ANTIGEN-BINDING ANTI-TL1A PROTEINS AND USES THEM

Description

[0001] This application claims the benefit of Application No. PCT / US2016 / 052006, filed September 15, 2016, and U.S. Provisional Application Nos.: 62 / 268,432, filed December 16, 2015 and 62 / 333,063, filed May 6, 2016.FIELD OF THE INVENTION

[0002] The invention relates to an antigen binding protein specific for TL1A, wherein the antigen binding protein comprises a combination of sequences for LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 selected from the following Table A, wherein the combination of sequences for LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 is selected from the rows shown in Table A: LCDR1 (SEQ ID NO) LCDR2 (SEQ ID NO) LCDR3 (SEQ ID NO) HCDR1 (SEQ ID NO) HCDR2 (SEQ ID NO) HCDR3 (SEQ ID NO) DASSLQS (100)SYGMH (164)ATSSLQS (106)SYFWS (170)AASSLQS (112)SYFWS (170)WASTRES (118)TNSVAWN (182)WASTRES (118)TNSVAWN (182)WASTRES (118)TNSVAWN (182)GASSRAT (124)GYYWN (188)GASSRAT (124)GYYWN (188)AASSLQS (112)SYGMH (164)LGSSRAS (685)TYYMS (777)WASTRES (118)TYGMH (783)WASTRES (118)SYGMH (164)GNNNRPS (699)SYVMS (792)GNSHRPS (705)NYAMS (798)GNSHRPS (705)NYAMN (804)AASSLQS (112)SSSATWN (809)TASSLQS (721)SNSATWN (815)SNNKRPS (725)GFYMH (819)VASSLQS (731)GYYWS (265)AASGLQG (737)SYGMH (164)AASSLQS (112)SYAMS (836)TASSLQS (749)GYYWS (265)KVSNWD S (755)AYYMH (847)AASRLQS (761)AYYMH (847)AASSLQS (112)SYAMS (836)EVAGAFDI (855)GASRLQS (769)GYYMH (857)AASSLQS (112)SYAMS (836)EVAGAFDI (855)

[0003] The invention further describes nucleic acids, expression vectors, a host cell, a method for the preparation, a pharmaceutical composition and medical uses related to the antigen binding protein specific for TL1A of the invention.BACKGROUND OF THE INVENTION

[0004] Cytokines are soluble, small proteins that mediate a variety of biological effects concerning the immune system. Such biological effects include induction of immune cell proliferation, development, differentiation, and / or migration; regulation of the growth and differentiation of many cell types; an inflammatory response through local or systemic accumulation of immune cells; and host-protective effects. See, for example, Arai et al., Annu. Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin. Immunol. 3:311 (1991); Paul et al., Cell, 76:241 (1994)). Such immune effects can produce pathological consequences when the effect leads to excessive and / or chronic inflammation, as in autoimmune disorders (such as multiple sclerosis) and cancer / neoplastic diseases. Oppenheim et al., eds., Cytokine Reference, Academic Press, San Diego, Calif. (2001); von Andrian et al., New Engl. J. Med., 343:1020 (2000); Davidson et al., New Engl. J. Med., 345:340 (2001); Lu et al., Mol. Cancer Res., 4:221 (2006); Dalgleish et al., Cancer Treat Res., 130:1 (2006).

[0005] TLIA (TNFSF15) is a cytokine, a TNF-α family member involved in T cell activation (Richard et al., J Leukoc Biol. 2015 Sep, 98(3):333-45). It is the ligand for Death Receptor 3 (DR3), also known as TNFRSF25. TL1A is mainly expressed at low basal level in monocytes, dendritic cells and endothelial cells, but highly induced after immune complex and cytokine and microbes stimulation. Multiple genome-wide association studies (GWAS) demonstrated TL1A single nucleotide polymorphisms (SNPs) associated with Crohn's disease in various ethnic populations. In addition, TL1A inflammatory bowel disease (IBD) risk SNPs were reported to be associated with Crohn's disease severity (Hirano, IBD 19: 526, 2013). In the preclinical studies, TL1A protein treatment exacerbated colitis development in the colitis prone mdr1a- / - mice, but not in the wild-type mice. Altogether, human genomic data and preclinical colitis model data demonstrate that TL1A plays an important role in IBD development, and its blockage will be beneficial for IBD treatment.

[0006] Tumor Necrosis Factor-α (TNF-α) is a cytokine involved in the regulation of various physiological and pathological process (Buetler, et al., J Rheumatol Suppl. 57: 16-21, 1999). It is implicated in tumor regression, septic shock, cachexia, and a number of inflammatory and autoimmune conditions. Fransen et al. (June 1985), "Molecular cloning of mouse tumor necrosis factor cDNA and its eukaryotic expression," Nucleic Acids Res. 13 (12) 4417-29; Kriegler et al. (April 1988), "A novel form of TNF-α / cachectin is a cell surface cytotoxic transmembrane protein: ramifications for the complex physiology of TNF-α ," Cell 53 (1): 45-53. TNF-α inhibitors are a class of therapeutics approved to treat rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, plaque psoriasis, Crohn's disease, and ulcerative colitis (Sethi et al., Adv Exp Med Biol. 2009;647:37∼51). TNF-α inhibitors include etanercept, adalimumab, certolizumab pegol, infliximab, and golimumab.

[0007] US 2012 / 114654 A1 relates to human antibodies to human TNF-like ligand 1A (TL1A).

[0008] WO 2014 / 144600 A2 relates to multivalent and monovalent multi-specific complexes and their uses.

[0009] WO 2014 / 161845 A1 relates to bispecific antibodies specific for FAP and DR5, antibodies specific for DR5 and methods of use.

[0010] Wu X. et al., mAbs, vol. 7:3, May / June 2015, pages 470-482, relates to Fab-based bispecific antibody formats with robust biophysical properties and biological activity.

[0011] Lewis et al., Nature Biotechnology, vol. 32(2), 26 January 2014, pages 191-198, relates to the generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface.

[0012] Cain et al., Science Business Exchange: SCIBX, Nature Publishing Group, vol. 4, no. 28, 21 July 2011, pages 1-3, relates to ways of producing bispecific antibodies.

[0013] Fischer et al., Arthritis & Rheumatology, vol. 67(1), January 2015, pages 51-62, describes the development and characterization of a novel bispecific antibody.

[0014] Approximately 50% of patients respond to treatment with TNF inhibitors. However, only approximately 40% of those patients maintain responses after one year of treatment. Therefore, there is strong need for therapeutics with large effect size with durable response. We hypothesize that targeting multiple inflammatory pathways, such as TNF and TL1A, will likely achieve larger effect size with anticipated safety profile.SUMMARY OF THE INVENTION

[0015] The invention is set out in the appended claims.

[0016] One aspect not covered by the claims relates to

[0017] bispecific antigen binding proteins, particularly bispecific antibodies. Bispecific antigen binding proteins of the invention comprise a TL1A binding entity and a TNF-α binding entity. TL1A blocking proteins and TNF-α blocking proteins have different effects in studies measuring inhibition of induction of cytokines IFNy, IL-5, IL-6, IL-8, and IL-10. TL1A but not TNF-α induces T cell activation in vivo. TL1A and TNF-α induce NF-κB in different cell types in human peripheral blood monocytes (PBMCs). TL1A and TNF-α further induce different cytokines in human PBMCs. TL1A and TNF-α induce some of the same genes but in different cell types (16 overlapping genes induced by TL1A in whole blood and TNF-α in NCM460 cells; 13 overlapping genes induced by TL1A in whole blood and TNF-α in PBMCs). There is a strong genetic association of TL1A with inflammatory bowel disease (IBD) and anti-TNF agents are clinically validated for treatment of IBD. Such bispecific antigen binding proteins thus are useful for treatment of IBD and other autoimmune and inflammatory conditions. For these and other reasons, there is a benefit to bispecific antigen binding proteins that specifically bind TL1A and TNF-α .

[0018] One format for such bispecific antigen binding proteins is heterodimeric immunoglobulins (hetero Ig). Such hetero Ig antigen binding proteins have one heavy chain-light chain pair directed to TL1A and another directed to TNF-α .

[0019] Another format for such bispecific antigen binding proteins is IgG-scFv molecules. In an IgG-scFv, each heavy chain of an antibody that specifically binds one target is linked to a single chain antibody (scFv) that specifically binds the other target. The scFv portion can be linked to the heavy chain of the IgG portion directly or through a peptide linker. In the IgG-scFv format, the IgG is directed to TL1A and the scFv portion to TNF-α or vice-versa. Bispecific antigen binding proteins in the IgG-scFv format have the advantage of being bivalent for both of their target antigens.

[0020] The invention is set out in the appended claims.

[0021] A further format for such bispecific antigen binding proteins is IgG-Fab molecules. In this format, the antigen binding protein has a structure such that a Fab molecule that binds to one target is fused to each heavy chain of an IgG molecule that binds to another target. One chain of a Fab portion can be linked directly or through a peptide linker to the C-terminus of a heavy chain of the IgG portion. The resulting molecule has the advantage of being tetravalent and bispecific. All variations of the IgG-Fab format preferably comprise the CDR sequences of Tables 21.2A and 21.2B hereinafter. Preferred heavy and light chain sequences of IgG-Fab molecules appear in Table 21.1 hereinafter.

[0022] Aspects not covered by the claims are bispecific antigen binding proteins, such as IgG-Fab molecules. In this format, each heavy chain of an antibody that specifically binds to one target is linked to a Fab fragment that specifically binds to the other target. In the IgG-Fab format, the IgG is directed to TL1A and the Fab portion to TNF-α or vice-versa. Bispecific antigen binding proteins in the IgG-Fab format have the advantage of being bivalent for both of their target antigens. Other formats for bispecific antigen binding proteins within the scope of this invention are described infra.

[0023] The invention also relates to isolated nucleic acids encoding the TL1A-specific antigen binding proteins of the invention, as well as vectors comprising the nucleic acids, host cells comprising the vectors, and methods of making and using the TL1A-specific antigen binding proteins.

[0024] The invention also relates to isolated nucleic acids encoding the antigen binding proteins of the invention, as well as vectors comprising the nucleic acids, host cells comprising the vectors, and methods of making and using the bispecific antigen binding proteins.

[0025] In other embodiments, the present invention provides compositions comprising the TL1A-specific antigen binding proteins, and kits comprising the anti-TL1A antigen binding proteins, as well as articles of manufacture comprising the anti-TL1A antigen binding proteins.

[0026] The TL1A-specific antigen binding proteins of the invention are for use in a method of treating an inflammatory disease characterized by the presence of elevated levels of TL1A in a patient in need thereof, as defined in the appended claims. The present invention also provides pharmaceutical compositions comprising a TL1A-specific antigen binding protein and a pharmaceutically acceptable diluent, excipient or carrier, as defined in the appended claims.

[0027] The references to the methods of treatment by therapy in this description are to be interpreted as references to the TL1A-specific antigen binding proteins and pharmaceutical compositions of the invention for use in those methods.

[0028] The invention further relates to methods of treatment using the TL1A-specific antigen binding proteins. The TL1A-specific antigen binding proteins of the present invention are useful for the inhibition of the proinflammatory cytokine TL1A. The antibodies can be used to reduce, limit, neutralize, or block the proinflammatory effects of TL1A. Thus, in some embodiments, the invention relates to the treatment of IBD and other autoimmune or inflammatory conditions using the TL1A-specific antigen binding proteins.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 shows a schematic representation of four bispecific hetero Ig formats used to generate anti-TL1A / anti-TNF-α bispecific antigen binding proteins. As shown, a heavy chain directed to one antigen is disulfide-bonded to a heavy chain directed to a different antigen. A light chain for each antigen is disulfide-bonded to the heavy chain for the corresponding antigen. Figure 1 shows a preferred embodiment, in which the heavy and light chains comprise charge mutations to aid in correct association of the heavy and light chains. The Kabat-Eu numbering scheme is used to denote the positions of charge pair mutations within each of the chains and for all sequences throughout this specification. This IgG-like bispecific antigen binding protein format is a heterotetramer comprising two different light chains and two different heavy chains. HC1 and LC1 refer to the heavy chain and light chain, respectively, of one Fab binding arm and HC2 and LC2 refers to the heavy chain and light chain, respectively, of the second Fab binding arm. For example, in the schematic, HC1 and LC1 correspond to the anti-TL1A receptor binding arm and HC2 and LC2 correspond to the anti-TNF-α binding arm. However, the two binding arms can be switched such that HC1 and LC1 correspond to the anti-TNF-α binding arm and HC2 and LC2 correspond to the anti-TL1A receptor binding arm. Figure 1 is for reference only.

[0030] Figure 2 shows a schematic representation of the IgG-scFv format used to generate anti-TL1A / anti-TNF-α bispecific antigen binding proteins. As shown, the structure incorporates a full tetrameric IgG directed to one antigen. A single-chain variable fragment (scFv), which comprises variable domains from a second antibody linked together by a glycine-serine linker, is fused to the carboxyl terminus of the heavy chain of a first antibody through a peptide linker to produce a modified heavy chain. Although the VH-VL orientation of the variable domains within the scFv is shown, the variable domains may also be organized in a VL-VH orientation. The complete molecule is a multimer comprising two heavy chains (but one unique heavy chain sequence) and two light chains (but one unique light chain sequence) from the first antibody. Figure 2 is for reference only.

[0031] Figure 3 concerns MabSelect SuRe affinity chromatography of an anti-TL1A / anti-TNF-α Hetero-Ig. It shows a representative FPLC protein A affinity capture chromatogram of an anti-TL1A / anti-TNF-α hetero-Ig. The protein was eluted with a step gradient of 100 mM acetic acid (conductivity: black trace, dashed), pH 3.6 and pooled based on the A280 (black trace, solid).

[0032] Figure 4 shows a representative FPLC SP high performance sepharose purification chromatogram of an anti-TL1A / anti-TNF-α hetero-Ig. Protein was eluted with an increasing salt gradient (conductivity : black trace, dashed) and was pooled based on the A280 elution profile (black trace, solid) and Caliper LabChip analysis of fractions.

