (ATCC #CRL 8287) that produce recombinant Ig. SP2 / 0 cells produce only immunoglobulins encoded by the transfected gene. Myeloma cells can be grown in mouse cultures or intraperitoneally, where secreted immunoglobulins can be obtained from ascites fluid. Other suitable recipient cells include lymphoid cells, such as human or non-human B lymphocytes, human or non-human hybridoma cells, or interspecies heterologous hybridoma cells. Expression vectors carrying the chimeric antibody constructs, humanized antibody constructs, or composite human antibody constructs or antibody peptides described herein can be introduced into suitable host cells by any of a variety of suitable methods, including biochemical methods such as transformation, transfection, conjugation, protoplast fusion, and calcium phosphate precipitation, as well as applications with polycations such as diethylaminoethyl (DEAE) dextran, and mechanical methods such as electroporation, direct microinjection, and microparticle bombardment. As is known to those skilled in the art, Johnston et al., 240 Science 1538 (1988).

[0353] Yeast offers certain advantages over bacteria in producing immunoglobulin H and L chains. Yeast undergoes post-translational peptide modifications, including glycosylation. Several recombinant DNA strategies exist that utilize strong promoter sequences and high-copy-number plasmids that can be used to produce the desired protein in yeast. Yeast recognizes the leader sequence of a cloned mammalian gene product and secretes a peptide (i.e., a propeptide) carrying the leader sequence. (Hitzman et al., 11th International Conference on Yeast Genetics & Molecular Biology (Montpelier, France), 1982) Antibody peptides or their antigen-binding fragments from yeast gene expression systems, as well as assembled chimeric antibodies, humanized antibodies, or complex human antibodies, their fragments and regions, production, secretion levels, and stability can be routinely evaluated. Any of a range of yeast gene expression systems can be utilized, incorporating promoter and terminator elements from genes encoding glycolytic enzymes that are produced in large quantities when yeast is grown in glucose-rich media. Known glycolysis genes can also provide highly efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. Several methods can be used to evaluate the optimal expression plasmid for expressing cloned immunoglobulin cDNA in yeast.

[0354] Bacterial strains can also be used as hosts to produce the antibody molecules or fragments thereof described herein, including *Escherichia coli* K12 strains such as *Escherichia coli* W3110 (ATCC 27325), *Bacillus* species, *Enterobacteria* species such as *Salmonella typhimurium* or *Serratia demersum*, and various *Pseudomonas* species. Plasmid vectors containing replicons and control sequences derived from species compatible with the host cell are used in conjunction with these bacterial hosts. The vector carries the replication site and specific genes that can provide phenotypic selection in the transformed cells. Several methods can be used to evaluate expression plasmids for producing chimeric antibodies, humanized antibodies, or complex humanized antibodies and fragments thereof encoded by immunoglobulin cDNA or CDR cloned from bacteria (see Glover, 1985; Ausubel, 1987, 1993; Sambrook, 1989; Colligan, 1992–1996).

[0355] Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin protein molecules, including leader peptide removal, folding and assembly of H and L chains, glycosylation of antibody molecules, and secretion of functional antibody proteins. In addition to the lymphoid-derived cells described above, mammalian cells that can be used as hosts for antibody protein production include fibroblast-derived cells such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells. Exemplary eukaryotic cells that can be used for peptide expression include, but are not limited to: COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; PER.C6® cells (Crucell); and NSO cells. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform desired post-translational modifications to variable heavy chains and / or variable light chains. For example, in some embodiments, peptides produced by CHO cells have a higher level of sialylation than the same peptides produced by 293 cells.

[0356] Expression builder

[0357] In some embodiments of this disclosure, it is contemplated to use an inducible promoter with an expression construct to be introduced into a host cell according to this disclosure in order to achieve an increased expression of a desired active gene product. Exemplary promoters are described herein and also in WO / 2016 / 205570, which are incorporated herein by reference in the relevant sections. As described herein, a cell containing one or more expression constructs may optionally include one or more inducible promoters to express a gene product of interest. In one embodiment, the gene product is a fusion protein. In other embodiments, the gene product is a protein, such as a therapeutic protein.

[0358] Expression constructs are polynucleotides designed to express one or more gene products of interest and are therefore not naturally occurring molecules. The use of any expression construct known in the art in the cells and methods of this disclosure is contemplated, including expression constructs that can be integrated into the host cell chromosome or maintained in the host cell as an extrachromosomally replicating polynucleotide molecule, i.e., an appendage having a replication origin independent of the host cell chromosome (such as a plasmid or artificial chromosome). Expression constructs according to this disclosure may also have one or more selectable markers to enable selection of cells containing the expression construct. Exemplary selectable markers confer resistance to antibodies that are lethal to host cells lacking said selectable markers or encoding enzymes required to produce essential nutrients. The use of any selectable markers known in the art in the expression constructs of this disclosure is contemplated. Expression markers may also contain an inducible promoter to provide the ability to induce expression of a coding region operatively linked to said inducible promoter. Exemplary inducible promoters contemplated in this disclosure include arabinose promoters (ParaBAD), ParaC, ParaE, propionate promoters (PprpBCDE), rhamnose promoters (PrhaSR), xylose promoters (PxylA), lactose promoters, and alkaline phosphatase promoters. Further information (including sequences thereof) regarding contemplated inducible promoters is provided in WO 2016 / 205570, which is incorporated herein by reference in the relevant sections. In addition to inducible promoters, this disclosure includes expression constructs comprising constitutive promoters. To ensure efficient translation of RNA transcribed from the expression construct, the construct may also include a ribosome binding site (RBS). Typically, in prokaryotes (archaea and bacteria), the RBS consensual sequence is GGAGG or GGAGGU, and in bacteria (such as Escherichia coli), the RBS consensual sequence is further defined as AGGAGG or AGGAGGU. To facilitate the incorporation of the coding region or gene of interest, expression constructs may include multiple cloning sites, wherein various restriction endonuclease cleavage sites are clustered to provide flexibility in incorporating exogenous polynucleotides, as known in the art. Some embodiments of the expression constructs disclosed herein further include a coding region for a signal peptide or leader peptide, wherein said coding region is directed to induce the expression of a fusion protein comprising the signal peptide and the active gene product of interest.

[0359] As used herein, the term "expression construct" refers to a polynucleotide designed to express one or more antigen-binding proteins of interest, and therefore is not a naturally occurring molecule. Expression constructs can be integrated into the host cell chromosome or maintained within the host cell as polynucleotide molecules that replicate independently of the host cell chromosome (such as plasmids or artificial chromosomes). Examples of expression constructs are polynucleotides produced by inserting one or more polynucleotide sequences into the host cell chromosome, wherein the inserted polynucleotide sequences alter the expression of chromosomal coding sequences. Expression vectors are plasmid expression constructs specifically designed to express one or more antigen-binding proteins. One or more expression constructs can be integrated into the host cell chromosome or maintained on extrachromosomal polynucleotides, such as plasmids or artificial chromosomes. The following describes specific types of polynucleotide sequences in expression constructs that can be used for the expression or co-expression of gene products, including fusion proteins as described herein.

[0360] Copy start point

[0361] Expression constructs must contain origins of replication, also known as replicons, to remain within the host cell while independently replicating polynucleotides. Different replicons using the same replication mechanism cannot remain together in a single host cell through repeated cell divisions. Therefore, as shown in Table 2 of International Publication WO 2016 / 205570, plasmids can be classified into incompatible groups based on the origins of replication they contain. Origins of replication can be selected for use in expression constructs based on other criteria such as incompatibility groups, copy number, and / or host range. As mentioned above, if two or more different expression constructs will be used for the co-expression of multiple gene products in the same host cell, it is preferable that the different expression constructs contain origins of replication from different incompatibility groups: for example, a pMB1 replicon in one expression construct and a p15A replicon in another. The average copy number of an expression construct in a cell relative to the number of host chromosomal molecules is determined by the origins of replication contained in the expression construct. Copy numbers can range from a few copies to hundreds per cell (Table 2 of WO / 2016 / 205570). In some embodiments, different expression constructs are used, each containing an inducible promoter activated by the same inducer but with different origins of replication. By selecting an origin of replication that maintains each different expression construct at an approximate copy number in the cell, the overall production level of the gene product expressed from one expression construct relative to the gene product expressed from another different expression construct can be adjusted. For example, to co-express subunits A and B of a multimeric protein, an expression construct is generated containing a colEl replicon, an am promoter, and a coding sequence for subunit A expressed from the am promoter: 'collet-Para-A'.

[0362] Another expression construct was created, comprising the pl5A replicon, the am promoter, and the coding sequence for subunit B: 'pl5A-Para-B'. Both expression constructs can be maintained together in the same host cell, and expression of both subunits A and B is induced by the addition of an inducer, arabinose, to the growth medium. For example, if a significant increase in subunit A expression level relative to subunit B expression level is desired, to bring the stoichiometric ratio of the two subunit expression levels closer to the desired ratio, a new expression construct for subunit A can be generated, possessing the modified pMB1 replicon found in the origin of replication of the pUC9 plasmid ('pUC9ori'): pUC9ori-Para-A. Expression of subunit A by a high copy number expression construct such as pUC9ori-Para-A should increase the amount of subunit A produced compared to subunit B expression by pl5A-Para-B. In a similar manner, using a replication origin that maintains the expression construct at a low copy number, such as pSOOl (WO / 2016 / 205570), can reduce the overall level of gene product expressed by the construct. Selecting the replication origin can also determine which host cells can maintain the expression construct containing the replicon. For example, expression constructs containing the colEl replication origin have a relatively narrow range of usable hosts, namely species within the Enterobacteriaceae family, while expression constructs containing the RK2 replicon can be maintained in Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Azotobacter vinelandii, and Alcaligenes eutrophus. Furthermore, if the expression construct contains the RK2 replicon and some regulatory genes from the RK2 plasmid, it can be maintained in diverse host cells such as Sinorhizobium meliloti, Agrobacterium tumefaciens, Caulobacter crescentus, Acinetobacter calcoaceticus, and Rhodobacter. In sphaeroides (Kiies and Stahl, Review of Microbiology, December 1989; 53(4): 491-516).