[0033] Figure 5 shows a representative FPLC SP high performance sepharose purification chromatogram of an anti-TL1A / anti-TNF-α hetero-Ig. Protein was eluted with decreasing ammonium sulfate gradient (conductivity : black trace, dashed) and pooled based on the A280 elution profile (black trace, solid) and Caliper LabChip analysis of fractions.

[0034] Figure 6 concems Caliper analysis of a TL1A / TNF-α hetero-Ig. It shows non-reduced and reduced Caliper analysis of an anti-TL1A / anti-TNF-α hetero-Ig.

[0035] Figure 7 concems SE-HPLC analysis of an anti-TL1A / anti-TNF-α hetero-Ig. It shows size exclusion chromatography on 30 µg of the final anti-TL1A / anti-TNF-α hetero-Ig product injected onto a Sepax Zenix-C SEC-300 column (7.8 x 300 mm) in 50 mM NaH 2 PO 4 , 250 mM NaCl, pH 6.9 at 1 ml / min, observing the absorbance at 280 nm (black trace).

[0036] Figure 8 concerns LC-MS of Non-Reduced Hetero-Ig (Theoretical mass: 145495 Da). Figure 8 shows mass analysis of 20 µg non-reduced anti-TL1A / anti-TNF-α hetero-Ig eluted from a reverse-phase HPLC gradient using an Agilent Zorbax 300SB-C8 column (2.1 x 50 mm 3.5 µm) and mobile phases of 0.1% TFA and 90% n-Propanol / 0.1% TFA (mobile phases A and B, respectively), equipped with an Agilent 6230 ESI-TOF Mass Spectrometer.

[0037] Figure 9 shows mass analysis of 20 µg TL1A / TNF-α hetero-Ig after limited Lysyl Endoproteinase C digestion for 30 minutes in 100 mM TRIS, pH 8. Reverse-phase HPLC was conducted using an Agilent Zorbax 300SB-C8 column (2.1 x 50 mm 3.5 µm) and mobile phases of 0.1% TFA and 90% n-propanol / 0.1% TFA (mobile phases A and B respectively) and mass detection on an Agilent 6230 ESI-TOF Mass Spectrometer. Theoretical masses for TL1A Fab, TNF Fab, and Fc are 47350 Da, 48002 Da, and 50175 Da, respectively.

[0038] Figure 10 concems MabSelect SuRe affinity chromatography of a anti-TL1A / anti-TNF-α IgG-scFv. It shows a representative FPLC protein A affinity capture chromatogram of an anti-TL1A / anti-TNF-α IgG-scFv. The protein was eluted with a step gradient of 100 mM acetic acid, pH 3.6 and pooled based on the A280 (black trace).

[0039] Figure 11 concems SP Sepharose High Performance chromatography of an anti-TL1A / anti-TNF-α IgG-scFv. It shows a representative FPLC Superdex 200 purification chromatogram of an anti-TL1A / anti-TNF-α IgG-scFv. Protein was eluted with isocratic gradient of buffer and pooled based on the A280 elution profile (black trace) and SE-HPLC analysis of fractions.

[0040] Figure 12 shows non-reduced and reduced Caliper analysis of an anti-TL1A / anti-TNF-α IgG-scFv.

[0041] Figure 13 concerns SE-HPLC analysis of an anti-TL1A / anti-TNF-α IgG-scFv. It shows size exclusion chromatography on 30 µg of the final anti-TL1A / anti-TNF-α IgG-scFv product injected onto a Sepax Zenix-C SEC-300 column (7.8 x 300 mm) in 50 mM NaH 2 PO 4 , 250 mM NaCl, pH 6.9 at 1 ml / min, observing the absorbance at 280 nm.

[0042] Figure 14 concerns IdeS Protease digested Ig-scFv. It shows mass analysis of 20 µg Ig-scFv using reverse-phase HPLC separation on an Agilent Zorbax 300SB column (2.1 x 50 mm, 3.5 µm) with mobile phases of 0.1% TFA and 90% n-propanol / 0.1% TFA (mobile phases A and B, respectively), and detection on an Agilent 6230 ESI-TOF Mass Spectrometer.

[0043] Figure 15 shows genotyping of TL1A SNPs. To evaluate potential TL1A genotype association with expression, genomic DNA (gDNA) was isolated from healthy PBMC donors using Gentra Puregene Tissue kit from Qiagen. Genomic DNA were genotyped using TaqMan SNP genotyping assays for rs7848647, rs6478109, rs6478108, and rs3810936 assays from LifeTech and standard protocols on the Bio-Rad droplet digital PCR platform. Donors were considered homozygous risk haplotype if only risk alleles were present at all 4 genotyped SNPs (rs7848647, rs6478109, rs6478108, and rs3810936). Donors were considered homozygous non-risk haplotype if only non-risk alleles were present at all 4 genotyped SNPs. Donors were considered heterozygous haplotype if both risk and non-risk alleles were present at all 4 genotyped SNPs. Donors that were considered "recombinant" had only homozygous risk alleles at rs7848647, rs6478109, and rs6478108, but were heterozygous (risk and non-risk alleles present) at rs3810936.

[0044] Figures 16A and 16B show higher fold induction of TL1A risk allele than non-risk allele in a heterozygous PBMC AEI Study. Frequency of risk vs. non-risk allele usage in heterozygous PBMC at basal level or after immune complex stimulation at various time points was examined by droplet digit PCR (ddPCR) using synonymous SNP (rs3810936) allelic specific fluorescent probes. The allelic expression ratio was calculated by dividing the copies / ml of the risk allele by the copies / ml of the non-risk allele. Total copy number of each allele was normalized by the input amount of cDNA (copies / ng), and the fold-induction for each allele at each time point was calculated by dividing the copies / ng at the time point of interest by the copies / ng at baseline (0 hr).

[0045] Figure 17 shows that TL1A SNPs in promoter and intron contribute to allelic expression imbalance regulation. The inventor(s) identified a small number of donors homozygous for risk alleles at rs7848647, rs6478109, and rs6478108, but heterozygous at rs3810936, likely due to recombination between rs6478109 and synonymous SNP rs3810936. Frequency of risk vs. non-risk allele usage in heterozygous or recombinant donor PBMC was examined by droplet digit PCR (ddPCR) using synonymous SNP (rs3810936) allelic specific fluorescent probes. Compared to heterozygous donors, no allelic expression imbalance was detected in these recombinant individuals before or after immune complex stimulation.

[0046] Figures 18A and 18B show that TL1A risk SNPs are associated with expression quantitative trait loci (eQTL), PBMC donors with homozygous TL1A risk allele, homozygous non-risk alleles or heterozygous alleles were treated with immune complex. Total expression of TL1A (TNFSF15) in each donor was determined by digital PCR. In brief, cDNA from each sample was mixed with ddPCRTM Supermix for Probes (Bio-Rad) and PrimeTime ®< qPCR assay ID Hs.PT.56a.41003970. Droplets were generated for each reaction using the QX100 ™< droplet generator (Bio-Rad) and subjected to thermal cycling on a C1000 Touch ™< thermal cycler (Bio-Rad). Following amplification, droplet fluorescence was read on a QX100 ™< droplet reader (Bio-Rad). Data were analyzed using QuantaSoft software (Bio-Rad) and copies / µl of TL1A was determined and normalized for the amount of input cDNA (copies / ng). P values for differences in TL1A expression levels between different TNFSF15 haplotypes were determined using the student t test. At basal level, PBMCs from donors homozygous for TL1A risk SNPs have lower TL1A mRNA compared to non-risk homozygous donors. However, after immune complex stimulation, PBMCs from donors homozygous for TL1A risk SNPs have higher TL1A expression compared to non-risk homozygous donors.

[0047] Figures 19A and 19B show cell-type specific regulation of TL1A synonymous SNP allelic expression imbalance. HUVEC cells from various donors were genotyped as described previously. Genotyped HUVEC cells from various donors were treated with IL-1 at various time points. Total copy number of each allele was measured as described previously. The allelic expression ratio was calculated by dividing the copies / ml of the risk allele by the copies / ml of the non-risk allele. No allelic expression imbalance was observed in HUVEC cells with or without IL-1 treatment.

[0048] Figures 20A and 20B show that prophylactivc treatment with anti-TL1A monoclonal antibody inhibited spontaneous colitis development in mdr1a - / -< mice. Mice (n=10, 6-7 weeks age) were randomized to different groups and treated intra-peritoneally once a week with 500 µg of anti-mouse TL1A antibody, anti-IL23p19 antibody, anti-mouse IL17RA antibody, or mouse isotype control or no treatment once per week for 8 weeks. Clinical disease activity was monitored by evaluating anal inflammation and stool consistency. Sections of the intestine were subjected to histopath analysis. Statistical analysis was performed using one-way ANOVA with Dunett's compared to mIgG1 group.

[0049] Figures 21A and 21B show that TL1A exacerbated colitis in mdr1a +< mice. Mdr1a - / -< mice or wild-type control mice at 6-8 weeks of age were treated intra-peritoneally once a week with 150 µg of recombinant mFc-TL1A fusion protein or isotype control three times each week for 4 weeks. Clinical disease activity was monitored by evaluating anal inflammation and stool consistency as described previously. After 4 weeks of treatment, mice were sacrificed, sections of the intestine were were subjected to histopath analysis. Statistical analysis was performed using one way ANOVA with Dunett's compared to mIgG1 group.

[0050] Figures 22A to 22F shows increased inflammatory cells in lamina propria from Fc-TL1A challenged mdr1a - / -< mice. Mdr1a - / -< mice or wild-type control mice at 6-8 weeks of age were treated intra-peritoneally once a week with 150 µg of recombinant mFc-TL1A fusion protein or isotype control three times each week for 4 weeks. Lamina propria lymphocytes were isolated and stained for surface antigens and analyzed by FACS. TL1A challenge in mdr1a - / -< mice resulted in increased inflammatory cells in lamina propria.

[0051] Figures 23A to 23E show distinct cytokine induction by TL1A and TNF challenge in mice. To evaluate if TL1A and TNF challenge result in similar or different pharmacodynamics effects, C57Bl / 6 mice (8 week, female) were first intraperitonially injected with 500 µg / mice of anti mouse TL1A, anti mouse TNF-α or PBS. After 4 hours, the mice were then challenged with 100 µg / mice of TL1A or 10 µg / mice of TNF-α or without challenging. The sera were collected after 24 hours. The cytokines were measured by MSD (IL-22 was measured by ELISA).

[0052] Figures 24A to 24F shows distinct cytokine induction by TL1A and TNF treatment in human PBMC. PBMC freshly isolated from human blood were cultured in media (RPMI1640 supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 5 X 10 -5< M 2-ME, and antibiotics) in the presence of 100 ng / ml of human TL1A or TNF-α. Supernatant was collected after 72 hours. The cytokines in the supernatant were measured by MSD (IL-22 was measured by ELISA).

[0053] Figure 25 depicts a schematic representation of a bispecific IgG-Fab format for an anti-TL1A anti-TNF-α bispecific antigen binding protein within the present invention. In this format, one polypeptide chain of a Fab fragment from a second antibody (e.g. the heavy chain (VH2-CH1) is fused to the carboxyl terminus of each heavy chain of a first antibody through a peptide linker to produce a modified heavy chain. The complete molecule is a homohexamer comprising two modified heavy chains, two light chains from the first antibody, and two polypeptide chains containing the other half of the Fab fragment from the second antibody (e.g. the light chain (VL2-CL)). Charge pair mutations (represented by the circles) can be introduced into the Fab regions of the first antibody (Fab 1) and / or second antibody (Fab 2) to promote correct heavy chain-light chain pairing.

[0054] Figure 26 depicts a schematic representation of a bispecific IgG-Fab format for an anti-TL1A anti-TNF-α bispecific antigen binding protein within the present invention using immunoglobulin domain crossover. In this variation of the IgG-Fab format, one polypeptide chain of a Fab fragment from a second antibody (e.g. the heavy chain (VH2-CH1) is fused to the carboxyl terminus of the heavy chain comprising a CL instead of a CH1 domain of a first antibody through a peptide linker to produce a modified heavy chain. In this way, the CH1 and the CL domains of Fab 1 are "swapped." This swap is referred to as a Fab1 swap or an N-terminal swap herein. The complete molecule is a homohexamer comprising two modified heavy chains, two light chains from the first antibody comprising a CH1 domain instead of a CL domain, and two polypeptide chains containing the other half of the Fab fragment from the second antibody (e.g. the light chain (VL2-CL)). Charge pair mutations (represented by the circles) can be introduced into the Fab regions of the first antibody (Fab 1) and / or second antibody (Fab 2) to promote correct heavy chain-light chain pairs. Not shown but also within the scope of this invention are IgG-Fab molecules in which (i) the CH1 and CL domains of Fab 2 are swapped instead of those of Fab 1 (referred to herein as a Fab 2 swap or a C-terminal swap) or (ii) both the CH1 and CL domains of Fab 1 are swapped and the CH1 and CL domains of Fab 2 are swapped (referred to herein as a dual swap).

[0055] Figure 27 compares the expression titer of IgG-Fabs based on domain swapping format.

[0056] Figure 28 compares the expression titer of IgG-Fabs based on type of charge pair mutation(s).

[0057] Figure 29 compares the purity of IgG-Fabs based on domain swapping format.

[0058] Figure 30 compares the anti-TL1A potency of IgG-Fabs based on domain swapping format.

[0059] Figure 31 compares the anti-TNF-α potency of IgG-Fabs based on domain swapping format.

[0060] Figure 32 compares the anti-TL1A potency of IgG-Fabs based on type of charge pair mutation(s).

[0061] Figure 33 compares the anti- TNF-α potency of IgG-Fabs based on type of charge pair mutation(s).DETAILED DESCRIPTION OF THE INVENTION Definition of terms

[0062] In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the invention.

[0063] Unless otherwise specified, "a", "an", "the", and "at least one" are used interchangeably and mean one or more than one.

[0064] A "binding entity" as used herein means any monomeric or multimeric protein or protein fragment that specifically binds a specified target antigen. The term "binding entity" includes but is not limited to antibodies and binding parts thereof, such as immunologically functional fragments. Peptibodies and peptides are other examples of binding entities. The term "immunologically functional fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy or light chain) binding entity, as used herein, is a species of binding entity comprising a portion (regardless of how that portion is obtained or synthesized) of an antibody that lacks at least some of the amino acids present in a full-length chain but which is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to the target antigen and can compete with other binding entities, including intact antibodies, for binding to a given epitope. In some embodiments, the fragments are neutralizing fragments. In some embodiments, the fragments can block or reduce the likelihood of the interaction between the target antigen (TL1A or TNF-α ) and its receptor. In one aspect, such a fragment will retain at least one CDR present in the full-length light or heavy chain, and in some embodiments will comprise a single heavy chain and / or light chain or portion thereof. These biologically active fragments can be produced by recombinant DNA techniques, or can be produced by enzymatic or chemical cleavage of binding entities, including intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, a diabody (heavy chain variable domain on the same polypeptide as a light chain variable domain, connected via a short peptide linker that is too short to permit pairing between the two domains on the same chain), Fab', F(ab') 2 , Fv, domain antibodies and single-chain antibodies, and can be derived from any mammalian source, including but not limited to human, mouse, rat, camelid or rabbit. It is further contemplated that a functional portion of the binding entities disclosed herein, for example, one or more CDRs, could be covalently bound to a second protein or to a small molecule to create a therapeutic agent directed to a particular target in the body, possessing bifunctional therapeutic properties, or having a prolonged serum half-life. As will be appreciated by one of skill in the art, a binding entity can include non-protein components. In some sections of the present disclosure, examples of binding entities are named herein in terms of "number / letter / number" (e.g., 23B3), with some binding entities further identified by additional letter / number combinations (e.g., VH4). In these cases, the exact name denotes a specific antibody. That is, a binding entity named 23B3 may have some degree of sequence identity with but is not the same as an antibody named 23B3 VH4 (unless they are explicitly taught to be the same in the specification).

[0065] A "TL1A binding entity" is a binding entity that specifically binds to human TL1A; i.e., a binding entity for which human TL1A is the target antigen.

[0066] A "TNF-α binding entity" is a binding entity that specifically binds to human TNF-α ; i.e., a binding entity for which human TNF-α is the target antigen.

[0067] "Antigen binding protein" refers to a protein or polypeptide that comprises an antigen-binding region or antigen-binding portion that has a strong affinity for another molecule to which it binds (antigen). Antigen-binding proteins encompass antibodies, peptibodies, antibody fragments, antibody derivatives, antibody analogs, fusion proteins, and antigen receptors including chimeric antigen receptors (CARs).

[0068] "Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas. Thus, as used herein, the term "antibody" or "antibody peptide(s)" refers to an intact antibody, an antibody that competes for specific binding with an antibody disclosed in this specification, or an antigen-binding fragment thereof that competes with the intact antibody for specific binding and includes chimeric, humanized, fully human, and bispecific antibodies. In certain embodiments, antigen-binding fragments are produced, for example, by recombinant DNA techniques. In additional embodiments, antigen-binding fragments are produced by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab) 2< , F(ab') 2< , Fv, and single-chain antibodies.

[0069] The term "isolated antibody" as used herein refers to an antibody that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0070] The term "agonist" refers to any compound including a protein, polypeptide, peptide, antibody, antibody fragment, large molecule, or small molecule (less than 10 kD), that increases the activity, activation or function of another molecule.

[0071] The term "antagonist" refers to any compound including a protein, polypeptide, peptide, antibody, antibody fragment, large molecule, or small molecule (less than 10 kD), that decreases the activity, activation or function of another molecule.

[0072] The term "bind(ing)" of an antigen or other polypeptide includes, but is not limited to, the binding of a ligand polypeptide of the present invention to a receptor; the binding of a receptor polypeptide of the present invention to a ligand; the binding of an antibody of the present invention to an antigen or epitope; the binding of an antigen or epitope of the present invention to an antibody; the binding of an antibody of the present invention to an anti-idiotypic antibody; the binding of an anti-idiotypic antibody of the present invention to a ligand; the binding of an anti-idiotypic antibody of the present invention to a receptor; the binding of an anti-anti-idiotypic antibody of the present invention to a ligand, receptor or antibody, etc.

[0073] A "bispecific" antigen binding protein is a molecule with binding entities derived from both a first antigen binding protein (e.g., antibody) that specifically binds a first target molecule of interest and a second antigen binding protein (e.g., antibody) that specifically binds a second target molecule of interest. Bispecific antigen binding proteins may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai et al., Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992), Molecules in the formats depicted in Figures 1 and 2 are bispecific antigen binding proteins in accordance with this definition. Various additional formats of bispecific antigen binding proteins within this definition are disclosed in WO 2014 / 159725; WO 2013 / 041687; US Pat. No. 8,945,553; US Pat. No. 8,945,553; US Pat. No. 8,258,268; US Pat. App. No. 2012 / 0195900; International patent application WO 2012 / 088302; US Prov. App. 62 / 218,977; Fischer and Leger (2007), Pathobiology 74:3-14;_van Spriel et al. (2000), Immunology Today 21(8): 391-7; Kufer et al. (2004), Trends in Biotechnology 22(5): 238-44; Byrne et al. (2013), Trends in Biotechnology 31(11): 621-31; Muller and Kontermann (2010), Biodrugs 24(2): 89-98; Chames and Baty (2009), http: / / dx.doi.org / 10.4161 / mabs.1.6.10015; Kontermann (2012), http: / / dx.doi.ore / 10.4161 / mabs.4.2.19000; Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-8; Speiss et al., Mol. Immunol. (2015), 67: 95-106, http: / / dx.doi.org / 10.1016 / j.molimm.2015.01.003; Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-8; Speiss et al., Mol. Immunol. (2015), http: / / dx.doi.org / 10.1016 / j.molimm.2015.01.003; Ridgway et al, (1996), Protein Eng. 9: 617; Gunasekaran et al (2010), J. Biol. Chem. 285:19637; Davis (2010), Protein Eng. Des. & Sel. 23:195; DiGiammarino et al. (2012), Methods Mol. Biol. 899:145, 2012); Lindhofer et al. (1995), J. Immunol. 155: 219: Schaefer et al. (2011), Proc. Natl. Acad. Sci. USA, 108: 11187; Regula et al, US Patent Application No: 2010 / 0322934; Bostrom et al. (2009), Science 323:1610; US Pat. App. 2011 / 0076722; Rossi et al. (2008), Cancer Res. 68:8384.

[0074] The term "epitope" refers to the portion of an antigen to which an antibody specifically binds. Thus, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. More specifically, the term "IL-17 epitope", "TNF-α epitope" and / or "TNF-α / p19 epitope" as used herein refers to a portion of the corresponding polypeptide having antigenic or immunogenic activity in an animal, preferably in a mammal, and most preferably in a mouse or a human. An epitope having immunogenic activity is a portion of, for example, an IL-17A or IL-17F or TNF-α / p19 polypeptide that elicits an antibody response in an animal. An epitope having antigenic activity is a portion of, for example, an IL-17A or IL-17F or TNF-α / p19 polypeptide to which an antibody immunospecifically binds as determined by any method well known in the art, for example, by immunoassays, protease digest, crystallography or HID-Exchange. Antigenic epitopes need not necessarily be immunogenic. Such epitopes can be linear in nature or can be a discontinuous epitope. Thus, as used herein, the term "conformational epitope" refers to a discontinuous epitope formed by a spatial relationship between amino acids of an antigen other than an unbroken series of amino acids.

[0075] The term "immunoglobulin" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions.

[0076] Full-length immunoglobulin "light chains" (about 25 Kd or about 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH-terminus. Full-length immunoglobulin "heavy chains" (about 50 Kd or about 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes (about 330 amino acids). Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG (such as IgG1, IgG2, IgG3 and IgG4), IgM, IgA, IgD and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. (See generally, Fundamental Immunology (Paul, W., ed., 2nd Edition, Raven Press, NY (1989)), Chapter 7

[0077] An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions. Thus, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region comprises amino acid residues from a "Complementarity Determining Region" or "CDR" (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those residues from a "hypervariable loop" (Chothia et al., J. Mol. Biol. 196: 901-917 (1987)) "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. Thus, a "human framework region" is a framework region that is substantially identical (about 85% or more, usually 90-95% or more) to the framework region of a naturally occurring human immunoglobulin. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDR's. The CDR's are primarily responsible for binding to an epitope of an antigen. Accordingly, the term "humanized" immunoglobulin refers to an immunoglobulin comprising a human framework region and one or more CDR's from a non-human (usually a mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDR's is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor". Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDR's, are substantially identical to corresponding parts of natural human immunoglobulin sequences. Further, one or more residues in the human framework region may be back mutated to the parental sequence to retain optimal antigen-binding affinity and specificity. In this way, certain framework residues from the non-human parent antibody are retained in the humanized antibody in order to retain the binding properties of the parent antibody while minimizing its immunogenicity. The term "human framework region" as used herein includes regions with such back mutations. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody as defined above, e.g., because the entire variable region of a chimeric antibody is non-human.

[0078] As used herein, the term "modified heavy chain" refers to a fusion protein comprising an immunoglobulin heavy chain, particularly a human IgG1 or human IgG2 heavy chain, and a functional antibody fragment (e.g., a Fab or scFv) or portion thereof (e.g. immunoglobulin light chain or Fd fragment), wherein the fragment or portion thereof is fused at its N-terminus, optionally through a peptide linker, to the C-terminus of the heavy chain.

[0079] The term "humanized" immunoglobulin refers to an immunoglobulin comprising a human framework region and one or more CDR's from a non-human, e.g., mouse, rat or rabbit, immunoglobulin. The non-human immunoglobulin providing the CDR's is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor". Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDR's and possibly a few back-mutated amino acid residues in the framework region (e.g., 1-15 residues), are substantially identical to corresponding parts of natural human immunoglobulin sequences. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody as defined above, e.g., because the entire variable region of a chimeric antibody is non-human.

[0080] The terms "human antibody" and "fully human antibody" each refer to an antibody that has an amino acid sequence of a human immunoglobulin, including antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins; for example, Xenomouse antibodies and antibodies as described by Kucherlapati et al. in U.S. Pat. No. 5,939,598.

[0081] The term "genetically altered antibodies" means antibodies wherein the amino acid sequence has been varied from that of a native antibody. Because of the relevance of recombinant DNA techniques in the generation of antibodies, one need not be confined to the sequences of amino acids found in natural antibodies; antibodies can be redesigned to obtain desired characteristics. The possible variations are many and range from changing just one or a few amino acids to complete redesign of, for example, the variable and / or constant region. Changes in the constant region will, in general, be made in order to improve or alter characteristics, such as complement fixation, interaction with membranes and other effector functions. Changes in the variable region will be made in order to improve the antigen binding characteristics.

[0082] A "Fab fragment" is comprised of one light chain and the C H1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0083] A "Fab' fragment" contains one light chain and one heavy chain that contains more of the constant region, between the C H1 and C H2 domains, such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab') 2 molecule.

[0084] A "F(ab') 2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the C H1 and C H2 domains, such that an interchain disulfide bond is formed between two heavy chains.

[0085] The term "native Fc" refers to molecule or sequence comprising the sequence of a non-antigen-binding fragment resulting from digestion of whole antibody, whether in monomeric or multimeric form. The original immunoglobulin source of the native Fc is preferably of human origin and may be any of the immunoglobulins, although IgG1, IgG2 and IgG4 are preferred. Native Fc's are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). The term "native Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms.

[0086] The term "Fc variant" refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn. International applications WO 97 / 34631 (published 25 September 1997) and WO 96 / 32478 describe exemplary Fc variants, as well as interaction with the salvage receptor, Thus, the term "Fc variant" comprises a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, the term "Fc variant" comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in further detail hereinafter.

[0087] The term "Fc domain" encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc's, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.

[0088] The term "multimer" as applied to Fc domains or molecules comprising Fc domains refers to molecules having two or more polypeptide chains associated covalently, noncovalently, or by both covalent and non-covalent interactions. IgG molecules typically form dimers; IgM, pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, or tetramers. Multimers may be formed by exploiting the sequence and resulting activity of the native Ig source of the Fc or by derivatizing (as defined below) such a native Fc.

[0089] The term "dimer" as applied to Fc domains or molecules comprising Fc domains refers to molecules having two polypeptide chains associated covalently or non-covalently. Thus, exemplary dimers within the scope of this invention are as shown in Figure 2.

[0090] The terms "Fv fragment" and "single chain antibody" refer to polypeptides containing antibody variable regions from both heavy and light chains but lacking constant regions. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only about 25 kDa, Fv fragments are much smaller than common antibodies (150-160 kD) which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments (about 50 kDa, one light chain and half a heavy chain).

[0091] A "single domain antibody" is an antibody fragment consisting of a single domain Fv unit, e.g., V H or V L . Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, single-domain antibodies are much smaller than common antibodies (150-160 kDa) which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments (.about.50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (.about.25 kDa, two variable domains, one from a light and one from a heavy chain). The first single-domain antibodies were engineered from heavy-chain antibodies found in camelids. Although most research into single-domain antibodies is currently based on heavy chain variable domains, light chain variable domains and nanobodies derived from light chains have also been shown to bind specifically to target epitopes.

[0092] The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0093] In some embodiments, the anti-TL1A antigen binding protein, bispecific antigen binding protein or functional fragment of either thereof from which the anti-TL1A binding domain is derived selectively inhibits human TL1A relative to TNF superfamily ligands. An antibody or functional fragment thereof "selectively inhibits" a specific receptor or ligand relative to other receptors or ligands when the IC50 of the antibody in an inhibition assay of the specific receptor is at least 50-fold lower than the IC50 in an inhibition assay of another "reference" ligand or receptor. An "IC50" is the dose / concentration required to achieve 50% inhibition of a biological or biochemical function. With radioactive ligands, IC50 is the concentration of a competing ligand that displaces 50% of the specific binding of the radioactive ligand. The IC50 of any particular substance or antagonist can be determined by constructing a dose-response curve and examining the effect of different concentrations of the drug or antagonist on reversing agonist activity in a particular functional assay. IC50 values can be calculated for a given antagonist or drug by determining the concentration needed to inhibit half of the maximum biological response of the agonist. Thus, the IC50 value for any anti-TL1A antibody or functional fragment thereof can be calculated by determining the concentration of the antibody or fragment needed to inhibit half of the maximum biological response of TL1A in activating the human TL1A receptor in any functional assay, such as the assay described in Example 14 hereinafter. An antibody or functional fragment thereof that selectively inhibits a specific ligand or receptor is understood to be a neutralizing antibody or neutralizing fragment with respect to that ligand or receptor. Thus, in some embodiments, the anti-TL1A antibody or functional fragment thereof from which the anti-TL1A binding domain of the bispecific antigen binding proteins of the invention is derived is a neutralizing antibody or fragment of human TL1A.