[0363] Similar considerations can be used to generate expression constructs for inducible or co-expression in eukaryotic cells. For example, the 2-micron circular plasmid of Saccharomyces cerevisiae is compatible with plasmids from other yeast strains such as pSRl (ATCC accessions 48233 and 66069; Araki et al., Journal of Molecular Biology, March 20, 1985; 182(2): 191-203) and pKDl (ATCC accession 37519; Chen et al., Nucleic Acid Research, June 11, 1986; 14(11): 4471-81).

[0364] In some embodiments, the expression construct contains a selection gene. A “selection gene,” also known as a selectable marker, encodes a protein essential for the survival or growth of host cells in a selective culture medium. Host cells that do not contain an expression construct containing a selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics or other toxins or compensate for nutritional deficiencies in the host cell. One example of a selection protocol utilizes a drug, such as an antibiotic, to inhibit the growth of host cells. Those cells containing an expression construct containing a selectable marker produce proteins that confer drug resistance and survive in the selection protocol. Some examples of antibiotics commonly used for selecting selectable markers (and the abbreviations indicating genes that provide an antibiotic resistance phenotype) are: ampicillin (AmpR), chloramphenicol (CmlR or CmR), kanamycin (KanR), spectinomycin (SpcR), streptomycin (StrR), and tetracycline (TetR). Many plasmids in Table 2 of WO / 2016 / 205570 contain selectable markers, such as pBR322 (AmpR, TetR); pMOB45 (CmR, TetR); pACYClW (AmpR, KanR); and pGBMl (SpcR, StrR). The natural promoter region used for gene selection is typically included as part of the selectable marker portion of the expression construct along with the coding sequence of its gene product. Alternatively, the coding sequence used for gene selection can be expressed by a constitutive promoter.

[0365] Exemplary optional biomarkers include, but are not limited to: neomycin phosphotransferase (NPT II), hygromycin phosphotransferase (HPT), dihydrofolate reductase (DHFR), zeocin, phleomycin, bleomycin resistance gene (BLE), gentamicin acetyltransferase, streptomycin phosphotransferase, mutant forms of acetyl-lactate synthase (ALS), bromobenzonitrile hydrolase, glufosinate acetyltransferase (Bar), and enolacetone-shikimate-3-phosphate (EPSP) synthase (ARO). A) Muscle-specific tyrosine kinase receptor molecule (MuSK-R), copper-zinc superoxide dismutase (sod1), metallothionein (cup1, MT1), β-lactamase (BLA), puromycin N-acetyltransferase (pac), blasticidin acetyltransferase (bls), blasticidin deaminase (bsr), histidine dehydrogenase (HDH), N-succinyl-5-aminoimidazolium-4-carboxamide ribonucleate (SAICAR) synthase (ade1), arginosuccinate lyase (arg4), β-isopropylmalate dehydrogenase (leu2), invertase (suc2), orotidine-5'-phosphate (OMP) decarboxylase (ura3), and any of the above orthologs.

[0366] Inducible promoters

[0367] As described herein, several different inducible promoters exist that can be included in an expression construct as part of the inducible co-expression system of this disclosure. In some embodiments, the inducible promoter shares at least 80% polynucleotide sequence identity (more preferably at least 90% and most preferably at least 95% identity) of at least 30 (more preferably at least 40 and most preferably at least 50) consecutive bases of a promoter polynucleotide sequence referencing the genome sequence of Escherichia coli K-12 substrain MG1655, as defined in Table 1 of International Publication WO 2016 / 205570, wherein the percentage of polynucleotide sequence identity is determined using the method of Example 11 of WO / 2016 / 205570. Under 'standard' induction conditions (see Example 5 of International Publication No. WO 2016 / 205570), the preferred inducible promoter has at least 75% (more preferably at least 100% and most preferably at least 110%) the strength of the corresponding 'wild-type' inducible promoter of Escherichia coli K-12 substrain MG1655, as determined using the quantitative PCR method of De Mey et al. (Example 6 of International Publication No. WO 2016 / 205570). In the expression construct, the inducible promoter is positioned 5' (or 'upstream') of the coding sequence of the gene product to be inducibly expressed, such that the presence of the inducible promoter will guide transcription of the gene product coding sequence in the 5' to 3' direction relative to the coding strand of the multinucleotide encoding the gene product.

[0368] Ribosome binding site

[0369] For polypeptide gene products, the nucleotide sequence of the region between the transcription start site and the start codon of the coding sequence of the gene product to be inducibly expressed corresponds to the 5' untranslated region ('UTR') of the mRNA for the polypeptide gene product. Preferably, the region of the expression construct corresponding to the 5'UT contains a polynucleotide sequence similar to the common ribosome binding site (RBS, also known as the Shine-Dalgarno sequence) present in the host cell species. In prokaryotes (archaea and bacteria), the common RBS sequence is GGAGG or GGAGGU, and in bacteria, such as Escherichia coli, the common RBS sequence is AGGAGG or AGGAGGU. The RBS is typically separated from the start codon by 5 to 10 intermediate nucleotides. In the expression construct, the RBS sequence preferably shares at least 55% identity with the AGGAGGU common sequence, more preferably at least 70% identity, and most preferably at least 85% identity, and is separated from the start codon by 5 to 10 intermediate nucleotides, more preferably 6 to 9 intermediate nucleotides, and most preferably 6 or 7 intermediate nucleotides. The ability of a given RBS to generate the desired translation initiation rate can be calculated using the RBS calculator at salis.psu.edu / software / RBSLibraryCalculatorSearchMode; this tool can be used to optimize synthetic RBSs with translation rates ranging from 100,000+ times (Salis, Methods Enzymol 2011; 498: 49-42).

[0370] Multiple cloning sites

[0371] Multiple cloning sites (MCS), also known as multiple adapters, are polynucleotides containing multiple restriction sites that are adjacent to or overlap each other. The restriction sites in the MCS typically occur once within the MCS sequence and preferably not within the rest of the plasmid or other polynucleotide construct, thereby allowing restriction enzymes to cleave the plasmid or other polynucleotide construct only within the MCS. Examples of MCS sequences are those sequences in the pBAD series expression vectors, including pBAD18, pBAD18-Cm, pBAD18-Kan, pBAD24, pBAD28, pBAD30, and pBAD33 (Guzman et al., J Bacteriol, July 1995; 177(14): 4121-30); or those sequences in the pPRO series expression vectors derived from pBAD vectors, such as pPR018, pPR018-Cm, pPR018-Kan, pPR024, pPRO30, and pPR033 (US Patent No. 8,178,338 B2; May 15, 2012; Keasling, Jay). Multiple cloning sites can be used to generate expression constructs: by placing the multiple cloning site at the 3' (or downstream) of the promoter sequence, the MCS can be used to insert the coding sequence of the gene product to be expressed or co-expressed into the construct at the appropriate position relative to the promoter, causing transcription of the coding sequence to occur. Depending on which restriction enzymes are used to cleave within the MCS, some portions of the remaining MCS sequence may remain within the expression construct after the coding sequence or other polynucleotide sequence is inserted. Any remaining MCS sequence can be upstream, downstream, or flanking the inserted sequence. Ribosome binding sites can be located upstream of the MCS, preferably immediately adjacent to the MCS or separated from the MCS by only a few nucleotides; in this case, the RBS is upstream of any coding sequence inserted into the MCS. Another alternative is to include a ribosome binding site within the MCS, in which case the choice of restriction enzymes used for cleavage within the MCS will determine whether the RBS is retained and its relationship to the inserted sequence. Another alternative is to include the RBS (inserted into the expression construct at the MCS) within the polynucleotide sequence, preferably in a proper relationship with any coding sequence to stimulate translation from the transcribed messenger RNA.

[0372] Expression from constitutive promoters

[0373] The expression constructs disclosed herein may also contain coding sequences expressed from constitutive promoters. Unlike inducible promoters, constitutive promoters initiate the production of sustained gene products under most growth conditions. An example of a constitutive promoter is the constitutive promoter of the Tn3 bla gene, which encodes a β-lactamase and is responsible for conferring the ampicillin resistance (AmpR) phenotype to host cells by a number of plasmids, including pBR322 (ATCC 31344), pACYClW (ATCC 37031), and pBAD24 (ATCC 87399). Another constitutive promoter that can be used in expression constructs is the promoter for the Escherichia coli lipoprotein gene Ipp, located at position 1755731-1755406 (positive strand) in Escherichia coli K-12 substrain MG1655 (Inouye and Inouye, Nucleic Acid Research, May 10, 1985; 13(9):3101-10). Another example of a constitutive promoter already used for heterologous gene expression in *E. coli* is the trpLEDCBA promoter, located at position 1321169-1321133 (negative strand) in *E. coli* substrain K-12 MG1655 (Windass et al., *Nucleic Acid Research*, 11 November 1982; 10(21): 6639-57). As described herein, constitutive promoters can be used in expression constructs for the expression of selectable markers, and also for the constitutive expression of other gene products useful for the co-expression of the desired product. For example, transcriptional regulators of inducible promoters, such as AraC, PrpR, RhaR, and XylR, can alternatively be expressed from constitutive promoters in the same or different expression constructs as the inducible promoters they regulate, if not from bidirectional inducible promoters. Similarly, to cite a few examples, gene products that can be used to produce or transport inducers, such as PrpEC, AraE, or Rha, or proteins that modify the cellular reductive-oxidative environment, can be expressed from constitutive promoters in vivo. Gene products that can be used to produce co-expressed gene products and the desired products derived therefrom also include chaperone proteins, cofactor transporters, and the like.

[0374] Incorporating regulatory factors cymR, luxR, vanR and AraC

[0375] VanR is a negative transcription factor belonging to the PadR family of bacteria, regulating the expression of the vanABK operon in response to vanillate. In the absence of vanillate, VanR inhibits the transcription of the vanABK gene by binding to the operator DNA located in the promoter region of the vanABK gene (Merkens et al., *Curr Microbiol*, 51: 59-65, 2005). Upon exposure to vanillate, VanR is released from the operator DNA, allowing RNA polymerase to initiate the transcription of the vanABK gene. One of VanR's roles is to transcriptionally regulate the catabolism of vanillate (phenolic acid) to utilize vanillate as a carbon or energy source (Morabbi et al., *Journal of Bacteriology*, 197: 959-72, 2015).