[0094] The variable regions or CDR regions of any anti-TL1A antibody or functional fragment thereof can be used to construct the anti-TL1A binding entity of any of the bispecific antigen binding proteins described herein. Likewise, the variable regions or CDR sequences of any anti-TNF-α antibody or functional fragment thereof can be used to construct the anti-TNF-α binding entity of any of the bispecific antigen binding proteins described herein. For instance, the anti-TL1A binding domain of the bispecific antigen binding proteins of the invention may comprise VH and / or VL regions or one or more CDRs from any of the anti-human TL1A antibodies described in US Pat. No. 7,820,798; US Pat. App. 2008 / 0003221; US Pat. No. 8,263,743; US Pat. App. 2011 / 0217310; US Pat. App. 2012 / 0263718; US Pat. App. 2014 / 0308271; WO 2012 / 161856; WO 2013 / 044298; WO 2005 / 018571; US Pat. App. 2014 / 0120109; US Pat. No. 6,521,422; US Pat. App. 2014 / 0255302; and US Pat. App. 2015 / 0132311 In some embodiments, the anti-TL1A antibody from which the anti-TL1A binding entity is derived cross-blocks one or more of the human anti-TL1A antibodies described in one of the references just mentioned or one or more of the human anti-TL1A antibodies described below. The terms "cross-block," "cross-blocked," and "cross-blocking" are used interchangeably herein to mean the ability of an antibody to interfere with the binding of other antibodies or binding fragments to a target (e.g. human TL1A). The extent to which an antibody or binding fragment is able to interfere with the binding of another to a target (e.g., human TL1A) and therefore whether it can be said to cross-block, can be determined using competition binding assays. In some embodiments, a cross-blocking antibody or binding fragment thereof reduces human TL1A binding of a reference antibody between about 40% and 100%, such as about 60% and about 100%, specifically preferably between about 70% and 100%, and more specifically preferably between about 80% and 100%. A particularly suitable quantitative assay for detecting cross-blocking uses a Biacore machine which measures the extent of interactions using surface plasmon resonance technology. Another suitable quantitative cross-blocking assay uses a FACS-based approach to measure competition between antibodies in terms of their binding to human TL1A. An exemplary assay regarding such cross-blocking appears in US Pat. App. 2015 / 0132311, example 2, paragraphs

[0922] -

[0924] ,

[0095] The term "nucleic acid" or "nucleic acid molecule" refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., .alpha.-enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and / or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids", which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.

[0096] The term "complement of a nucleic acid molecule" refers to a nucleic acid molecule having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence.

[0097] The term "degenerate nucleotide sequence" denotes a sequence of nucleotides that includes one or more degenerate codons as compared to a reference nucleic acid molecule that encodes a polypeptide. Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).

[0098] An "isolated nucleic acid molecule" is a nucleic acid molecule that is not integrated in the genomic DNA of an organism. For example, a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism. A nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome from that species

[0099] A "nucleic acid molecule construct" is a nucleic acid molecule, either single- or double-stranded, that has been modified through human intervention to contain segments of nucleic acid combined and juxtaposed in an arrangement not existing in nature

[0100] "Complementary DNA (cDNA)" is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription. Those skilled in the art also use the term "cDNA" to refer to a double-stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand. The term "cDNA" also refers to a clone of a cDNA molecule synthesized from an RNA template.

[0101] A "promoter" is a nucleotide sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' non-coding region of a gene, proximal to the transcriptional start site of a structural gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee et al., Mol. Endocrinol., 7:551 (1993)), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman, Seminars in Cancer Biol., 1:47 (1990)), glucocorticoid response elements (GREs), and binding sites for other transcription factors, such as CRE / ATF (O'Reilly et al., J. Biol. Chem., 267:19938 (1992)), AP2 (Ye et al., J. Biol. Chem., 269:25728 (1994)), SP1, cAMP response element binding protein (CREB; Loeken, Gene Expr., 3:253 (1993)) and octamer factors (see, in general, Watson et al., eds., Molecular Biology of the Gene, 4th Edition, The Benjamin / Cummings Publishing Company, Inc. (1987), and Lemaigre et al., Biochem. J., 303:1 (1994)). If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter. Repressible promoters are also known.

[0102] A "regulatory element" is a nucleotide sequence that modulates the activity of a core promoter. For example, a regulatory element may contain a nucleotide sequence that binds with cellular factors enabling transcription exclusively or preferentially in particular cells, tissues, or organelles. These types of regulatory elements are normally associated with genes that are expressed in a "cell-specific", "tissue-specific", or "organelle-specific" manner.

[0103] An "enhancer" is a type of regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.

[0104] "Heterologous DNA" refers to a DNA molecule, or a population of DNA molecules, that does not exist naturally within a given host cell. DNA molecules heterologous to a particular host cell may contain DNA derived from the host cell species (i.e., endogenous DNA) so long as that host DNA is combined with non-host DNA (i.e., exogenous DNA). For example, a DNA molecule containing a non-host DNA segment encoding a polypeptide operably linked to a host DNA segment comprising a transcription promoter is considered to be a heterologous DNA molecule. Conversely, a heterologous DNA molecule can comprise an endogenous gene operably linked with an exogenous promoter. As another illustration, a DNA molecule comprising a gene derived from a wild-type cell is considered to be heterologous DNA if that DNA molecule is introduced into a mutant cell that lacks the wild-type gene.

[0105] An "expression vector" is a nucleic acid molecule encoding a gene that is expressed in a host cell. Typically, an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be "operably linked to" the promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.

[0106] A "recombinant host" is a cell that contains a heterologous nucleic acid molecule, such as a cloning vector or expression vector. In the present context, an example of a recombinant host is a cell that produces an antagonist of the present invention from an expression vector. In contrast, such an antagonist can be produced by a cell that is a "natural source" of said antagonist, and that lacks an expression vector.

[0107] The terms "amino-terminal" and "carboxyl-terminal" are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.

[0108] A "fusion protein" is a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes. For example, a fusion protein can comprise at least part of a IL-17RA polypeptide fused with a polypeptide that binds an affinity matrix. Such a fusion protein provides a means to isolate large quantities of IL-17A using affinity chromatography.

[0109] The term "receptor" denotes a cell-associated protein that binds to a bioactive molecule termed a "ligand." This interaction mediates the effect of the ligand on the cell. Receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor). Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. In certain membrane-bound receptors, the extracellular ligand-binding domain and the intracellular effector domain are located in separate polypeptides that comprise the complete functional receptor. In general, the binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell, which in turn leads to an alteration in the metabolism of the cell. Metabolic events that are often linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.

[0110] The term "expression" refers to the biosynthesis of a gene product. For example, in the case of a structural gene, expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.

[0111] The term "complement / anti-complement pair" denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement / anti-complement pair. Other exemplary complement / anti-complement pairs include receptor / ligand pairs, antibody / antigen (or hapten or epitope) pairs, sense / antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement / anti-complement pair is desirable, the complement / anti-complement pair preferably has a binding affinity of less than 10 9< M -1< .

[0112] A "detectable label" is a molecule or atom which can be conjugated to an antibody moiety to produce a molecule useful for diagnosis. Examples of detectable labels include chelators, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, or other marker moieties.

[0113] The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a polyhistidine tract, protein A (Nilsson et al., EMBO J., 4:1075 (1985); Nilsson et al., Methods Enzymol., 198:3 (1991)), glutathione S transferase (Smith et al., Gene, 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA, 82:7952 (1985)), substance P, FLAG ®< peptide (Hopp et al., Biotechnology, 6:1204 (1988)), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification, 2:95 (1991). DNA molecules encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

[0114] The term "acidic residue" refers to amino acid residues having sidechains comprising acidic groups. Exemplary acidic residues include D and E.

[0115] The term "amide residue" refers to amino acids having sidechains comprising amide derivatives of acidic groups. Exemplary residues include N and Q.

[0116] The term "aromatic residue" refers to amino acid residues having sidechains comprising aromatic groups. Exemplary aromatic residues include F, Y, and W.

[0117] The term "basic residue" refers to amino acid residues having sidechains comprising basic groups. Exemplary basic residues include H, K, and R.

[0118] The term "hydrophilic residue" refers to amino acid residues having sidechains comprising polar groups. Exemplary hydrophilic residues include C, S, T, N, and Q.

[0119] The term "non-functional residue" refers to amino acid residues having sidechains that lack acidic, basic, or aromatic groups. Exemplary non-functional amino acid residues include M, G, A, V, I, L and norleucine (Nle).

[0120] Both the EU index as in Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD and AHo numbering schemes (Honegger and Plückthun (2001), J Mol Biol. 8;309(3): 657-70) can be used in the present invention. Amino acid positions and CDRs and FRs of a given antibody may be identified using either system. For example, EU heavy chain positions 39, 44, 183, 356, 357, 370, 392, 399, and 409 are equivalent to AHo heavy chain positions 46, 51, 230, 484, 485, 501, 528, 535, and 551, respectively. Similarly, EU light chain positions 38, 100, and 176 are equivalent to AHO light chain positions 46 141, and 230, respectively. Tables (i), (ii) and (iii) below demonstrate the equivalence between numbering positions for v1, v2, and v3 versions of charge positions to aid correct assembly of, for example, IgG-Fab bispecific antigen binding proteins.

[0121] The invention is defined in the appended claims. Bispecific antigen binding protein formats are not covered by the claims.

[0122] One aspect of the invention concems novel TL1A-specific antigen binding proteins and antibodies. Such antibodies are useful to treat conditions known in the art and described hereinafter that are amenable to treatment by inhibition of TL1A biological activity, as defined in the claims. Another aspect not covered by the claims concerns bispecific antigen binding proteins in which one light chain-heavy chain pair specifically binds TL1A and another light chain-heavy chain pair binds TNF-α . Such bispecific antigen binding proteins can be whole antibodies (see Figure 1) or F(ab') 2 fragments. In this aspect the TL1A binding entity is monovalent for TL1A and the TNF-α binding entity is monovalent for TNF-α.

[0123] In another bispecific antigen binding protein aspect, the TL1A binding entity and the TNF-α binding entity are arranged in an overall structure referred to herein as IgG-scFv. In this embodiment, a first binding entity has the structure of an antibody-- i.e., two pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. A second binding entity comprises the structure of a pair of Fv units-- i.e., each member of the pair has a variable domain from a heavy chain and a variable domain from a light chain linked in tandem as a single chain. In the IgG-scFv configuration, each Fv unit is covalently bound to the C-terminus of a heavy chain constant domain (Fc) of the first binding entity (see Figure 2). Each Fv unit can be linked to the Fc domain of the first binding entity directly or through a linker. In this embodiment of the invention, each binding entity is bivalent for its target antigen. In a preferred embodiment, the TL1A binding entity has the structure of an antibody and the TNF-α binding entity has the structure of a pair of Fv units.

[0124] One further aspect not covered by the claims concerns bispecific antibodies with mutations to aid correct heavy-heavy and heavy-light chain pairing. Such mutations are described in US 8,592,562; WO 2009 / 089004; WO 2006 / 106905; WO 2014 / 4082179; WO 2014 / 081955; Speiss et al., Mol. Immunol. (2015), http: / / dx.doi.org / 10.1016 / j.molimm.2015.01.003.

[0125] Not covered by the claims is a TL1A binding entity and a TNF-α binding entity arranged in other bispecific antigen binding protein formats known in the art. Specifically, the invention concems the TL1A and TNF-α binding entities in the formats described in: WO 2014 / 159 725; WO 2013 / 041687; US Pat. No. 8,945,553; US Pat. No. 8,945,553; US Pat. No. 8,258,268; US Pat. App. No. 2012 / 0195900; International patent application WO 2012 / 088 302; US Prov. App. 62 / 218,977; Fischer and Leger (2007), Pathobiology 74:3-14;_van Spriel et al. (2000), Immunology Today 21(8): 391-7; Kufer et al. (2004), Trends in Biotechnology 22(5): 238-44; Byrne et al. (2013), Trends in Biotechnology 31(11): 621-31; Muller and Kontermann (2010), Biodrugs 24(2): 89-98; Chames and Baty (2009), http: / / dx.doi.org / 10.4161 / mabs.1.6.10015; Kontermann (2012), http: / / dx.doi.org / 10.4161 / mabs.4.2.19000; Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-8; Speiss et al., Mol. Immunol. (2015), http: / / dx.doi.org / 10.1016 / j.molimm.2015.01.003; WO 2009 / 089004, published 16 July 2009; Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-8; Speiss et al., Mol. Immunol. (2015), http: / / dx.doi.org / 10.1016 / j.molimm.2015.01.003; Ridgway et al, (1996), Protein Eng. 9: 617; Gunasekaran et al (2010), J. Biol. Chem. 285:19637; Davis (2010), Protein Eng. Des. & Sel. 23:195; DiGiammarino et al. (2012), Methods Mol. Biol. 899:145, 2012); Lindhofer et al. (1995), J. Immunol. 155: 219: Schaefer et al. (2011), Proc. Natl. Acad. Sci. USA, 108: 11187; Regula et al, US Patent Application No: 2010 / 0322934; Bostrom et al. (2009), Science 323:1610; US Pat. App. 2011 / 0076722; Rossi et al. (2008), Cancer Res. 68:8384.Preferred embodiments

[0126] The invention is defined in the appended claims. The amino acid sequences of the antigen binding proteins and binding entities are preferably based upon the sequences of human and / or humanized monoclonal antibodies against TL1A The invention also comprises sequences having at least 90%, at least 95%, or at least 99% sequence identity to the preferred sequences set forth hereinafter. The amino acid sequences shown in the following tables are preferred for the antigen binding proteins of this invention, including bispecific antigen binding proteins in any format.TL1A-specific antigen Binding Proteins

[0127] TL1A-specific antigen binding proteins and antigen binding entities preferably comprise the complementarity determining region (CDR) sequences derived from preferred TL1A antibodies disclosed herein. Table A shows the preferred CDR sequences, alongside the preferred antibodies from which they were derived. Throughout, LCDR1, LCDR2, and LCDR3 refer to the light chain CDRs; HCDR1, HCDR2, and HCDR3 to the heavy chain CDRs, Throughout, the sequence identification number (SEQ ID NO) for each sequence appears in parentheses after the sequence in the table. Table A: Preferred TL1A-binding CDR sequences Source Antibody designation LCDR1 (SEQ ID NO) LCDR2 (SEQ ID NO) LCDR3 (SEQ ID NO) HCDR1 (SEQ ID NO) HCDR2 (SEQ ID NO) HCDR3 (SEQ ID NO) 3C6DASSLQS (100)SYGMH (164)2G11ATSSLQS (106)SYFWS (170)9C8AASSLQS (112)SYFWS (170)23B3WASTRES (118)TNSVAWN (182)23B3 VH4WASTRES (118)TNSVAWN (182)23B3 VH3WASTRES (118)TNSVAWN (182)3B3GASSRAT (124)QQYGSSPT (126)GYYWN (188)5G4GASSRAT (124)QQYGSSPT (126)GYYWN (188)17E9AASSLQS (112)SYGMH (164)53D3LGSSRAS (685)TYYMS (777)54E5WASTRES (118)TYGMH (783)56E1WASTRES (118)SYGMH (164)57A8GNNNRPS (699)SYVMS (792)58G5GNSHRPS (705)NYAMS (798)60G11GNSHRPS (705)NYAMN (804)73C2AASSLQS (112)SSSATWN (809)76A4TASSLQS (721)SNSATWN (815)77D12SNNKRPS (725)GFYMH (819)EGIAVALTY (823)87H11VASSLQS (731)GYYWS (265)88H9AASGLQG (737)SYGMH (164)89H9AASSLQS (112)QQSYSSIT (745)SYAMS (836)EMAGAFDI (840)91D7TASSLQS (749)GYYWS (265)91F10_LC1KVSNWDS (755)MQGTHWP (757)AYYMH (847)91F10_LC2AASRLQS (761)AYYMH (847)91G8AASSLQS (112)QQSFSTIT (767)SYAMS (836)EVAGAFDI (855)92D3GASRLQS (769)GYYMH (857)92E5AASSLQS (112)QQSFSSIT (775)SYAMS (836)EVAGAFDI (855)

[0128] Each of the foregoing sequences is encoded by the nucleic acid sequence immediately preceding it in the Sequence Listing. Throughout, the sequences from antibodies 9C8 and 3B3 are preferred.

[0129] In the antigen binding proteins of this invention, it is preferable to avoid isomerization sites. The two-amino acid sequences DG, DH, DS, and DT are known isomerization sites. The present invention relates also to antigen binding proteins in which the source antibody sequences, including the sequences of CDRs, are modified to eliminate isomerization sites. With that consideration, the invention relates to antigen binding proteins wherein HCDR2 is a modified form of the HCDR2 from source antibody 3C6 having the sequence VISYDXNNKLYTDXVKG (SEQ ID NO: 204) wherein each X is independently a residue other than G, H, S, or T (i.e., A, C, D, E, F, I, K, L, M, N, P, Q, R, V, W, or Y), with A preferred. The invention further relates to antigen binding proteins wherein HCDR2 is a modified form of the HCDR2 from source antibody 3C6 in which the acidic residue D is replaced with E so as to remove the isomerization sites, resulting in the sequence VISYEGNNKLYTESVKG (SEQ ID NO: 678). The invention further relates to antigen binding proteins wherein HCDR3 is a modified form of HCDR3 from source antibody 23B3 having the sequence EDGDXYYRYGMDV (SEQ ID NO: 676). The invention further relates to antigen binding proteins wherein the isomerization site of HCDR3 from source antibody 23B3 is removed by replacing the acidic residue D with E, resulting in the sequence EEGESYYRYGMDV (SEQ ID NO: 657).

[0130] In the antigen binding proteins of this invention, it is preferable to avoid deamidation sites. The two-amino acid sequences NG, NH, NS, and NT are known deamidation sites. The present invention relates also to antigen binding proteins in which the source antibody sequences, including the sequences of CDRs, are modified to eliminate deamidation sites. One way to eliminate the deamidation sites is to replace the second amino acid residue in the sites NG, NH, NS, and NT with a residue other than G, H, S, or T (i.e., A, C, D, E, F, I, K, L, M, N, P, Q, R, V, W, or Y). A preferred way to eliminate deamidation sites is to replace the N in NG, NH, NS and NT with Q. With that consideration, the invention relates to antigen binding proteins having the following sequences in addition to those listed in Table A:

[0131] LCDR3 may have the sequence LQHQSYPPT (SEQ ID NO: 631) based on modification of the sequence from the source antibody 3C6;

[0132] HCDR1 may have the sequences TQSVAWN (SEQ ID NO: 632) based on modification of the sequence from the source antibody 23B3 VH4;

[0133] HCDR2 may have the sequences YIYYSGQTKYNPSLKS (SEQ ID NO: 633) or EIQHAGQTNYNPSLKS (SEQ ID NO: 677) based on modification of the sequences from the source antibodies 9C8 and 3B3, respectively.

[0134] Antigen binding proteins of this invention may further result from substitution of an acidic residue for another acidic residue, an amide residue for another amide residue, and likewise for aromatic residues, basic residues, hydrophilic residues, non-functional residues, neutral polar residues, and polar hydrophobic residues. Such substitutions of non-functional residues of the source antibody CDRs results in the sequences of the invention shown in Table B, wherein each X is independently M, G, A, V, I, or L. Table B: TL1A-binding CDR sequences with substitutions Source Antibody designation LCDR1 (SEQ ID NO) LCDR2 (SEQ ID NO) LCDR3 (SEQ ID NO) HCDR1 (SEQ ID NO) HCDR2 (SEQ ID NO) HCDR3 (SEQ ID NO) 3C6DXSSLQS (636)SYXXH (639)2G11XTSSXQS (643)SYFWS (170)9C8XXSSXQS (647)SYFWS (170)23B3WXSTRES (652)23B3 VH4WXSTRES (652)23B3 VH3WXSTRES (652)3B3XXSSRXT (662)QQYXSSPT (663)XYYWN (664)5G4XXSSRXT (662)QQYXSSPT (663)XYYWN (664)17E9XXSSXQS (647)SYXXH (639)53D3XXSSRXS (789)TYYXS (955)54E5WXSTRES (652)TYXXH (979)56E1WXSTRES (652)SYXXH (639)57A8XNNNRPS (988)SYXXS (994)58G5NYXXS (1010)60G11NYXXN (1017)73C2[none]SSSXTWN (1022)76A4[none]77D12[none]XFYXH (1037)87H11XXSSXQS (647)[none]XYYWS (1045)88H9XXSXXQX (1054)SYXXH (639)89H9XXSSXQS (647)QQSYSSXT (1065)SYXXS (994)EXXXXFDX (1071)91D7TXSSXQS (1073)[none]XYYWS (1045)91F10_LC1XQXTHWP (1086)XYYXH (1087)91F10_LC2XXSRXQS (1092)XYYXH (1087)91G8XXSSXQS (647)QQSFSTXT (1094)SYXXS (994)EXXXXFDX (1071)92D3XXSRXQS (1098)XYYXH (1087)92E5XXSSXQS (647)QQSFSSXT (1107)SYXXS (994)EXXXXFDX (1071)

[0135] Also preferred are antigen binding proteins comprising the CDR germline sequences related to the antibodies shown in Table A. Such sequences are shown in Table C. Table C: Related germline CDR sequences of anti-TL1A antibodies Antibody designation related to germline LCDR1 (SEQ ID NO) LCDR2 (SEQ ID NO) LCDR3 (SEQ ID NO) HCDR1 (SEQ ID NO) HCDR2 (SEQ ID NO) HCDR3 (SEQ ID NO) 3C6AASSLQS (112)SYAMH (253)2G11AASSLQS (112)SYYWS (256)SYYYFD (258)9C8AASSLQS (112)SYYWS (259)LTGYFD (261)23B3WASTRES (118)SNSAAWN (262)23B3 VH4AASTLQS (242)SGSYYWS (268)23B3 VH3AASTLQS (242)SNYMS (271)3B3GASSRAT (124)GYYWS (265)5G4GASSRAT (124)GYYWS (265)17E9AASSLQS (112)SYAMH (253)53D3LGSNRAS (935)SYSMN (950)54E5WASTRES (118)SYGMH (164)56E1WASTRES (118)SYGMH (164)57A8GNSNRPS (148)SYAMS (836)58G5GNSNRPS (148)SYAMS (836)60G11GNSNRPS (148)SYAMS (836)73C2AASSLQS (112)SNSAAWN (262)76A4AASSLQS (112)SNSAAWN (262)77D12SNNQRPS (948)GYYMH (857)87H11AASSLQS (112)GYYWS (265)88H9AASSLQS (112)SYGMH (164)89H9AASSLQS (112)SYAMS (836)91D7AASSLQS (112)GYYWS (265)91F10_LC1KVSNRDS (943)GYYMH (857)91F10_LC2AASSLQS (112)GYYMH (857)91G8AASSLQS (112)SYAMS (836)92D3AASSLQS (112)GYYMH (857)92E5AASSLQS (112)SYAMS (836)88H9AASSLQS (112)SYGMH (164)54E5WASTRES (118)SYGMH (164)56E1WASTRES (118)SYGMH (164)53D3LGSNRAS (935)SYSMN (950)89H9AASSLQS (112)SYAMS (836)91G8AASSLQS (112)SYAMS (836)92E5AASSLQS (112)SYAMS (836)92D3AASSLQS (112)GYYMH (857)91F10_LC2AASSLQS (112)GYYMH (857)91D7AASSLQS (112)GYYWS (265)76A4AASSLQS (112)SNSAAWN (262)87H11AASSLQS (112)GYYWS (265)73C2AASSLQS (112)SNSAAWN (262)91F10_LC1KVSNRDS (943)GYYMH (857)60G11GNSNRPS (148)SYAMS (836)58G5GNSNRPS (148)SYAMS (836)57A8GNSNRPS (148)SYAMS (836)77D12SNNQRPS (948)GYYMH (857)

[0136] Further preferred are antigen binding proteins comprising the variable domain sequences of the preferred anti-TL1A antibodies, as shown in Table D. Throughout, "VH" as shown in Table D refers to the variable domain of the heavy chain, "VL" to that of the light chain. Molecules within this invention may incorporate modifications to the sequences shown in Table D, including truncations or substitutions for stability or other functionality or removal of hotspots (chemical or physical modification of amino acids). Also included within the invention are molecules having at least 90% sequence identity with the sequences shown in Table D. Table D: Variable domain sequences of preferred anti-TL1A antibodies Antibody designation Amino acid sequence (SEQ ID NO) 3C6 VL3C6 VH2G11 VL2G11 VH9C8 VL9C8 VH23B3 VL23B3 VH3B3 VL3B3 VH23B3 VH4 VL23B3 VH4 VH23B3 VH3 VL23B3 VH3 VH5G4 VL5G4 VH17E9 VL17E9 VH53D3 VL53D3 VH54E5 VL54E5 VH56E1 VL56E1 VH57A8VL57A8VH58G5VL58G5VH60G11VL60G11VH73C2VL73C2VH76A4VL76A4VH77D12VL77D12VH87H11VL87H11VH88H9VL88H9VH89H9VL89H9VH91D7VL91D7VH91F10_LC1V L91F10_LC1V H91F10_LC2V L91F10_LC2V H91G8VL91G8VH92D3VL92D3VH92E5VL92E5VH

[0137] Each of the foregoing polypeptides is encoded by a nucleic acid having the nucleotide sequence immediately preceding the polypeptide sequence in the Sequence Listing. The sequences from antibodies 9C8 and 3B3 are preferred.

[0138] The full anti-TL1A antibody sequences shown in Table E are preferred for the anti-TL1A antibodies. LC refers to the antibody light chain, HC to the heavy chain. Also encompassed within the invention are molecules having at least 90% sequence identity with either or both of the light chain and heavy chain sequences in Table E. Table E: Preferred anti-TL1A Antibodies Antibody designation Light chain SEQ ID NO Heavy chain SEQ ID NO iPS (molecule designation) 3C650522841122G1154562853969C8586028541223B362642900433B3666830884423B3 HC4707232524623B3 HC374763253365G445545728451017E945946128407888H91116111842748554E51120112242757660G111124112642749353D31128113042750489H91132113442750991D71136113842751457A81140114242751991G81144114642752476A41148115042752987H111152115442753458G51156115842753957A81160116242751973C21164116642754477D121168117042754956E11172117442755492E51176117842755992D31180118242756491F10_LC11184118642758091F10_LC211881190427572

[0139] Each of the amino acid sequences in Table E is encoded by the nucleic acid sequence immediately preceding it in the Sequence Listing. The sequences from antibodies 9C8 and 3B3 are preferred.

[0140] Bispecific antigen proteins are not covered by the claimsBinding affinity and biological activity

[0141] In another embodiment of the foregoing aspects of the invention, an anti-TL1A antibody (or an antigen-binding fragment thereof) binds TL1A with a binding affinity (K D1 ) of at least 1 x 10 -10< M -1< , at least 5 x 10 -10< M -1< , at least 1 x 10 -10< M -1< , at least 5 x 10 -10< M -1< , at least 8 x 10 -10< M -1< , or at least 1 x 10 -10< M -1< wherein the binding affinity is measured by surface plasmon resonance, such as Biacore. More particularly, the invention relates to the following embodiments: an anti-TL1A antibody (or an antigen-binding fragment thereof) binds to human TL1A when the antibody is immobilized on an SCM5 sensor chip at a binding affinity of about 1 to about 5 KD pM or to cynomolgus TL1A when the antibody is immobilized on an SCM5 sensor chip at a binding affinity of about 1 -10< to about 7.5 -10< M -1< KD;

[0142] Further details on TL1A and binding appear in Examples 13 and 15 hereinafter.