[0376] CymR is a TetR-type regulatory factor. The sequences of members of the TetR family are highly conserved in terms of their DNA-binding domains. These regulators primarily act as repressors that bind to their operators to suppress target genes and are released from DNA upon binding to their homologous ligands (Ramos et al., *Microbiol Mol Biol Rev.*, 69: 326-56, 2005). CymR can respond to the inducer coumarate. CymR is a transcriptional repressor involved in controlling gene expression targeting the degradation of p-cymene (cym) and p-cumate (cmt) (Eaton et al., *Journal of Bacteriology*, 179: 3171-80, 1997). CymR proteins are dimers in solution.

[0377] LuxI and LuxR are quorum sensing regulatory genes. LuxI is the synthase of the self-inducer N-3-oxohexanoyl-L-homoserine lactone (OC6), which interacts with its homologous receptor LuxR to form the transcriptional activator of the lux operon (Perez et al., BMC Systems Biology, 5: 153, 2011; Hao et al., Journal of Bacteriology, 188:2173-83, 2006).

[0378] AraC responds to the inducer arabinose; it is a gene regulator that positively influences the synthesis of other ara proteins in *E. coli*. AraC is homologous to P… BAD The promoter also responds to the presence of arabinose; it is stimulated by CAP and is inhibited by the AraC protein itself via DNA circularization or direct binding to the Pc polymerase-binding region (Casadaban et al., Journal of Molecular Biology, 104: 557-66, 1971).

[0379] In some embodiments, one or more of the aforementioned sensors are combined into a single sensor operon. In some embodiments, all four sensors are combined into a single sensor operon. In some embodiments, the sensor operon comprises, in sequence, cymR, luxR, vanR, and araC. In some embodiments, the sensor operon further comprises a J23100 constitutive promoter. In some embodiments, the sensor operon is inserted into the genome of an *E. coli* strain at the yciA locus.

[0380] Δ(cusS-argU) Δ(cdsbC-galM)

[0381] Bacterial genomes can contain a significant proportion (>20%) of functional and non-functional phage genes. These phage or phage-like gene clusters in the bacterial genome are referred to as “prophage regions” (Casjens et al., *Molecular Microbiology*, 49: 277–300, 2003). Prophage regions in bacterial strains of interest can be identified by (i) experimental methods or (ii) computational methods. Experimental methods involve inducing the host bacteria to release phage particles by exposure to UV light or other DNA-damaging conditions, but this method may not reveal defective phages or certain prophages induced by other conditions. Computational methods involve sequence comparisons with known phage or prophage genes, comparisons with known bacterial genes, tRNA and dinucleotide analysis, and hidden Markov scans for attachment site identification. Programs that can perform this type of analysis include Phage_Finder, Prophinder, Prophage Finder, and PHASTER (Lima-Mendez et al., Bioinformatics, 24: 863–865, 2008).

[0382] In some embodiments, the prophage region is identified and removed from the *E. coli* genome to minimize prophage reactivation and induced changes. In some embodiments, the prophage region is replaced with a cassette containing a resistance marker. In some embodiments, the cassette containing the resistance marker is a kanamycin resistance marker.

[0383] Recombinant engineering is a highly efficient method for in vivo genetic engineering of E. coli replicons, enabling the insertion or deletion of DNA sequences without the need for restriction sites. In contrast to traditional techniques utilizing restriction enzymes and DNA ligases, recombinant engineering is the preferred method for inserting large DNA molecules, as these molecules are more likely to have restriction sites within their sequences that can be cleaved by restriction enzymes. Recombinant engineering allows modification of clonal DNA in E. coli via homologous recombination. Through recombinant engineering, virtually any type of mutation can now be introduced into bacterial artificial chromosomes (BACs) by targeting cassettes or single-stranded oligonucleotides with short homologous regions at their ends using PCR-amplified linear double-stranded DNA.

[0384] In one embodiment, the replaced prephage region is an approximately 18,000 bp region located between the cusS CDS and argU tRNA genes. In another embodiment, an approximately 44,000 bp region is located between the cytosol dsbC CDS and the remaining portion of galM CDS. In some embodiments, both the approximately 18,000 bp region between the cusS CDS and argU tRNA and the approximately 44,000 bp region between the cytosol dsbC CDS and the remaining portion of galM CDS are replaced.

[0385] 6-Phosphogluconolactone (pgl)

[0386] Minimizing post-translational modifications of heterologously expressed proteins, especially when preparing proteins for pharmaceutical and medical applications, is a key objective of bacterial engineering. Glucosylation of heterologously expressed proteins is commonly observed in *E. coli*, likely due to the accumulation of 6-phosphogluconide in the absence of phosphogluconide lactone (pgl) in the pentose phosphate pathway (Aon et al., *Applied and Environmental Microbiology*, 74(4): 950-58, 2008). In some embodiments, pgl is added to the *E. coli* genome. In other embodiments, heterologous pgl is added to the *E. coli* genome.

[0387] clpB

[0388] ClpB is an ATP-dependent chaperone protein belonging to the Hsp100 family of ATPases associated with various cellular activities. ClpB is a key protein mediating the heat shock response, possessing the ability to rescue stress-damaged proteins from aggregated states (Vale et al., *Journal of Cell Biology*, 150: F13-F19, 2000). Unlike other Hsp100 proteins, ClpA, ClpB does not associate with structurally and functionally unrelated ClpP proteases and does not guide the degradation of its substrate proteins (Wickner et al., *Science*, 286: 1888-93, 1999). Conversely, ClpB rescues proteins from aggregated states by mediating the disaggregation of stress-damaged proteins, where complete protein recovery requires the additional involvement of the DnaK / Hsp70 chaperone protein system (Parsell et al., Nature, 372(b): 475-78, 1994; Zolkiewski et al., Protein Science, 8: 1899-1903, 1999). In some embodiments, engineered E. coli are further engineered with plasmids to express clpB.

[0389] PDI (protein disulfide isomerase)

[0390] Secreted proteins and cell surface proteins are typically stabilized by disulfide bonds. Early in protein folding, disulfide formation is prone to errors: incorrect cysteine ​​residues may link together, or the correct cysteine ​​residues may pair, but the timing of pairing inhibits folding. Cells correct these errors through a specialized redox environment in the endoplasmic reticulum (ER), where catalysts for disulfide formation and isomerization exist (Wilkinson et al., *Biochim Biophys Acta*, 1699(1-2): 35-44, 2004). Protein disulfide isomerases (PDIs) are essential folding catalysts and chaperone proteins of the ER, acting by introducing disulfides into proteins (oxidase activity), catalyzing incorrect disulfide rearrangements (isomerase activity), and stabilizing misfolded proteins (redox-dependent chaperone activity). In some embodiments, engineered *E. coli* are further engineered with plasmids to express PDIs. In other embodiments, the engineered E. coli were further engineered using two plasmids, one for expressing clpB and the other for expressing PDI.

[0391] signal peptide

[0392] The polypeptide gene products expressed or co-expressed by the methods of this disclosure may contain or lack a signal peptide, depending on whether such gene products are expected to be exported from the host cytoplasm to the periplasm or retained in the cytoplasm. The signal peptide (also known as a signal sequence, leader sequence, or leader peptide) is structurally characterized by a hydrophobic amino acid segment of about five to twenty amino acids long, and typically about ten to fifteen amino acids long, which tends to form a single α-helix. This hydrophobic segment usually precedes a shorter segment enriched with positively charged amino acids (especially lysine). Signal peptides cleaved from mature polypeptides typically end with an amino acid segment recognized and cleaved by a signal peptidase. Functionally, the signal peptide is typically characterized by its ability to directly transport the co-translated or translated polypeptide across the plasma membrane of prokaryotes (or the inner membrane of Gram-negative bacteria such as *Escherichia coli*) or into the endoplasmic reticulum of eukaryotic cells. For example, the extent to which a signal peptide enables a polypeptide to be transported into the periplasmic space of a host cell (such as Escherichia coli) can be determined by means of the method described in Example 12 of International Publication No. WO 2016 / 205570, by separating the periplasmic protein from the protein retained in the cytoplasm.

[0393] Unless otherwise stated, these examples of inducible promoters and related genes are derived from *Escherichia coli* strain MG1655 (ATCC 700926, American Center for Type Culture Collection), a substrain of *Escherichia coli* K-12 (ATCC 10798, American Center for Type Culture Collection). Table 1 of International Publication No. WO 2016 / 205570 lists the genomic locations of these examples of inducible promoters and related genes in *E. coli* MG1655. Nucleotides and other gene sequences mentioned by genomic location in Table 1 of International Publication No. WO 2016 / 205570 are explicitly incorporated herein by reference. Further information about the *E. coli* promoters, genes, and strains described herein is available in numerous public sources, including the online EcoliWiki resource at ecoliwiki.net.