[0143] In other embodiments of the foregoing aspects of the invention: an anti-TL1A antibody (or an antigen-binding fragment thereof) neutralizes or inhibits human TL1A in a TF-1 NF-κB reporter cell line with an IC 50 of about 0.08 to about 3 nM, cynomolgus TL1A TL1A in a TF-1 NF-κB reporter cell line with an IC 50 of about 0.375 to about 10 nM;

[0144] Further details on TL1A inhibition appear in Examples 14 and 16 hereinafter. Bispecific antibodies are not covered by the claims.Immunoconjugates, derivatives, variants

[0145] The antibodies of the invention may be used alone or as immunoconjugates with a therapeutic agent (e.g., a cytotoxic agent). In some embodiments, the agent is a chemotherapeutic agent. In some embodiments, the agent is a radioisotope, including but not limited to Lead-212, Bismuth-212, Astatine-211, Iodine-131, Scandium-47, Rhenium-186, Rhenium-188, Yttrium-90, Iodine-123, Iodine-125, Bromine-77, Indium-111, and fissionable nuclides such as Boron-10 or an Actinide. In other embodiments, the agent is a toxin or cytotoxic drug, including but not limited to ricin, abrin, modified Pseudomonas enterotoxin A, Pseudomonas exotoxin, calicheamicin, adriamycin, 5-fluorouracil, diphtheria toxin, and the like. Methods of conjugation of antibodies to such agents are known in the literature, and include direct and indirect conjugation.

[0146] Suitable detectable molecules may be directly or indirectly attached to the antibodies and bispecific antibodies of the present invention. Suitable detectable molecules include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with a member of a complementary / anti-complementary pair, where the other member is bound to the binding polypeptide or antibody portion. For these purposes, biotin / streptavidin is an exemplary complementary / anti-complementary pair.

[0147] The antibodies of the invention also include derivatives that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to its epitope. Examples of suitable derivatives include but are not limited to antibodies or bispecific antibodies that are fucosylated, glycosylated, acetylated, pegylated, phosphorylated, or amidated. The antibodies and derivatives thereof of the invention may themselves by derivatized by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other proteins, and the like. In some embodiments of the invention, at least one heavy chain of the antibody or bispecific antigen binding protein is PEGylated. In some embodiments, the PEGylation is N-linked or is linked through the sidechain of an amino acid (e.g., lysine).

[0148] Glycosylation can contribute to the effector function of antibodies, particularly IgG1 antibodies. Thus, in some embodiments, the antigen binding proteins of the invention may comprise one or more amino acid substitutions that affect the level or type of glycosylation of the binding proteins. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tri-peptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tri-peptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0149] In certain embodiments, glycosylation of the antigen binding proteins described herein is increased by adding one or more glycosylation sites, e.g., to the Fc region of the binding protein. Addition of glycosylation sites to the antigen binding protein can be conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tri-peptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites). For ease, the antigen binding protein amino acid sequence may be altered through changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.

[0150] The invention also encompasses production of antigen binding proteins with altered carbohydrate structure resulting in altered effector activity, including antigen binding proteins with absent or reduced fucosylation that exhibit improved ADCC activity. Various methods are known in the art to reduce or eliminate fucosylation. For example, ADCC effector activity is mediated by binding of the antibody molecule to the FcγRIII receptor, which has been shown to be dependent on the carbohydrate structure of the N-linked glycosylation at the N297 residue of the CH2 domain. Non-fucosylated antibodies bind this receptor with increased affinity and trigger FcyRIII-mediated effector functions more efficiently than native, fucosylated antibodies. For example, recombinant production of non-fucosylated antibody in CHO cells in which the alpha-1,6-fucosyl transferase enzyme has been knocked out results in antibody with 100-fold increased ADCC activity (see Yamane-Ohnuki et al., Biotechnol Bioeng. 87(5):614-22, 2004). Similar effects can be accomplished through decreasing the activity of alpha-1,6-fucosyl transferase enzyme or other enzymes in the fucosylation pathway, e.g., through siRNA or antisense RNA treatment, engineering cell lines to knockout the enzyme(s), or culturing with selective glycosylation inhibitors (see Rothman et al., Mol Immunol. 26(12):1113-23, 1989). Some host cell strains, e.g. Lec13 or rat hybridoma YB2 / 0 cell line naturally produce antibodies with lower fucosylation levels (see Shields et al., J Biol Chem. 277(30):26733-40, 2002 and Shinkawa et al., J Biol Chem. 278(5):3466-73, 2003). An increase in the level of bisected carbohydrate, e.g. through recombinantly producing antibody in cells that overexpress GnTIII enzyme, has also been determined to increase ADCC activity (see Umana et al., Nat Biotechnol. 17(2):176-80, 1999).

[0151] In other embodiments, glycosylation of the antigen binding proteins described herein is decreased or eliminated by removing one or more glycosylation sites, e.g., from the Fc region of the binding protein. Amino acid substitutions that eliminate or alter N-linked glycosylation sites can reduce or eliminate N-linked glycosylation of the antigen binding protein. In certain embodiments, the bispecific antigen binding proteins described herein comprise a mutation at position N297 (EU numbering), such as N297Q, N297A, or N297G. In one particular embodiment, the bispecific antigen binding proteins of the invention comprise a Fc region from a human IgG1 antibody with a N297G mutation. To improve the stability of molecules comprising a N297 mutation, the Fc region of the molecules may be further engineered. For instance, in some embodiments, one or more amino acids in the Fc region are substituted with cysteine to promote disulfide bond formation in the dimeric state. Residues corresponding to V259, A287, R292, V302, L306, V323, or I332 (EU numbering) of an IgG1 Fc region may thus be substituted with cysteine. In one embodiment, specific pairs of residues are substituted with cysteine such that they preferentially form a disulfide bond with each other, thus limiting or preventing disulfide bond scrambling. In certain embodiments pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C. In particular embodiments, the bispecific antigen binding proteins described herein comprise a Fc region from a human IgG1 antibody with mutations at R292C and V302C. In such embodiments, the Fc region may also comprise a N297G mutation.

[0152] Modifications of the antigen binding proteins of the invention to increase serum half-life also may desirable, for example, by incorporation of or addition of a salvage receptor binding epitope (e.g., by mutation of the appropriate region or by incorporating the epitope into a peptide tag that is then fused to the antigen binding protein at either end or in the middle, e.g., by DNA or peptide synthesis; see, e.g., WO96 / 32478) or adding molecules such as PEG or other water soluble polymers, including polysaccharide polymers. The salvage receptor binding epitope preferably constitutes a region wherein any one or more amino acid residues from one or two loops of a Fc region are transferred to an analogous position in the antigen binding protein. In one embodiment, three or more residues from one or two loops of the Fc region are transferred. In one embodiment, the epitope is taken from the CH2 domain of the Fc region (e.g., an IgG Fc region) and transferred to the CH1, CH3, or VH region, or more than one such region, of the antigen binding protein. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the CL region or VL region, or both, of the antigen binding protein. See International applications WO 97 / 34631 and WO 96 / 32478 for a description of Fc variants and their interaction with the salvage receptor.

[0153] The antibodies of the invention include variants having single or multiple amino acid substitutions, deletions, additions, or replacements that retain their biological properties (e.g., blocking the binding of TL1A to their respective receptors, inhibiting the biological activity of TL1A). A person of ordinary skill in the art can produce variants having single or multiple amino acid substitutions, deletions, additions or replacements. These variants may include, inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to or deleted from the polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the polypeptide, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like. Antibodies and bispecific antibodies of the invention may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or non-conserved positions. In another embodiment, amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues. The techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to the person having ordinary skill in the art.Nucleic acids, vectors, host cells

[0154] The invention also includes isolated nucleic acids encoding the bispecific antibodies of the invention, which includes, for instance, the light chain, light chain variable region, light chain constant region, heavy chain, heavy chain variable region, heavy chain constant region, linkers, and any and all components and combinations thereof of the bispecific antibodies disclosed herein. Nucleic acids of the invention include nucleic acids having at least 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 98% homology to nucleic acids of the invention. The terms "percent similarity", "percent identity" and "percent homology" when referring to a particular sequence are used as set forth in the University of Wisconsin GCG ®< software program. Nucleic acids of the invention also include complementary nucleic acids. In some instances, the sequences will be fully complementary (no mismatches) when aligned. In other instances, there may be up to about a 20% mismatch in the sequences. In some embodiments of the invention are provided nucleic acids encoding both a heavy chain and a light chain of an antibody of the invention.

[0155] Nucleic acids of the invention can be cloned into a vector, such as a plasmid, cosmid, bacmid, phage, artificial chromosome (BAC, YAC) or virus, into which another genetic sequence or element (either DNA or RNA) may be inserted so as to bring about the replication of the attached sequence or element. In some embodiments, the expression vector contains a constitutively active promoter segment (such as but not limited to CMV, SV40, Elongation Factor or LTR sequences) or an inducible promoter sequence such as the steroid inducible pIND vector (Invitrogen), where the expression of the nucleic acid can be regulated. Expression vectors of the invention may further comprise regulatory sequences, for example, an internal ribosomal entry site. The expression vector can be introduced into a cell by transfection, for example.

[0156] In another embodiment, the present invention provides an expression vector comprising the following operably linked elements; a transcription promoter; a first nucleic acid molecule encoding the heavy chain of a bispecific antigen binding protein, antibody or antigen-binding fragment of the invention; a second nucleic acid molecule encoding the light chain of a bispecific antigen binding protein, antibody or antigen-binding fragment of the invention; and a transcription terminator. In another embodiment, the present invention provides an expression vector comprising the following operably linked elements; a first transcription promoter; a first nucleic acid molecule encoding the heavy chain of a bispecific antigen binding protein, antibody or antigen-binding fragment of the invention; a first transcription terminator; a second transcription promoter a second nucleic acid molecule encoding the light chain of a bispecific antigen binding protein, antibody or antigen-binding fragment of the invention; and a second transcription terminator.

[0157] A secretory signal peptide sequence can also, optionally, be encoded by the expression vector, operably linked to the coding sequence of interest, so that the expressed polypeptide can be secreted by the recombinant host cell, for more facile isolation of the polypeptide of interest from the cell, if desired. For instance, in some embodiments, signal peptide sequences may be appended / fused to the amino terminus of any of the polypeptide sequences listed in Tables E, J, K, and L. In certain embodiments, a signal peptide having the amino acid sequence of MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 499) is fused to the amino terminus of any of the polypeptide sequences in Tables D, I, J, and K. In other embodiments, a signal peptide having the amino acid sequence of METPAQLLFLLLLWLPDTTG (SEQ ID NO: 501) is fused to the amino terminus of any of the polypeptide sequences in Tables E, J, K, and L. In still other embodiments, a signal peptide having the amino acid sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 503) is fused to the amino terminus of any of the polypeptide sequences in Tables E, J, K, and L. Each of the foregoing signal peptides is encoded by the nucleic acid having a sequence immediately preceding it in the Sequence Listing. Other suitable signal peptide sequences that can be fused to the amino terminus of the polypeptide sequences described herein include: MEAPAQLLFLLLLWLPDTTG (SEQ ID NO: 504), MEWTWRVLFLVAAATGAHS (SEQ ID NO: 505), and MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 506). Other signal peptides are known to those of skill in the art and may be fused to any of the polypeptide chains listed in Tables E, J, K and L, for example, to facilitate or optimize expression in particular host cells.

[0158] Recombinant host cells comprising such vectors and expressing the heavy and light chains are also provided.

[0159] Antibody-producing cells and bispecific antigen binding protein producing cells contain, depending on the bispecific antigen binding protein format, nucleic acids encoding the heavy chain, light chain, heavy chain-scFv construct, heavy chain-Fab heavy chain variable domain construct, and Fab light chain variable domain. Such nucleic acids can be used to produce the antibodies or bispecific antibodies of the invention in accordance with techniques known in the art. The present invention, in one embodiment, provides a method of producing a bispecific antigen binding protein or antibody comprising culturing a recombinant host cell expressing the heavy and light chains or other constructs noted above and isolating the bispecific antigen binding protein or antibody produced by the cell.

[0160] The recombinant host cell may be a prokaryotic cell, for example an E. coli cell, or a eukaryotic cell, for example a mammalian cell or a yeast cell. Yeast cells include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris cells. Mammalian cells include VERO, HeLa, Chinese hamster Ovary (CHO), W138, baby hamster kidney (BHK), COS-7, MDCK, human embryonic kidney line 293, normal dog kidney cell lines, normal cat kidney cell lines, monkey kidney cells, African green monkey kidney cells, COS cells, and non-tumorigenic mouse myoblast G8 cells, fibroblast cell lines, myeloma cell lines, mouse NIH / 3T3 cells, LMTK31 cells, mouse sertoli cells, human cervical carcinoma cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor cells, TRI cells, MRC 5 cells, and FS4 cells. Antibody-producing and bispecific antigen binding protein-producing cells of the invention also include any insect expression cell line known, such as for example, Spodoptera frugiperda cells. In a preferred embodiment, the cells are mammalian cells. In a most preferred embodiment, the mammalian cells are CHO cells.

[0161] The antibody-producing cells preferably are substantially free of TL1A and TNF-α binding competitors. In preferred embodiments, the antibody-producing cells comprise less than about 10%, preferably less than about 5%, more preferably less than about 1%, more preferably less than about 0.5%, more preferably less than about 0.1%, and most preferably 0% by weight TL1A or TNF-α binding competitors. In some embodiments, the antibodies and bispecific antibodies produced are substantially free of TL1A and TNF-α competitors. In preferred embodiments, the antibodies and bispecific antibodies produced comprise less than about 10%, preferably less than about 5%, more preferably less than about 1%, more preferably less than about 0.5%, more preferably less than about 0.1%, and most preferably 0% by weight both TL1A and TNF-α binding competitors.Purification

[0162] Methods of antibody purification are known in the art and can be employed with production of the antibodies and bispecific antibodies of the present invention. In some embodiments of the invention, methods for antibody purification include filtration, affinity column chromatography, cation exchange chromatography, anion exchange chromatography, and concentration. The filtration step preferably comprises ultrafiltration, and more preferably ultrafiltration and diafiltration. Filtration is preferably performed at least about 5-50 times, more preferably 10 to 30 times, and most preferably 14 to 27 times. Affinity column chromatography, may be performed using, for example, PROSEP ®< Affinity Chromatography (Millipore, Billerica, Mass.). In a preferred embodiment, the affinity chromatography step comprises PROSEP ®< -vA column chromatography. Eluate may be washed in a solvent detergent. Cation exchange chromatography may include, for example, SP-Sepharose Cation Exchange Chromatography. Anion exchange chromatography may include, for example but not limited to, Q-Sepharose Fast Flow Anion Exchange. The anion exchange step is preferably non-binding, thereby allowing removal of contaminants including DNA and BSA. The antibody product is preferably nanofiltered, for example, using a Pall DV 20 Nanofilter. The antibody product may be concentrated, for example, using ultrafiltration and diafiltration. The method may further comprise a step of size exclusion chromatography to remove aggregates. Further parameters of purification appear in the working examples hereinafter.

[0163] The bispecific antibodies, antibodies or antigen-binding fragments may also be produced by other methods known in the art, for example by chemical coupling of antibodies and antibody fragments.

[0164] The references to the methods of treatment by therapy in this description are to be interpreted as references to the TL1A-specific antigen binding proteins and pharmaceutical compositions of the invention for use in those methods.Uses of the monospecific antibodies of the invention

[0165] The monospecific antibodies of the present invention are useful, for example, for the inhibition of proinflammatory cytokines, such as TL1A. The monospecific antibodies of the invention can be used to treat inflammatory disorders and autoimmune diseases, such as inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome (IBS), bladder syndrome / intersticial cystitis, urinary bowel disfunction, sepsis, uveitis, encephalomyelitis, myasthenia gravis, Sjogren's syndrome (SS), scleroderma, multiple sclerosis (MS), cystic fibrosis (CF), inflammation in chronic kidney disease (CKD), psoriasis (Pso), psoriatic arthritis (PsA), ankylosing spondylitis (AS), rheumatoid arthritis (RA), juvenile rheumatoid arthritis (JRA), osteoarthritis (OA), spondyloarthropathy, primary sclerosing cholangitis, primary biliary cirrhosis, atherosclerosis, splenomegaly, inflammation in chronic kidney disease (CKD), atopic dermatitis (AD), eczematous dermatitis, contact dermatitis systemic sclerosis, systemic lupus erythematosus (SLE), lupus nephritis (LN), cutaneous lupus erythematosus, autoimmune thyroiditis, IgA nephropathy, diabetic kidney disease, antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV), minimal change disease (lipoid nephrosis), focal segmental glomerulosclerosis (FSGS), nephrogenic systemic fibrosis (NSF), nephrogenic fibrosing dermopathy, fibrosing cholestatic hepatitis, eosinophilic fasciitis (Shulman's syndrome), scleromyxedema (popular mucinosis), scleroderma, lichen sclerosusetatrophicus, inflammatory lung injury such as idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease (COPD), airway hyper-responsiveness, chronic bronchitis, allergic asthma, eczema, Helicobacter pylori infection, intraabdominal adhesions and / or abscesses as results of peritoneal inflammation (e.g., from infection, injury, etc.), nephrotic syndrome, idiopathic demyelinating polyneuropathy, Guillain-Barre syndrome, transplant rejection, organ allograft rejection, graft vs. host disease (GVHD) (e.g., from a transplant, such as blood, bone marrow, kidney, pancreas, liver, orthotopic liver, lung, heart, intestine, small intestine, large intestine, thymus, allogeneic stem cell, reduced-intensity allogeneic, bone, tendon, cornea, skin, heart valves, veins, arteries, blood vessels, stomach and testis), IgA nephropathy, diabetic kidney disease, diabetes mellitus, minimal change disease (lipoid nephrosis), nephrogenic systemic fibrosis (NSF), nephrogenic fibrosing dermopathy, fibrosing cholestatic hepatitis, eosinophilic fasciitis (Shulman's syndrome), scleromyxedema (popular mucinosis), scleroderma, lichen sclerosusetatrophicus, Takatsuki disease (or PEP syndrome), nephrotic syndrome, POEMs syndrome, Crow-Fukase syndrome, nephrotic syndrome, , antineutrophil cytoplasmic antibodies, vasculitis, giant cell arteritis and multiple-myeloma-induced lytic bone disease, streptococcal cell wall (SCW)-induced arthritis, gingivitis / periodontitis, herpetic stromal keratitis, gluten-sensitive enteropathy restenosis, Kawasaki's disease, and immune-mediated renal diseases. The monospecific antibodies described herein can also be used to treat cancer, including angiogenesis.

[0166] In one embodiment, the invention concerns methods of treating one or more of the aforementioned diseases and disorders in a mammal in need of such treatment by administering a therapeutically effective amount of a monospecific antigen binding protein of the present invention. In a preferred embodiment the mammal is a human. In another preferred embodiment, the disease or disorder is IBD, CD, or UC.

[0167] The TL1A-specific antigen binding proteins of the invention are for use in a method of treating an inflammatory disease characterized by the presence of elevated levels of TL1A in a patient in need thereof, as defined in the appended claims.

[0168] Accordingly, in one embodiment, the present invention provides a method of inhibiting one or more of proinflammatory cytokines, e.g., TL1A, in a mammal in need of such treatment comprising administering a therapeutically effective amount of a bispecific or monospecific antibody of the invention to a mammal in need of such treatment. In a preferred embodiment, the mammal is a human. The method may be used to treat a disorder characterized by elevated expression or activity of TL1A. The monospecific antigen binding protein may be administered with another pharmaceutical agent, either in the same formulation or separately.

[0169] In another embodiment, the present invention provides a composition comprising a monospecific antigen binding protein as described herein and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising an antibody, of the invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic antibodies are combined in a mixture with a pharmaceutically acceptable carrier. A composition is said to comprise a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well-known to those in the art. See, for example, Getman), ed., Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company (1995).

[0170] For pharmaceutical use, an antibody, of the present invention is formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. Intravenous administration may be by bolus injection, controlled release, e.g., using mini-pumps or other appropriate technology, or by infusion over a typical period of one to several hours. In general, pharmaceutical formulations will include an antibody, of the invention in combination with a pharmaceutically acceptable carrier, such as saline, buffered saline, 5% dextrose in water or the like. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. When utilizing such a combination therapy, the antibodies, may be combined in a single formulation or may be administered in separate formulations. Methods of formulation are well known in the art and are disclosed, for example, in Gennaro, ed., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa. (1990), which is incorporated herein by reference. Therapeutic doses will generally be in the range of 0.1 to 100 mg / kg of patient weight per day, preferably 0.5-20 mg / kg per day, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art. More commonly, the antibodies will be administered over one week or less, often over a period of one to three days. Generally, the dosage of administered antibodies will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. Typically, it is desirable to provide the recipient with a dosage of antibodies which is in the range of from about 1 pg / kg to 10 mg / kg (amount of agent / body weight of patient), although a lower or higher dosage also may be administered as circumstances dictate.

[0171] Administration of an antibody, of the invention to a subject can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection. When administering therapeutic antibodies by injection, the administration may be by continuous infusion or by single or multiple boluses.

[0172] Additional routes of administration include oral, mucosal-membrane, pulmonary, and transcutaneous. Oral delivery is suitable for polyester microspheres, zein microspheres, proteinoid microspheres, polycyanoacrylate microspheres, and lipid-based systems (see, for example, DiBase et al., "Oral Delivery of Microencapsulated Proteins", in Sanders et al., eds., Protein Delivery: Physical Systems, pp. 255-288, Plenum Press (1997)). The feasibility of an intranasal delivery is exemplified by such a mode of insulin administration (see, for example, Hinchcliffe et al., Adv. Drug Deliv. Rev., 35:199 (1999)). Dry or liquid particles comprising antibodies of the invention can be prepared and inhaled with the aid of dry-powder dispersers, liquid aerosol generators, or nebulizers (e.g., Pettit et al., TIBTECH, 16:343 (1998); Patton et al., Adv. Drug Deliv. Rev., 35:235 (1999)). This approach is illustrated by the AERX ®< diabetes management system, which is a hand-held electronic inhaler that delivers aerosolized insulin into the lungs. Studies have shown that proteins as large as 48,000 kDa have been delivered across skin at therapeutic concentrations with the aid of low-frequency ultrasound, which illustrates the feasibility of transcutaneous administration (Mitragotri et al., Science, 269:850 (1995)). Transdermal delivery using electroporation provides another means to administer a molecule having IL-17 and TNF-α / p19 binding activity (Potts et al., Pharm. Biotechnol., 10:213 (1997)).

[0173] For purposes of therapy, compositions comprising an antibody, of the invention and a pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective amount. A combination of an antibody, of the present invention and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient. For example, an agent used to treat inflammation is physiologically significant if its presence alleviates the inflammatory response. Effective treatment may be assessed in a variety of ways. In one embodiment, effective treatment is determined by reduced inflammation. In other embodiments, effective treatment is marked by inhibition of inflammation. In still other embodiments, effective therapy is measured by increased well-being of the patient including such signs as weight gain, regained strength, decreased pain, thriving, and subjective indications from the patient of better health.

[0174] A pharmaceutical composition comprising an antibody, of the invention can be furnished in liquid form, in an aerosol, or in solid form. Liquid forms, are illustrated by injectable solutions and oral suspensions. Exemplary solid forms include capsules, tablets, and controlled-release forms. The latter form is illustrated by miniosmotic pumps and implants (Bremer et al., Pharm. Biotechnol., 10:239 (1997); Ranade, "Implants in Drug Delivery", in Ranade et al., eds., Drug Delivery Systems, pp. 95-123, CRC Press (1995); Bremer et al., "Protein Delivery with Infusion Pumps", in Sanders et al., eds., Protein Delivery: Physical Systems, pp. 239-254, Plenum Press (1997); Yewey et al., "Delivery of Proteins from a Controlled Release Injectable Implant", in Sanders et al., eds., Protein Delivery: Physical Systems, pp. 93-117, Plenum Press (1997).

[0175] Liposomes provide one means to deliver therapeutic polypeptides to a subject intravenously, intraperitoneally, intrathecally, intramuscularly, subcutaneously, or via oral administration, inhalation, or intranasal administration. Liposomes are microscopic vesicles that consist of one or more lipid bilayers surrounding aqueous compartments (see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol. Infect. Dis., 12(Suppl. 1):561 (1993), Kim, Drugs, 46:618 (1993), and Ranade, "Site-Specific Drug Delivery Using Liposomes as Carriers", in Ranade et al., eds., Drug Delivery Systems, pp. 3-24, CRC Press (1995)). Liposomes are similar in composition to cellular membranes and as a result, liposomes can be administered safely and are biodegradable. Depending on the method of preparation, liposomes may be unilamellar or multilamellar, and liposomes can vary in size with diameters ranging from 0.02 .mu.m to greater than 10 .mu.m. A variety of agents can be encapsulated in liposomes: hydrophobic agents partition in the bilayers and hydrophilic agents partition within the inner aqueous space(s) (see, for example, Machy et al., Liposomes in Cell Biology and Pharmacology, John Libbey (1987), and Ostro et al., American J. Hosp. Pharm., 46:1576 (1989)). Moreover, it is possible to control the therapeutic availability of the encapsulated agent by varying liposome size, the number of bilayers, lipid composition, as well as the charge and surface characteristics of the liposomes.

[0176] Liposomes can absorb to virtually any type of cell and then slowly release the encapsulated agent. Alternatively, an absorbed liposome may be endocytosed by cells that are phagocytic. Endocytosis is followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci., 446:368 (1985)). After intravenous administration, small liposomes (0.1 to 1.0 .mu.m) are typically taken up by cells of the reticuloendothelial system, located principally in the liver and spleen, whereas liposomes larger than 3.0 .mu.m are deposited in the lung. This preferential uptake of smaller liposomes by the cells of the reticuloendothelial system has been used to deliver chemotherapeutic agents to macrophages and to tumors of the liver.

[0177] The reticuloendothelial system can be circumvented by several methods including saturation with large doses of liposome particles, or selective macrophage inactivation by pharmacological means (Claassen et al., Biochim. Biophys. Acta, 802:428 (1984)). In addition, incorporation of glycolipid- or polyethelene glycol-derivatized phospholipids into liposome membranes has been shown to result in a significantly reduced uptake by the reticuloendothelial system (Allen et al., Biochim. Biophys. Acta, 1068:133 (1991); Allen et al., Biochim. Biophys. Acta, 1150:9 (1993)).

[0178] Liposomes can also be prepared to target particular cells or organs by varying phospholipid composition or by inserting receptors or ligands into the liposomes. For example, liposomes, prepared with a high content of a nonionic surfactant, have been used to target the liver (Hayakawa et al., Japanese Patent No. 04-244,018; Kato et al., Biol. Pharm. Bull., 16:960 (1993)). These formulations were prepared by mixing soybean phospatidylcholine, alpha. - tocopherol, and ethoxylated hydrogenated castor oil (HCO-60) in methanol, concentrating the mixture under vacuum, and then reconstituting the mixture with water. A liposomal formulation of dipalmitoylphosphatidylcholine (DPPC) with a soybean-derived sterylglucoside mixture (SG) and cholesterol (Ch) has also been shown to target the liver (Shimizu et al., Biol. Pharm. Bull., 20:881 (1997)).