[0394] Arabic sugar promoter

[0395] (As used herein, 'arabinose' refers to L-arabinose.) Several E. coli operons involved in arabinose utilization are arabinose-inducible—araBAD, araC, arciE, and araFGH—but the terms 'arabinose promoter' and 'ara promoter' are generally used to specify the araBAD promoter. Several other terms have been used to refer to the E. coli araBAD promoter, such as Para, ParaB, ParaBAD, and PBAD—as used herein, 'ara promoter' or any alternative term given above refers to the E. coli araBAD promoter. As can be seen from the use of the other term 'araC-araBAD promoter', the araBAD promoter is considered to be part of a bidirectional promoter, where the araBAD promoter controls the expression of the araBAD operon in one direction, and the araC promoter, very close to and on the opposite strand of the araBAD promoter, controls the expression of the araC coding sequence in the other direction. The AraC protein is both a positive and negative transcriptional regulator of the araBAD promoter. In the absence of arabinose, the AraC protein inhibits transcription from PBAD, but in the presence of arabinose, the AraC protein, upon binding to arabinose, alters its configuration to become a positive regulatory element allowing transcription from PBAD. The araBAD operon encodes a protein that metabolizes L-arabinose by converting it to D-xylinose-5-phosphate via the intermediates L-ribulose and L-ribulose-phosphate. To maximize the induction of expression by arabinose-inducible promoters, it is extremely useful to eliminate or attenuate the function of AraA, which catalyzes the conversion of L-arabinose to L-ribulose, and optionally also eliminate or attenuate the function of at least one of AraB and AraD. Eliminating or attenuating the ability of the host cell to reduce the effective concentration of arabinose in the cell by eliminating or attenuating the cell's ability to convert arabinose to other sugars makes it possible to have more arabinose available for induction by arabinose-inducible promoters. The genes encoding the transport proteins that move arabinose into host cells are araE, which encodes a low-affinity L-arabinose proton cotransporter; and the araFGH operon, which encodes a subunit of the ABC superfamily high-affinity L-arabinose transporter. Other proteins that can transport L-arabinose into cells are certain mutants of LacY lactose permease: LacY(AlWC) and LacY(AlWV) proteins, which have either alanine or cysteine ​​amino acid substitutions at position 177 (Morgan-Kiss et al., Proceedings of the National Academy of Sciences, May 28, 2002; 99(11): 7373-77). To achieve homogeneous induction of arabinose-inducible promoters, it is useful to transport arabinose into cells independently of arabinose regulation.This can be achieved by eliminating or attenuating the activity of the AraFGH transporter and altering the expression of araE, thereby making it transcribed solely by a constitutive promoter. Constitutive expression of araE can be achieved by eliminating or attenuating the function of the native araE gene and introducing an expression construct that includes the coding sequence for the AraE protein expressed by a constitutive promoter into the cell. Alternatively, in cells lacking AraFGH function, the promoter controlling the expression of the araE gene on the host cell chromosome can be changed from an arabinose-inducible promoter to a constitutive promoter. In a similar manner, as another alternative to homogeneous induction by an arabinose-inducible promoter, host cells lacking AraE function can allow any functional AraFGH coding sequence present in the cell to be expressed by a constitutive promoter. As another alternative, the araE gene and araFGH operon can be expressed by constitutive promoters by replacing the native araE and araFGH promoters with constitutive promoters from the host chromosome. The activity of the AraE and AraFGH arabinose transporters can also be eliminated or attenuated, and in this case, a mutation in LacY lactose permease can be used, which allows this protein to transport arabinose. Since the expression of the lacY gene is generally not regulated by arabinose, using LacY mutants such as LacY(A177C) or LacY(A177V) will not result in an "all or none" induction when the arabinose-inducible promoter is induced by the presence of arabinose. Because the LacY(A177C) protein appears to be more efficient at transporting arabinose into the cell, using a polynucleotide encoding the LacY(A177C) protein is preferred over using a polynucleotide encoding the LacY(A177V) protein.

[0396] propionate promoter

[0397] The propionate promoter, or prp promoter, is the promoter of the *E. coli* pprBCDE operon. Similar to the ara promoter, the ppr promoter is part of a bidirectional promoter, controlling the expression of the pprBCDE operon in one direction, while the pprR promoter controls the expression of the pprR coding sequence in the other. The pprR protein is a transcriptional regulator of the ppr promoter, and transcription from the ppr promoter is activated when the pprR protein binds to 2-methylcitrate ('2-MC'). Propionate (also known as propanoate) is propionic acid (or 'propanoic acid'). The CH3CH2COO- ion of propionate is the smallest of the fatty acids with the general formula H(CH2)COOH, which share certain properties with such molecules: producing an oily layer when salted out of water and having soap-like potassium salts. Commercially available propionates are generally sold as monovalent cationic salts of propionate, such as sodium propionate (CH3CH2COONa), or as divalent cationic salts, such as calcium propionate (Ca(CH3CH2COO)2). Propionates are membrane-permeable and are metabolized to 2-MC by PrpE (propionyl-CoA synthase) to convert propionate to propionyl-CoA and then by PrpC (2-methylcitrate synthase) to convert propionyl-CoA to 2-MC. Other proteins encoded by the prpBCDE operon, PrpD (2-methylcitrate dehydratase), and PrpB (2-methylisocitrate lyase) are involved in further catabolizing 2-MC into smaller products, such as... Pyruvate and succinate. To maximize the induction of propionate-inducible promoters by propionate added to cell growth medium, host cells with PrpC and PrpE activities are required to convert propionate to 2-MC, and said host cells also have eliminated or attenuated PrpD activity and optionally eliminated or attenuated PrpB activity to prevent 2-MC from being metabolized. Another operon encoding proteins involved in 2-MC biosynthesis is the scpA-argK-scpBC operon, also known as the sbm-yg / DGH operon. These genes encode proteins required to convert succinate to propionyl-CoA, which can then be converted to 2-MC via PrpC. Eliminating or attenuating the function of these proteins removes the parallel pathways that produce 2-MC inducers and thus reduces the background expression level of propionate-inducible promoters and increases the sensitivity of propionate-inducible promoters to exogenously supplied propionate.It has been found that introducing the deletion of sbm-ygfD-ygfG-ygfH-ygfl into *E. coli* BL21(DE3) to produce strain JSB (Lee and Keasling, “A propionate-inducible expression system for enteric bacteria”, *Applied and Environmental Microbiology*, November 2005; 71(11): 6856-62) helps reduce background expression in the absence of exogenously supplied inducers, but this deletion also reduces overall expression in strain JSB via the pprp promoter. However, it should be noted that the deletion of sbm-ygfD-ygfG-ygfH-ygfl also significantly affects ygfl encoding a putative LysR family transcriptional regulator with an unknown function. The gene sbm-gyn / DGH is transcribed as an operon, and gulf is transcribed by the opposite strand. The 3' ends of the ygfti and ygfl coding sequences overlap by several base pairs, so removing all deletions of the sbm-yg / DGH operon also significantly removes the ygfl coding function. Eliminating or attenuating the function of a subset of the sbm-ygfDGH gene product, such as YgfG (also known as ScpB, methylmalonyl-CoA decarboxylase), or deleting most of the sbm-yg / DGH (or scpA-argK-scpBC) operon, while leaving enough of the 3' end of the ygfli (or scpC) gene to allow ygfl expression to remain unaffected, can be sufficient to adequately reduce background expression from the propionate-inducible promoter without reducing the maximum level of induced expression.

[0398] Rhamnose promoter

[0399] As used herein, 'rhamnose' refers to L-rhamnose. The 'rhamnose promoter' or 'rha promoter' or PrhaSR is the promoter for the *E. coli* rhaSR operon. Similar to the ara and ppr promoters, the rha promoter is part of a bidirectional promoter that controls the expression of the rhaSR operon in one direction and the rhaBAD promoter controls the expression of the rhaBAD operon in the other direction. However, the rha promoter has two transcriptional regulators involved in regulating expression: RhaR and RhaS. The RhaR protein activates the expression of the rhaSR operon in the presence of rhamnose, while the RhaS protein activates the expression of the L-rhamnose catabolism and transport operons rhaBAD and rhaT, respectively (Wickstrum et al., *Journal of Bacteriology*, January 2010; 192(1): 225-32). Although the RhaS protein can also activate the expression of the rhaSR operon, RhaS actually negatively self-regulates this expression by interfering with the ability of the cyclic AMP receptor protein (CRP) to co-activate and express RhaR to much larger levels. The rhaBAD operon encodes the rhamnose catabolism proteins RhaA (L-rhamnose isomerase), which converts L-rhamnose to L-rhamnose; RhaB (rhamnose glycokinase), which phosphorylates L-rhamnose to form L-rhamnose-1-P; and RhaD (rhamnose-1-phosphate aldolase), which converts L-rhamnose-1-P to L-lactic acid and DHAP (dihydroxyacetone phosphate). To maximize the amount of rhamnose available in cells for expression induced by a rhamnose-inducible promoter, it is necessary to reduce the amount of rhamnose catalytically broken down by eliminating or attenuating the function of RhaA or optionally at least one of RhaA, RhaB, and RhaD. *E. coli* cells can also synthesize L-rhamnose from α-D-glucose-1-P via the activity of proteins RmlA, RmlB, RmlC, and RmlD (also known as RfbA, RfbB, RfbC, and RfbD, respectively) encoded by the rmlBDACX (or rfbBDACX) operon. Eliminating or attenuating the function of one or more of RmlA, RmlB, RmlC, and RmlD may be useful to reduce background expression from the rhamnose-inducible promoter and to enhance the sensitivity of rhamnose-inducible promoter induction by exogenously supplied rhamnose.

[0400] RmlD protein

[0401] L-rhamnose is transported into the cell via RhaT, rhamnose permease, or the L-rhamnose:proton cotransporter. As mentioned above, RhaT expression is activated by the transcriptional regulator RhaS. To make RhaT expression independent of rhamnose induction (which induces RhaS expression), host cells can be altered so that all functional RhaT coding sequences in the cell are expressed by constitutive promoters. Alternatively, the RhaS coding sequence can be deleted or inactivated, resulting in no functional RhaS. By eliminating or attenuating the function of RhaS in the cell, the expression level from the rhaSR promoter increases due to the absence of negative self-regulation via RhaS, and the expression level of the rhamnose catalytic operon rhaBAD decreases, thereby further increasing the ability of rhamnose to induce expression from the rha promoter.

[0402] Xylose promoter

[0403] As used herein, 'xylose' refers to D-xylose. The xylose promoter, or 'xyl promoter', or PxyiA, refers to the promoter used for the *E. coli* xylAB operon. The xylose promoter region is tissue-similar to other inducible promoters because the xylAB and xylFGHR operons are expressed in opposite directions on the *E. coli* chromosome by adjacent xylose-inducible promoters (Song and Park, *Journal of Bacteriology*, Nov 1997; 179(22): 7025-7032). The transcriptional regulators of both the PxyiA and PxyiF promoters are XylR, which activates the expression of these promoters in the presence of xylose. The xylR gene is expressed either as part of the xylFGHR operon or from its weak, non-xylose-inducible self-promoter located between the xylH and xylR protein-coding sequences. D-xylose is converted to D-xylulose by XylA (D-xylose isomerase), which then phosphorylates it by XylB (xylulose kinase) to form D-xylulose-5-P, thus catabolizing D-xylose. To maximize the amount of xylose in cells that can be induced to express from xylose-inducible promoters, it is necessary to reduce the amount of xylose catalytically broken down by eliminating or attenuating the function of at least XylA or optionally both XylA and XylB. The xylFGHR operon encodes XylF, XylG, and XylH, which are subunits of high-affinity D-xylose transporters in the ABC superfamily. The xylE gene, which encodes a low-affinity xylose proton cotransporter in *E. coli*, represents an independent operon whose expression is also xylose-inducible. To make xylose transporter expression independent of xylose induction, host cells can be altered to express all functional xylose transporters from constitutive promoters. For example, the xylFGHR operon can be modified to delete the xylFGH coding sequence, leaving XylR as the sole active protein expressed by the xylose-inducible PxyiF promoter, wherein the xylE coding sequence is expressed from the constitutive promoter rather than its native promoter. As another example, the xylR coding sequence is expressed by the PxyiA or promoter in the expression construct, while the xylFGHR operon is deleted and xylE is constitutively expressed; or alternatively, the xylFGH operon (lacking the xylR coding sequence because it is present in the expression construct) is expressed from the constitutive promoter, and the xylE coding sequence is deleted or modified such that it does not produce an active protein.

[0404] lactose promoter

[0405] The term 'lactose promoter' refers to a lactose-inducible promoter for the lacZYA operon, also known as lacZpl; this lactose promoter is located at approximately 365603-365568 (negative strand, with an NA polymerase binding ('-35') site at approximately 365603-365598, a Pribnow box ('-10') at 365579-365573, and a transcription start site at 365567) in the genome sequence of *E. coli* substrain K-12 MG1655 (NCBI reference sequence NC000913.2, January 11, 2012). In some embodiments, the inducible co-expression system of this disclosure may comprise a lactose-inducible promoter, such as the lacZYA promoter. In other embodiments, the inducible co-expression system of this disclosure comprises one or more inducible promoters that are not lactose-inducible promoters.

[0406] alkaline phosphatase promoter

[0407] The terms 'alkaline phosphatase promoter' and 'phoA promoter' refer to the promoters used for the phoApsiF operon, which are induced under phosphate starvation conditions. The phoA promoter region is located at approximately 401647–401746 in the genome sequence of *E. coli* substrain K-12 MG1655 (positive strand, with a Pribno box ('-10') at 401695–401701 (Kikuchi et al., *Nucleic Acid Research*, 11 Nov 1981; 9(21): 5671–78) (NCBI reference sequence NC000913.3, 16 Dec 2014). The transcriptional activator of the phoA promoter is PhoB, a transcriptional regulator that, together with the sensor protein PhoR, forms a two-component signal transduction system in *E. coli*. PhoB and PhoR are transcribed from the phoBR operon (positive strand, with the PhoB coding sequence located at approximately 417050–419300 in the genome sequence of *E. coli* substrain MG1655, where the PhoB coding sequence is located at 417,142–417,831 and the PhoR coding sequence is located at 417,889–419,184) (NCBI reference sequence NC 000913.3, December 16, 2014). The phoA promoter differs from the inducible promoters described above in that it is induced by the absence of a substance, namely intracellular phosphate, rather than by the addition of an inducer. For this reason, the phoA promoter is generally used to guide the transcription of gene products that will be produced at a stage where the host cell is depleting phosphate (such as in late fermentation). In some embodiments, the inducible co-expression system of this disclosure may include the phoA promoter. In other embodiments, the inducible co-expression system of this disclosure includes one or more inducible promoters that are not the phoA promoter.

[0408] As described herein, removal of the expression construct described herein (e.g., by means of an inducible or constitutive “hardening” mechanism) may be advantageous or desirable, for example, if the cell line carrying the expression construct is used or will be used for commercial purposes. Therefore, in some embodiments, the expression construct may include a “kill switch.” For example, in one embodiment, the expression construct includes a temperature-sensitive replication origin. Other hardening methods are known in the art and include the use of detergents and embedding agents, pharmaceuticals, and antibiotics (Buckner, MMC et al., FEMS Microbiology Reviews, fuy031,42, 2018, 781-804).

[0409] In some embodiments, the antibodies or antigen-binding fragments of the peptides disclosed herein can be produced in animals that have been engineered or transfected with one or more nucleic acid molecules encoding the peptides, according to any suitable method.

[0410] In some embodiments, antibodies or antigen-binding fragments thereof are generated in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods in Molecular Biology 498: 229-44 (2009); Spirin, Trends in Biotechnology 22: 538-45 (2004); and Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0411] Many vector systems are available for expressing H and L chain nucleic acid sequences in mammalian cells (see Glover, 1985). Different methods can be used to obtain complete H2L2 antibodies. As mentioned above, it is possible to co-express the H and L chains in the same cells to achieve intracellular association and linkage of the H and L chains into complete tetrameric H2L2 antibody and / or antigen-binding fragment peptides. Co-expression can be achieved by using the same or different plasmids in the same host. The genes for the H and L chains and / or CDR3 region peptides can be placed in the same plasmid, which is then transfected into cells, thereby directly selecting cells expressing both chains. Alternatively, cells can be first transfected with a plasmid encoding one chain (e.g., the L chain), followed by transfection of the resulting cell line with an H chain plasmid containing a second selectable marker. Cell lines that produce antigen-binding peptide fragments and / or H2L2 molecules via any of these pathways can be transfected with additional optional markers by plasmids encoding peptides, H, L, or H plus L chains to produce cell lines with enhanced properties (such as the production of more assembled H2L2 antibody molecules or enhanced stability of the transfected cell lines).

[0412] Furthermore, plants have become a convenient, safe, and economical alternative to mainstream expression systems for producing recombinant antibodies, which are based on large-scale cultures of microorganisms or animal cells. Antibodies can be expressed in plant cell cultures or conventionally grown plants. Expression in plants can be systemic, limited to subcytoplasm, or limited to seeds (endosperm). Several plant-derived antibodies are in late-stage development (see, for example, Biolex, NC).

[0413] In some respects, this document provides methods and systems for generating humanized antibodies prepared by a process comprising: maintaining a host transformed with a first expression vector encoding the light chain of the humanized antibody and a second expression vector encoding the heavy chain of the humanized antibody, wherein each chain is expressed, and isolating the humanized antibody formed by assembly. The first and second expression vectors may be the same vector. This document also provides a DNA sequence encoding the light or heavy chain of the humanized antibody; an expression vector incorporated into the DNA sequence; and a host transformed with the expression vector. Those skilled in the art can generate humanized antibodies based on the sequences and information provided herein without excessive experimentation. In one method, four general steps are employed to humanize a monoclonal antibody. The steps are: (1) determining the nucleotide and predicted amino acid sequences of the light and heavy variable domains of the starting antibody; (2) designing the humanized antibody, i.e., deciding which antibody framework region to use in the humanization process; (3) the actual humanization method / technique; and (4) transfection and expression of the humanized antibody.

[0414] Antibodies generated using AI methods

[0415] This article provides the peptide and nucleic acid sequences of TL1A antibodies that have been generated using AI and machine learning technologies.

[0416] As used herein, “de novo design” (e.g., AI-based de novo design of a single antibody CDR) refers to the structure-based design of antigen-targeting antibodies. In one embodiment, for de novo design, regions of the designed antibody are not provided as sequence input, and the experimental structure of the antibody complexed with the antigen is not provided as structural input. The method is generally described in WO 2023 / 154829, which is incorporated herein by reference in its entirety. Briefly, the method addresses the need for artificial intelligence (AI) and machine learning (ML) models trained to predict improved sequences or properties of biomolecules (e.g., antibodies or antibody fragments) using known biomolecules / binding coupler complexes as provided herein, in some embodiments, for several light chain element portions of the overall completed antibody. Specifically, the technique involves de novo design of antibodies against three different targets using a de novo deep learning model, where all designs are the result of a single-round model generation without subsequent optimization. The described technique demonstrates zero-sample antibody design through extensive wet laboratory testing. As a first step in fully de novo antibody design, the described techniques demonstrate the use of a model system of trastuzumab and its target antigen, human epidermal growth factor receptor 2 (HER2), as a model system to design HCDR3 via a generative AI approach. The techniques disclosed herein may include the de novo design of numerous (e.g., approximately 440,000 or more) unique HCDR3 variants of trastuzumab and screening for binding to HER2 using an activity-specific cell enrichment (“qaACE”) assay. As used herein, the terms “quantitative affinity activity-specific cell enrichment” or “qaACE assay” refer to a high-throughput assay used to obtain affinity and sequence data for biomolecular variants (U.S. Provisional Application No. 63 / 371,474, filed August 15, 2022, and PCT / US23 / 60167 and PCT / US23 / 72153, filed January 5, 2023, each of which is incorporated herein by reference in its entirety).

[0417] For example, quantitative affinity ACE (“qaACE”) and the ACE analysis further described herein, as well as so-called “de novo ACE” or “dnACE”, are methods for sampling the binding of antibody variants at high throughput using flow cytometry and next-generation sequencing. The primary goal of this method is to generate high-throughput binding information and / or training data for AI models to perform sequence-based binding predictions. This method can be applied to any antibody form, including but not limited to mab, Fab, scFv, scFab, VHH, nanobodies, and other conjugated drug forms.

[0418] As used herein, “backfolding” (e.g., AI-based design of a single antibody CDR based on backfolding) refers to designing antibody sequences using the experimental crystal structure or predicted antibody-antigen complex structure, where the prediction depends on the sequences of both the antibody and the antigen as input. In short, and similar to the above, this approach addresses the need for artificial intelligence (AI) and machine learning (ML) models trained to predict biomolecules (e.g., antibodies or antibody fragments) using known biomolecules / binding partner complexes. Specifically, the technique may include predicting the sequence of a biomolecule from its crystal structure, the sequence of the binding partner biomolecule, and / or the structure of the binding partner biomolecule.

[0419] By predicting biomolecular sequences that can fold into biomolecular structures, the described techniques can be applied both downstream and upstream in drug discovery, for biomolecular design and / or biomolecular validation. For example, optimized models can output sequences by determining how they fold based on desired specifications of the resulting structures (e.g., non-immunogenic structures, structures that are robust at different pH levels, more compact structures, and / or structures of a specific size, etc.).

[0420] Similarly, the sequences or structures of effective and proprietary biomolecules may not be publicly available. Biomolecule (e.g., antibodies or antibody fragments) sequence folding techniques can allow for diversification and further discovery of drugs (biomolecules) that share similar structures but have different and non-proprietary sequences. Alternatively or alternatively, downstream drug discovery can utilize biomolecule structure prediction to perform computer simulation validation of AI-designed biomolecule sequences, for example, by comparing a reference structure of the sequence with a biomolecule structure predicted using the techniques disclosed herein.

[0421] As used herein, “AI-guided affinity optimization” refers to the design of biomolecular (e.g., antibody) sequence variants with pre-specified properties. The methods are generally described in WO 2023 / 133462 and PCT application PCT / US23 / 72153, which are incorporated herein by reference in their entirety. In short, this method addresses the need for artificial intelligence (AI) and machine learning (ML) models trained using mappings between antibody sequence variants and experimental measurements (e.g., binding affinity, pH sensitivity, and other data types). Once trained, the model is able to predict the binding affinity of unseen sequence variants. The described techniques include deep contextual language models, which, combined with high-throughput and low-throughput binding affinity data, can predict the binding affinity of unseen antibody sequence variants spanning several (e.g., four) orders of magnitude KD. The techniques are also capable of measuring the “naturalness” of biomolecular (e.g., antibody) sequence variants, a widely applicable metric shown herein to be relevant to downstream issues related to drug development and immunogenicity. Therefore, the technology accelerates and improves the engineering of biomolecules (e.g., antibodies) and increases the success rate of practical applications (e.g., developing antibody drug candidates).

[0422] The aforementioned methods require experimental measurements (such as binding affinity) using or otherwise. WO 2021 / 146626 describes an activity-specific cell enrichment (ACE) assay for identifying host cells expressing an active gene product of interest (e.g., an antibody or fragment thereof as used herein) rather than an inactive substance; this literature is incorporated herein by reference in the relevant section. As an example, an active gene product can be distinguished from an inactive substance by its ability to specifically bind to a binding partner molecule, or by its ability to participate in a chemical or enzymatic reaction. The presence of properly formed disulfide bonds in a polypeptide gene product indicates its correct folding and presumes activity. In cell enrichment methods, the active gene product of interest is detected by utilizing an appropriate labeled complex that specifically binds to it. This could be, for example, a labeled antigen if the gene product of interest is an antibody or Fab; a labeled ligand if the gene product of interest is a receptor or receptor fragment, wherein the ligand specifically binds to the active conformation of the receptor; or a labeled substrate or labeled substrate analog if the gene product of interest is an enzyme, as an example. For any gene product of interest, if an available antibody or antibody fragment is available that specifically binds to the active gene product but not to the inactive gene product, the antibody or antibody fragment can be used to label the active gene product of interest when linked to a detectable portion.

[0423] A key advantage of ACE is its ability to screen tens of thousands of “variant units” in a single run. However, ongoing AI work for drug discovery places additional demands on wet-only screening, requiring further optimization of ACE to generate datasets suitable for AI. Wet-only screening, designed to select the best-performing variants, does not require quantitative analysis of the string from the assay. In fact, the iterative nature of this screening allows for re-screening of hits in step n-1, effectively eliminating n-1 false positives. Furthermore, wet-only screening is typically tailored to select only the population of interest (e.g., variants with higher affinity), and thus the assay does not need to be quantified over a large dynamic range of the parameter of interest (e.g., antibody affinity). However, AI models used to predict quantification benefit from training data that quantifies sequence variants. Thus, the training data for quantifying sequence variants needs to be accurate for the model to produce meaningful predictions. This disclosure addresses these needs and drawbacks.

[0424] As described herein, the aforementioned method can therefore utilize an enhanced version of ACE assay – quantitative affinity qaACE (“qaACE”) – as a method for high-throughput sampling of antibody variant affinity using flow cytometry and next-generation sequencing to generate a KD-related qaACE score. The primary goal of this method is to generate highly quantitative, high-throughput training data for AI models to perform sequence-based affinity prediction. This method can be applied to any antibody form, including but not limited to mab, Fab, scFv, scFab, VHH, nanobodies, etc., and can also be conceived to be appli...

Claims

1. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising at least one of the following: (a) Variable heavy chain complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein: (i) CDR-H1 contains a polypeptide sequence selected from any of the sequences of SEQ ID NO: 4, 14, 23, 28, 35, 39, 43, 46, 48, 64, 78, 84, 88, 94, 98, 109, 112, 115, 118, 120, 122, 125, 127, 130, and 137. (ii) CDR-H2 comprises a polypeptide sequence selected from any one of the sequences of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, and 133, and (iii) CDR-H3 comprises a polypeptide sequence selected from any one of the sequences of SEQ ID NO: 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128, and 135; and (b) Variable light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein: (i) CDR-L1 contains a polypeptide sequence selected from any of the sequences of SEQ ID NO: 1, 12, 19, 27, 41, 53, 61, and 69. (ii) CDR-L2 comprises a polypeptide sequence including a sequence of GAS, ATS, SAS, or YAS, or a sequence selected from any of SEQ ID NO: 62, 70, 72, 77, and 87, and (iii) CDR-L3 contains a polypeptide sequence selected from either SEQ ID NO: 3 or 63.

2. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising at least one of the following: (a) A variable heavy chain, wherein the variable heavy chain comprises a polypeptide sequence having at least 90% sequence identity with an amino acid sequence selected from any one of SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136, and 138; and (b) A variable light chain, wherein the variable light chain comprises a polypeptide sequence having at least 90% sequence identity with an amino acid sequence selected from any one of SEQ ID NO: 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93 and 97.

3. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising: (a) Variable heavy chain complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises a polypeptide sequence selected from any of SEQ ID NO: 4, 14, 23, 28, 35, 39, 43, 46, 48, 64, 78, 84, 88, 94, 98, 109, 112, 115, 118, 120, 122, 125, 127, 130, and 137; CDR-H2 comprises a polypeptide sequence selected from any of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, and 133; and CDR-H3 comprises a polypeptide sequence selected from any of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, and 133. The polypeptide sequence of any one of the following: 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128, and 135; and (b) Variable light chain complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, wherein CDR-L1 contains a polypeptide sequence selected from any of SEQ ID NO: 1, 12, 19, 27, 41, 53, 61 and 69, CDR-L2 contains a polypeptide sequence including a sequence of GAS, ATS, SAS or YAS or a sequence selected from any of SEQ ID NO: 62, 70, 72, 77 and 87, and CDR-L3 contains a polypeptide sequence selected from any of SEQ ID NO: 3 and 63.

4. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a variable heavy chain, wherein said variable heavy chain comprises a polypeptide sequence selected from any one of SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136 or 138; and a variable light chain, wherein said variable light chain comprises a polypeptide sequence selected from any one of SEQ ID NO: 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93 or 97.

5. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a variable heavy chain complementarity-determining region (CDR-H1), CDR-H2, and CDR-H3, wherein CDR-H1 comprises a polypeptide sequence selected from any one of SEQ ID NO: 4, 14, 23, 28, 35, 39, 43, 46, 48, 64, 78, 84, 88, 94, 98, 109, 112, 115, 118, 120, 122, 125, 127, 130, and 137; CDR-H2 comprises a polypeptide sequence selected from any one of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, and 133; and CDR-H3 comprises a polypeptide sequence selected from any one of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, and 133. The polypeptide sequence of any one of the following: 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128, and 135.

6. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a variable light chain complementarity-determining region (CDR-L1), CDR-L2, and CDR-L3, wherein CDR-L1 comprises a polypeptide sequence selected from any one of SEQ ID NO: 1, 12, 19, 27, 41, 53, 61, and 69; CDR-L2 comprises a polypeptide sequence comprising a sequence of GAS, ATS, SAS, or YAS or a sequence selected from any one of SEQ ID NO: 62, 70, 72, 77, and 87; and CDR-L3 comprises a polypeptide sequence selected from any one of SEQ ID NO: 3 and 63.

7. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising: The variable heavy chain complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3) are wherein CDR-H1 contains a polypeptide sequence selected from any one of SEQ ID NO: 4, 14, 23, 28, 35, 39, 43, 46, 48, 64, 78, 84, 88, 94, 98, 109, 112, 115, 118, 120, 122, 125, 127, 130, and 137; CDR-H2 contains a polypeptide sequence selected from any one of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, and 133; and CDR-H3 contains a polypeptide sequence selected from any one of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, and 133. The polypeptide sequence of any one of the following: 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128, and 135; and The variable light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 contains a polypeptide sequence selected from any of SEQ ID NO: 1, 12, 19, 27, 41, 53, 61, and 69; CDR-L2 contains a polypeptide sequence including a sequence of GAS, ATS, SAS, or YAS, or a sequence selected from any of SEQ ID NO: 62, 70, 72, 77, and 87; and CDR-L3 contains a polypeptide sequence selected from any of SEQ ID NO: 3 and 63.

8. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a variable heavy chain, said variable heavy chain comprising a polypeptide sequence selected from any one of SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136 and 138.

9. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a variable light chain, said variable light chain comprising a polypeptide sequence selected from any one of the sequences of SEQ ID NO: 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93 and 97.

10. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising: (a) A variable heavy chain, wherein the variable heavy chain comprises a polypeptide sequence selected from any one of SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136, or 138; and (b) An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a variable light chain, said variable light chain comprising a polypeptide sequence selected from any of the sequences of SEQ ID NO: 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93 and 97.

11. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising: (i) Variable heavy chain complementarity determination regions CDR-H1, CDR-H2 and CDR-H3, wherein (a) The CDR-H1 contains the amino acid sequence of SEQ ID NO:

4. (b) The CDR-H2 contains the amino acid sequence of SEQ ID NO: 5, and (c) The CDR-H3 contains the amino acid sequence of SEQ ID NO: 6; (ii) Variable light chain complementarity determination regions CDR-L1, CDR-L2, and CDR-L3, wherein: (a) CDR-L1 contains the amino acid sequence of SEQ ID NO:

1. (b) CDR-L2 contains the amino acid sequence of GAS, and (c) CDR-L3 contains the amino acid sequence of SEQ ID NO: 3; or (iii) The variable heavy chain complementarity determination regions CDR-H1, CDR-H2 and CDR-H3 of (i) and the variable light chain complementarity determination regions CDR-L1, CDR-L2 and CDR-L3 of (ii).

12. The antibody according to any one of claims 1 to 11, which is capable of binding to human TL1A.

13. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising: (a) A variable heavy chain comprising a polypeptide sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 9; (b) A variable light chain comprising a polypeptide sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 7; or (c) The variable heavy chain of (a) and (b) the variable light chain.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the antibody comprises an IgG, IgA, IgM or IgE antibody.

15. The antibody or antigen-binding fragment thereof according to claim 14, wherein the IgG comprises IgG1, IgG2, IgG3, IgG4, IgGA1 or IgGA2.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the antibody comprises a bispecific antibody, a multispecific antibody, a multivalent antibody, a chimeric antibody, a human antibody, a humanized antibody, a monoclonal antibody, a deimmunizing antibody, or a combination thereof.

17. The antibody or antigen-binding fragment thereof according to claim 16, wherein the antibody is a human antibody.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the antigen-binding fragment comprises Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, biantibody, linear antibody, single-domain antibody (sdAb), Camelidae V H H domain or multispecific antibodies formed from antibody fragments.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, wherein the antibody or antigen-binding fragment thereof is recombinant or synthetic.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the antibody or antigen-binding fragment thereof further comprises an enzyme, a substrate, a cofactor, a fluorescent marker, a chemiluminescent marker, a peptide tag, a magnetic particle, a drug, a toxin, or a combination thereof.

21. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, wherein the antibody or antigen-binding fragment thereof binds to TL1A.

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the antibody or antigen-binding fragment thereof inhibits inflammation.

23. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, wherein the antibody or antigen-binding fragment thereof inhibits the binding of TL1A to DR3 on host cells.

24. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, wherein the antibody or antigen-binding fragment is used to treat a disease, condition, or inflammation, including, for example, autoimmune diseases, including rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, systemic lupus erythematosus, asthma, ulcerative colitis, Crohn's disease, psoriasis, primary biliary cirrhosis, primary biliary cholangitis, ankylosing spondylitis, and fibrosis, including intestinal fibrosis, pulmonary fibrosis, and liver fibrosis.

25. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, wherein the antibody or antigen-binding fragment is capable of binding to human TL1A with an affinity of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times that of the affinity of the reference antibody for binding to the human TL1A.

26. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, wherein the antibody or antigen-binding fragment is capable of binding to human TL1A with an affinity at least 8.30 times greater than the affinity of a reference antibody for binding to said human TL1A.

27. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, wherein the antibody or antigen-binding fragment is capable of binding to human FcRn with a certain affinity, said affinity being at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the affinity of a reference antibody for binding to said human FcRn.

28. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, wherein the antibody or antigen-binding fragment is capable of binding to human FcRn with an affinity at least 6.6 to 9.1 times that of a reference antibody binding to said human FcRn.

29. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, wherein the antibody or antigen-binding fragment is in the form of approximately 0.05 nM, 0.10 nM, 0.15 nM, 0.20 nM, 0.25 nM, 0.3 nM, 0.25 nM, 0.40 nM, 0.45 nM, 0.50 nM, 0.55 nM, 0.60 nM, 0.65 nM, 0.70 nM, 0.75 nM, 0.80 nM, 0.85 nM, 0.90 nM, 0.95 nM, 1.00 nM, 1.10 nM, 1.20 nM, 1.30 nM, 1.40 nM, 1.50 nM, 1.60 nM, 1.70 nM, 1.80 nM, 1.90 nM, 2.0 nM, 3.0 nM, 4.0 nM. Affinities of nM, 5.0 nM, 6.0 nM, 7.0 nM, 8.0 nM, 9.0 nM, or 10.0 nM bind to human TL1A.

30. The antibody or antigen-binding fragment thereof according to any one of claims 25 to 28, wherein the affinity is determined by SPR.

31. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, wherein the antibody or antigen-binding fragment is capable of binding to HEK293 cells overexpressing TL1A on the surface of HEK293 cells at a certain gMFI, wherein the gMFI is at least 1-2 times the gMFI of a reference antibody binding to the HEK293 cells.

32. The antibody or antigen-binding fragment thereof according to claim 31, wherein gMFI is determined by flow cytometry.

33. A pharmaceutical composition or drug comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 32, and a pharmaceutically acceptable carrier, excipient, or diluent.

34. The pharmaceutical composition or medicament according to claim 33, formulated for administration by means of subcutaneous, intravenous, intradermal, intraperitoneal, intramuscular, intravenous, intracranial, intracavitary, or intracerebellar administration.

35. The pharmaceutical composition or preparation according to any one of claims 33 to 34, wherein it is in aqueous or lyophilized form.

36. The pharmaceutical composition or medicament according to any one of claims 33 to 35, comprising a delivery device selected from the group consisting of: syringes, blunt-tipped syringes, catheters, and implantable pumps.

37. The pharmaceutical composition or medicament according to any one of claims 33 to 36, wherein it comprises an additional therapeutic agent.

38. The pharmaceutical composition or medicament according to claim 37, wherein the additional therapeutic agent is a nonsteroidal anti-inflammatory drug, a corticosteroid, a dietary supplement such as an antioxidant, a small molecule, a therapeutic vaccine, an immunomodulator, an antiviral agent, acetaminophen, an anticancer agent, chemotherapy, or another anti-TL1A antibody.

39. A method for preventing a disease, condition, or inflammation in a subject of need, said disease, condition, or inflammation including, for example, autoimmune diseases including rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, systemic lupus erythematosus, asthma, ulcerative colitis, Crohn's disease, psoriasis, primary biliary cirrhosis, primary biliary cholangitis, ankylosing spondylitis, and fibrosis, including intestinal fibrosis, pulmonary fibrosis, and liver fibrosis, said method comprising administering to said subject a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 32 or a pharmaceutical composition according to any one of claims 33 to 38.

40. A method for treating a disease, condition, or inflammation in a subject in need, said disease, condition, or inflammation including, for example, autoimmune diseases including rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, systemic lupus erythematosus, asthma, ulcerative colitis, Crohn's disease, psoriasis, primary biliary cirrhosis, primary biliary cholangitis, ankylosing spondylitis, and fibrosis, including intestinal fibrosis, pulmonary fibrosis, and liver fibrosis, said method comprising administering to the subject the following: (a) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 32; or (b) The pharmaceutical composition or drug according to any one of claims 33 to 38.

41. The method according to any one of claims 39 to 40, wherein the antibody or its antigen-binding fragment binds to TL1A.

42. The method according to any one of claims 39 to 41, wherein the antibody or its antigen-binding fragment inhibits the binding of TL1A to DR3 on host cells.

43. The method according to any one of claims 39 to 42, wherein the application reduces one or more symptoms.

44. The method according to any one of claims 39 to 43, wherein the antibody or its antigen-binding fragment is administered to the subject together with an additional therapeutic agent, wherein the additional therapeutic agent is one or more of the following: aminosalicylate, balsalazide, olsalazine, prednisone, azathioprine, 6-mercaptopurine, methotrexate, infliximab, adalimumab, certolizumab pegol, and natalizumab.

45. The method of claim 44, wherein the additional therapeutic agent is a nonsteroidal anti-inflammatory drug, a corticosteroid, a dietary supplement such as an antioxidant, a small molecule, a therapeutic vaccine, an immunomodulator, an antiviral agent, acetaminophen, an anticancer agent, chemotherapy, or another anti-TL1A antibody.

46. ​​A hybridoma that produces an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 32.

47. A fusion protein comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 32.

48. An immunoconjugate comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 32, and a therapeutic agent.

49. An isolated nucleic acid encoding one or more or all CDRs of an antibody according to any one of claims 1 to 32.

50. An isolated nucleic acid comprising at least one of the following: (a) The nucleic acid sequence of CDR-H1 encoding the sequence selected from SEQ ID NO: 4, 14, 23, 28, 35, 39, 43, 46, 48, 64, 78, 84, 88, 94, 98, 109, 112, 115, 118, 120, 122, 125, 127, 130 and 137; (b) The nucleic acid sequence encoding CDR-L1 of the sequences selected from SEQ ID NO: 1, 12, 19, 27, 41, 53, 61 and 69; (c) The nucleic acid sequence of CDR-H2 encoding the sequence selected from SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131 and 133; (d) A nucleic acid sequence encoding a sequence selected from GAS, ATS, SAS or YAS or a sequence selected from any of SEQ ID NO: 62, 70, 72, 77 and 87; (e) A nucleic acid sequence encoding CDR-H3 of the sequence selected from SEQ ID NO: 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128, and 135; or (f) The nucleic acid sequence encoding CDR-L3 selected from the sequences of SEQ ID NO: 3 and 63.

51. An isolated nucleic acid comprising at least one of the following: (a) A nucleic acid sequence encoding a heavy chain polypeptide of an antibody, said nucleic acid sequence being selected from any one of the sequences of SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136, or 138, and (b) A nucleic acid sequence encoding a light chain polypeptide of an antibody, said nucleic acid sequence being selected from any one of the sequences of SEQ ID NO: 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93 and 97.

52. An isolated nucleic acid comprising a nucleic acid sequence encoding a heavy chain polypeptide of an antibody, said nucleic acid sequence being selected from any one of the sequences of SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136, or 138.

53. An isolated nucleic acid comprising a nucleic acid sequence encoding a light chain polypeptide of an antibody, said nucleic acid sequence being selected from any one of the sequences of SEQ ID NO: 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93 and 97.

54. An expression vector comprising the isolated nucleic acid molecule according to any one of claims 49 to 53.

55. The expression vector of claim 50, wherein the isolated nucleic acid is operatively linked to a regulatory control sequence.

56. A host cell comprising an expression vector according to claim 54 or 55 or an isolated nucleic acid molecule according to any one of claims 49 to 53.

57. The host cell of claim 56, wherein the host cell is a mammalian cell or a bacterial cell.

58. The host cell according to claim 56 or claim 57, wherein the bacterial cell is an Escherichia coli cell.

59. The host cell according to any one of claims 56 to 58, wherein the expression of the nucleic acid is controlled by one or more inducible promoters.

60. A method for inhibiting the binding of TL1A to host cells expressing DR3, the method comprising contacting the host cells with an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 32.

61. A method for generating an antibody or an antigen-binding fragment thereof, the method comprising: (a) culturing host cells according to any one of claims 56 to 59 in a culture medium under conditions that allow expression of a polypeptide encoded by the isolated nucleic acid and assembly of the antibody or its antigen-binding fragment; and (b) Purify the antibody or its antigen-binding fragment from cultured cells or cell culture medium.

62. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising at least one of the following: (a) Variable heavy chain complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein: (i) CDR-H1 contains a polypeptide sequence selected from any one of SEQ ID NO: 4, 14, 23, 28, 35, 39, 43, 46, 48, 64, 78, 84, 88, 94, 98, 109, 112, 115, 118, 120, 122, 125, 127, 130, and 137. (ii) CDR-H2 contains a polypeptide sequence selected from any one of SEQ ID NO: 5, 15, 29, 36, 49, 65, 79, 89, 99, 104, 131, and 133, and (iii) CDR-H3 comprises a polypeptide sequence selected from any one of SEQ ID NO: 6, 16, 20, 24, 30, 37, 44, 50, 59, 66, 73, 80, 95, 100, 102, 105, 107, 110, 113, 123, 128, and 135; and (b) Variable light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein: (i) CDR-L1 contains a polypeptide sequence selected from any one of SEQ ID NO: 1, 12, 19, 27, 41, 53, 61, and 69. (ii) CDR-L2 comprises a polypeptide sequence including a sequence of GAS, ATS, SAS, or YAS, or a sequence selected from any of SEQ ID NO: 62, 70, 72, 77, and 87, and (iii) CDR-L3 contains a polypeptide sequence selected from either SEQ ID NO: 3 or 63.

63. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising at least one of the following: (c) A variable heavy chain, wherein the variable heavy chain comprises a polypeptide sequence having at least 90% sequence identity with an amino acid sequence selected from any one of SEQ ID NO: 9, 18, 22, 26, 32, 38, 40, 45, 47, 52, 55, 60, 68, 75, 82, 91, 96, 101, 103, 106, 108, 111, 114, 119, 121, 124, 126, 129, 132, 134, 136, or 138; and (d) A variable light chain, wherein the variable light chain comprises a polypeptide sequence having at least 90% sequence identity with an amino acid sequence selected from any one of SEQ ID NO: 7, 17, 21, 25, 31, 34, 42, 51, 54, 57, 58, 67, 71, 74, 76, 81, 83, 85, 86, 90, 92, 93 or 97.

64. A method for preparing an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is capable of binding to TL1A with higher affinity than a reference antibody, the method comprising: (a) Identify a reference antibody or fragment thereof containing one or more CDR sequences associated with TL1A binding activity; (b) Optional identification of frame sequences; (c) Modifying one or more amino acids in one or more CDR sequences in the CDR sequence to produce a variant antibody, wherein one or more amino acid modifications are predicted to make the variant antibody have a higher affinity for TL1A than the reference antibody for TL1A, and wherein the prediction is generated by a computational system.

65. The method of claim 64, wherein the computing system comprises: One or more processors; and One or more non-transitory computer-readable media, wherein the one or more non-transitory computer-readable media stores thereon: A machine learning model trained using training data, wherein the training data includes one or more training antibody sequence variants, each training antibody sequence variant having a corresponding measured binding property, the corresponding measured binding property representing the ability of each training antibody sequence variant to bind to a corresponding corresponding binding partner, and wherein the machine learning model is configured to output predicted antibody binding properties of input antibody sequence variants. as well as Instructions, which, when executed by the one or more processors, cause the computing system to: The machine learning model is used to process one or more antibody sequence variants to generate one or more predicted binding properties, each predicted binding property corresponding to a specific antibody sequence variant among the one or more antibody sequence variants; Analyze the one or more predicted binding properties to identify one or more antibody sequence variants of interest from the one or more antibody sequence variants of interest, each of the one or more antibody sequence variants of interest having a corresponding one or more desired properties; and Provide one or more antibody sequence variants of interest as output.

66. The method of claim 64, wherein the computing system comprises: One or more processors; and One or more non-transitory computer-readable media, wherein the one or more non-transitory computer-readable media stores thereon: A machine learning antibody prediction model, wherein the machine learning antibody prediction model is trained to predict the structural information of antibodies based on input; and Instructions, which, when executed by the one or more processors, cause the computing system to: (1) Receiving target input, the target input comprising one or more of the following: a primary sequence of the target binding mate, three-dimensional coordinates of the target binding mate, a primary sequence of the target binding mate epitope, or three-dimensional coordinates of the primary sequence of the target binding mate epitope, or a fragment or portion thereof; and (2) The structural information of the target antibody is predicted by processing the target input with the machine learning antibody prediction model.

67. The method of claim 64, wherein the computing system comprises: One or more processors; and One or more non-transitory computer-readable media, wherein the one or more non-transitory computer-readable media stores thereon: A machine learning affinity prediction artificial neural network, wherein the machine learning affinity prediction artificial neural network comprises: (i) One or more antibody prediction layers, which are trained to predict antibody structure information from target input; (ii) One or more docking layers, said one or more docking layers being trained to generate docking complexes from two or more input three-dimensional antibodies; as well as (iii) One or more affinity prediction layers, which are trained to predict affinity based on the input docking complex; The one or more antibody prediction layers, the one or more docking layers, and the one or more affinity prediction layers are connected; and Instructions, which, when executed by the one or more processors, cause the computing system to: Receives a target input containing one or more of the following: a target-binding mating body sequence, a target-binding mating body, or a target epitope; and The affinity prediction artificial neural network is used to process the target input to generate a docking complex corresponding to the target input and a corresponding structural affinity value.

68. The method of claim 64, wherein the computing system performs antigen-sensing antibody folding, and comprises: One or more processors; and One or more memories, wherein the one or more memories store: A machine learning model trained to predict numerical representations of output antibodies corresponding to output antibodies using one or more training inputs, the one or more training inputs comprising one or more training antibody numerical representations, each of the one or more training antibody numerical representations corresponding to a corresponding one of a plurality of training antibodies; and A set of computer-executable instructions that, when executed by the one or more processors, cause the computing system to: Receive multiple input antibody digital representations, each of which corresponds to a specific input antibody among a plurality of input antibodies; and The machine learning model is used to process one or more of the multiple input antibody digital representations to produce one or more predicted output antibody digital representations.

69. An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is capable of binding to TL1A with higher affinity than a reference antibody, said antibody or the antigen-binding fragment thereof comprising one or more of the following: (a) At least one HCDR sequence, which, compared to the reference antibody, comprises one or more amino acid modifications, and wherein the one or more amino acid modifications are predicted to give the antibody a higher affinity for TL1A than the reference antibody for TL1A, and wherein the prediction is generated by a computational system; and / or (b) At least one LCDR sequence, which, compared to the reference antibody, comprises one or more amino acid modifications, and wherein the one or more amino acid modifications are predicted to give the antibody a higher affinity for TL1A than the reference antibody for TL1A, and wherein the prediction is generated by a computational system; At least one LCDR is unmodified compared to the reference antibody.

70. The antibody or antigen-binding fragment thereof according to claim 69, wherein the antibody or antigen-binding fragment thereof comprises amino acid modifications of HCDR1, HCDR2 and / or HCDR3 compared to the reference antibody.

71. The antibody or antigen-binding fragment thereof according to claim 69 or 70, wherein the antibody or antigen-binding fragment thereof comprises amino acid modifications of LCDR1, LCDR2 and / or LCDR3 compared to the reference antibody.

72. The antibody or antigen-binding fragment thereof according to any one of claims 69 to 71, comprising a frame region different from the frame region of the reference antibody.

73. The antibody or antigen-binding fragment thereof according to any one of claims 69 to 72, which is produced by the method according to any one of claims 64 to 68.

74. A formulation comprising an antibody according to any one of claims 1 to 32 or 69 to 73 in a solution comprising histidine, arginine hydrochloride and an emulsifier.

75. The formulation according to claim 74, comprising 20 mM His, 140 mM Arg HCl, pH 6.0 and 0.04% PS80 (w / v).

76. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 32 or 69 to 73, comprising a half-life of about 10 days.

77. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 32 or 69 to 73, comprising at least one of the following: (a) The variable heavy chain complementarity determination regions CDR-H1, CDR-H2 and CDR-H3 for the following: i. HCDR1 (SEQ ID NO: 4), HCDR2 (SEQ ID NO: 5), HCDR3 (SEQ ID NO: 20), ii. HCDR1 (SEQ ID NO: 28), HCDR2 (SEQ ID NO: 29), HCDR3 (SEQ ID NO: 30), and / or iii. HCDR1 (SEQ ID NO: 4), HCDR2 (SEQ ID NO: 5), HCDR3 (SEQ ID NO: 20); and (b) The variable light chain complementarity determination regions CDR-L1, CDR-L2, and CDR-L3 for the following: i. LCDR1 (SEQ ID NO: 19), LCDR2 (GAS), LCDR3 (SEQ ID NO: 3), ii. LCDR1 (SEQ ID NO: 27), LCDR2 (GAS), LCDR3 (SEQ ID NO: 3), and / or iii. LCDR1 (SEQ ID NO: 41), LCDR2 (SAS), LCDR3 (SEQ ID NO: 3).

78. The antibody or antigen-binding fragment thereof according to claim 77, wherein the antibody or antigen-binding fragment thereof is capable of binding to human TL1A with an affinity at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times that of a reference antibody binding to human TL1A, and / or the antibody or antigen-binding fragment thereof is stable in a formulation comprising 20 mM His, 140 mM Arg HCl, pH 6.0, and 0.04% PS80 (w / v), and / or the antibody or antigen-binding fragment thereof has a half-life of 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days or longer compared to a reference antibody.

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