[0179] Alternatively, various targeting ligands can be bound to the surface of the liposome, such as antibodies, antibody fragments, carbohydrates, vitamins, and transport proteins. For example, liposomes can be modified with branched type galactosyllipid derivatives to target asialoglycoprotein (galactose) receptors, which are exclusively expressed on the surface of liver cells (Kato et al., Crit. Rev. Ther. Drug Carrier Syst., 14:287 (1997); Murahashi et al., Biol. Pharm. Bull., 20:259 (1997)). Similarly, Wu et al., Hepatology, 27:772 (1998), have shown that labeling liposomes with asialofetuin led to a shortened liposome plasma half-life and greatly enhanced uptake of asialofetuin-labeled liposome by hepatocytes. On the other hand, hepatic accumulation of liposomes comprising branched type galactosyllipid derivatives can be inhibited by preinjection of asialofetuin (Murahashi et al., Biol. Pharm. Bull., 20:259 (1997)). Polyaconitylated human serum albumin liposomes provide another approach for targeting liposomes to liver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA, 94:11681 (1997)). Moreover, Geho et al. U.S. Pat. No. 4,603,044, describe a hepatocyte-directed liposome vesicle delivery system, which has specificity for hepatobiliary receptors associated with the specialized metabolic cells of the liver.

[0180] In a more general approach to tissue targeting, target cells are prelabeled with biotinylated antibodies specific for a ligand expressed by the target cell (Harasym et al., Adv. Drug Deliv. Rev., 32: 99 (1998)). After plasma elimination of free antibody, streptavidin-conjugated liposomes are administered. In another approach, targeting antibodies are directly attached to liposomes (Harasym et al., Adv. Drug Deliv. Rev., 32: 99 (1998)).

[0181] Antibodies can be encapsulated within liposomes using standard techniques of protein microencapsulation (see, for example, Anderson et al., Infect. Immun., 31:1099 (1981), Anderson et al., Cancer Res., 50:1853 (1990), and Cohen et al., Biochim. Biophys. Acta, 1063:95 (1991), Alving et al. "Preparation and Use of Liposomes in Immunological Studies", in Gregoriadis, ed., Liposome Technology, 2nd Edition, Vol. III, p. 317, CRC Press (1993), Wassef et al., Meth. Enzymol., 149:124 (1987)). As noted above, therapeutically useful liposomes may contain a variety of components. For example, liposomes may comprise lipid derivatives of poly(ethylene glycol) (Allen et al., Biochim. Biophys. Acta, 1150:9 (1993)).

[0182] Degradable polymer microspheres have been designed to maintain high systemic levels of therapeutic proteins. Microspheres are prepared from degradable polymers such as poly(lactide-co-glycolide) (PLG), polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetate polymers, in which proteins are entrapped in the polymer (Gombotz et al., Bioconjugate Chem., 6:332 (1995); Ranade, "Role of Polymers in Drug Delivery", in Ranade et al., eds., Drug Delivery Systems, pp. 51-93, CRC Press (1995); Roskos et al., "Degradable Controlled Release Systems Useful for Protein Delivery", in Sanders et al., eds., Protein Delivery: Physical Systems, pp. 45-92, Plenum Press (1997); Bartus et al., Science, 281:1161 (1998); Putney et al., Nature Biotechnology, 16:153 (1998); Putney, Curr. Opin. Chem. Biol., 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres can also provide carriers for intravenous administration of therapeutic proteins (see, for example, Gref et al., Pharm. Biotechnol., 10:167 (1996).

[0183] The formulation can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0184] In one embodiment, an antibody, of the invention is administered in combination therapy, i.e., combined with other agents, e.g., therapeutic agents, that are useful for treating pathological conditions or disorders, such as autoimmune disorders and inflammatory diseases. The term "in combination" in this context means that the agents are given substantially contemporaneously, either simultaneously or sequentially. If given sequentially, at the onset of administration of the second compound, the first of the two compounds is preferably still detectable at effective concentrations at the site of treatment.

[0185] For example, the combination therapy can include one or more an antibodies, of the invention coformulated with, and / or coadministered with, one or more additional therapeutic agents, e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxic or cytostatic agents, as described in more detail below. Furthermore, one or more antibodies, described herein may be used in combination with two or more of the therapeutic agents described herein. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.

[0186] Preferred therapeutic agents used in combination with an antibody, of the invention are those agents that interfere at different stages in an inflammatory response. In one embodiment, one or more antibodies, e.g., bispecific antibodies, described herein may be co-formulated with, and / or co-administered with, one or more additional agents such as other cytokine or growth factor antagonists (e.g., soluble receptors, peptide inhibitors, small molecules, ligand fusions); or antibodies or antigen binding fragments thereof that bind to other targets (e.g., antibodies that bind to other cytokines or growth factors, their receptors, or other cell surface molecules); and anti-inflammatory cytokines or agonists thereof. Non-limiting examples of the agents that can be used in combination with the antibodies described herein, include, but are not limited to, antagonists of one or more interleukins (ILs) or their receptors, e.g., antagonists of IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL17A-F, IL-18, IL-20, IL-21, IL-22, IL-25 and IL-31; antagonists of cytokines or growth factors or their receptors, such as, LT, EMAP-II, GM-CSF, FGF and PDGF. Antibodies of the invention can also be combined with inhibitors of e.g., antibodies to, cell surface molecules such as CD2, CD3, CD4, CD8, CD20 (e.g., the CD20 inhibitor rituximab (RITUXAN ®< ), CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, or their ligands, including CD154 (gp39 or CD40L), or LFA-1 / ICAM-1 and VLA-4 / VCAM-1 (Yusuf-Makagiansar et al., Med. Res. Rev., 22:146-167 (2002)). Preferred antagonists that can be used in combination with one or more antibodies, e.g., bispecific antibodies, described herein include antagonists of IL-1, IL-6, IL-12, TNF-α, IL-15, IL-18, IL-20, IL-22 and IL-31.

[0187] Examples of those agents include IL-12 antagonists, such as chimeric, humanized, human or in vitro-generated antibodies (or antigen binding fragments thereof) that bind to IL-12 (preferably human IL-12), e.g., the antibody disclosed in WO 00 / 56772; IL-12 receptor inhibitors, e.g., antibodies to human IL-12 receptor; and soluble fragments of the IL-12 receptor, e.g., human IL-12 receptor. Examples of IL-15 antagonists include antibodies (or antigen binding fragments thereof) against IL-15 or its receptor, e.g., chimeric, humanized, human or in vitro-generated antibodies to human IL-15 or its receptor, soluble fragments of the IL-15 receptor, and IL-15-binding proteins. Examples of IL-17 antagonists include brodalumab, secukinumab, and ixekizumab. Examples of IL-18 antagonists include antibodies, e.g., chimeric, humanized, human or in vitro-generated antibodies (or antigen binding fragments thereof), to human IL-18, soluble fragments of the IL-18 receptor, and IL-18 binding proteins (IL-18BP). Examples of IL-1 antagonists include Interleukin-1-converting enzyme (ICE) inhibitors, such as Vx740, IL-1 antagonists, e.g., IL-1 RA (anakinra, Kineret ®< ), sIL1RII, and anti-IL-1 receptor antibodies (or antigen binding fragments thereof).

[0188] In other embodiments, one or more antibodies, described herein may be administered in combination with one or more of the following: IL-13 antagonists, e.g., soluble IL-13 receptors (sIL-13) and / or antibodies against IL-13; IL-2 antagonists, e.g., DAB 486-IL-2 and / or DAB 389-IL-2 (IL-2 fusion proteins, Seragen), and / or antibodies to IL-2R, e.g., anti-Tac (humanized anti-IL-2R, Protein Design Labs). Yet another combination includes one or more antibodies, of the invention, antagonistic small molecules, and / or inhibitory antibodies in combination with nondepleting anti-CD4 inhibitors (DEC-CE9.1 / SB 210396; non-depleting primatized anti-CD4 antibody; IDEC / SmithKline). Yet other preferred combinations include antagonists of the costimulatory pathway CD80 (B7.1) or CD86 (B7.2), including antibodies, soluble receptors or antagonistic ligands; as well as p-selectin glycoprotein ligand (PSGL), anti-inflammatory cytokines, e.g., IL-4 (DNAX / Schering); IL-10 (SCH 52000; recombinant IL-10 DNAX / Schering); IL-13 and TGF-beta, and agonists thereof (e.g., agonist antibodies).

[0189] In other embodiments, one or more antibodies, of the invention can be co-formulated with, and / or co-administered with, one or more anti-inflammatory drugs, immunosuppressants, or metabolic or enzymatic inhibitors. Non-limiting examples of the drugs or inhibitors that can be used in combination with the antibodies described herein, include, but are not limited to, one or more of: nonsteroidal anti-inflammatory drug(s) (NSAIDs), e.g., ibuprofen, tenidap, naproxen, meloxicam, piroxicam, diclofenac, and indomethacin; sulfasalazine; corticosteroids such. as prednisolone; cytokine suppressive anti-inflammatory drug(s) (CSAIDs); inhibitors of nucleotide biosynthesis, e.g., inhibitors of purine biosynthesis, folate antagonists (e.g., methotrexate (N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-glutamic acid); and inhibitors of pyrimidine biosynthesis, e.g., dihydroorotate dehydrogenase (DHODH) inhibitors. Preferred therapeutic agents for use in combination with one or more antibodies, e.g., bispecific antibodies, of the invention include NSAIDs, CSAIDs, (DHODH) inhibitors (e.g., leflunomide), and folate antagonists (e.g., methotrexate).

[0190] Examples of additional inhibitors include one or more of: corticosteroids (oral, inhaled and local injection); immunosuppresants, e.g., cyclosporin, tacrolimus (FK-506); and mTOR inhibitors, e.g., sirolimus (rapamycin--RAPAMUNE ®< or rapamycin derivatives, e.g., soluble rapamycin derivatives (e.g., ester rapamycin derivatives, e.g., CCI-779); agents which interfere with signaling by proinflammatory cytokines such as IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors); COX2 inhibitors, e.g., celecoxib, rofecoxib, and variants thereof; phosphodiesterase inhibitors, e.g., R973401 (phosphodiesterase Type IV inhibitor); phospholipase inhibitors, e.g., inhibitors of cytosolic phospholipase 2 (cPLA2) (e.g., trifluoromethyl ketone analogs); inhibitors of vascular endothelial cell growth factor or growth factor receptor, e.g., VEGF inhibitor and / or VEGF-R inhibitor; and inhibitors of angiogenesis. Preferred therapeutic agents for use in combination with the antibodies of the invention are immunosuppresants, e.g., cyclosporin, tacrolimus (FK-506); mTOR inhibitors, e.g., sirolimus (rapamycin) or rapamycin derivatives, e.g., soluble rapamycin derivatives (e.g., ester rapamycin derivatives, e.g., CCI-779); COX2 inhibitors, e.g., celecoxib and variants thereof; and phospholipase inhibitors, e.g., inhibitors of cytosolic phospholipase 2 (cPLA2), e.g., trifluoromethyl ketone analogs.

[0191] Additional examples of therapeutic agents that can be combined with an antibody, of the invention include one or more of: 6-mercaptopurines (6-MP); azathioprine sulphasalazine; mesalazine; olsalazine; chloroquine / hydroxychloroquine (PLAQUENIL ®< ); pencillamine; aurothiornalate (intramuscular and oral); azathioprine; coichicine; beta-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeteral); xanthines (theophylline, aminophylline); cromoglycate; nedocromil; ketotifen; ipratropium and oxitropium; mycophenolate mofetil; adenosine agonists; antithrombotic agents; complement inhibitors; and adrenergic agents.

[0192] Anti-TL1A antibodies of the invention may be combined with TNF-α antagonists for treatment of the same conditions as noted herein for anti-TL1A / anti-TNF-α bispecific antigen binding proteins. Such TNF-α antagonists include, for example, etanercept, adalimumab, infliximab, golimumab, and certolizumab pegol.

[0193] Non-limiting examples of agents for treating or preventing arthritic disorders (e.g., rheumatoid arthritis, inflammatory arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis and psoriatic arthritis), with which an antibody, e.g., bispecific antigen binding protein, of the invention may be combined include one or more of the following: IL-12 antagonists as described herein; NSAIDs; CSAIDs; nondepleting anti-CD4 antibodies as described herein; IL-2 antagonists as described herein; anti-inflammatory cytokines, e.g., IL-4, IL-10, IL-13 and TGF-α, or agonists thereof; IL-1 or IL-1 receptor antagonists as described herein; phosphodiesterase inhibitors as described herein; Cox-2 inhibitors as described herein; iloprost: methotrexate; thalidomide and thalidomide-related drugs (e.g., Celgen); leflunomide; inhibitor of plasminogen activation, e.g., tranexamic acid; cytokine inhibitor, e.g., T-614; prostaglandin E1; azathioprine; an inhibitor of interleukin-1 converting enzyme (ICE); zap-70 and / or 1 ck inhibitor (inhibitor of the tyrosine kinase zap-70 or 1 ck); an inhibitor of vascular endothelial cell growth factor or vascular endothelial cell growth factor receptor as described herein; an inhibitor of angiogenesis as described herein; corticosteroid anti-inflammatory drugs (e.g., SB203580); TNF-α -convertase inhibitors; IL-1; IL-13; IL-17 inhibitors; gold; penicillamine; chloroquine; hydroxy chloroquine; chlorambucil; cyclophosphamide; cyclosporine; total lymphoid irradiation; antithymocyte globulin; CD5-toxins; orally administered peptides and collagen; lobenzarit disodium; cytokine regulating agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP 10; T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycan polysulphate; minocycline (MINOCIN ®< ); anti-IL2R antibodies; marine and botanical lipids (fish and plant seed fatty acids); auranofin; phenylbutazone; meclofenamic acid; flufenamic acid; intravenous immune globulin; zileuton; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose (therafectin); cladribine (2-chlorodeoxyadenosine); and azaribine. Preferred combinations include one or more antibodies, e.g., bispecific antibodies, of the invention in combination with methotrexate or leflunomide, and in moderate or severe rheumatoid arthritis cases, cyclosporine.

[0194] Preferred examples of inhibitors to use in combination with one or more antigen binding proteins, e.g., bispecific antigen binding proteins, of the invention to treat arthritic disorders include antagonists of IL-12, IL-15, IL-18, IL-22; T cell and B cell-depleting agents (e.g., anti-CD4 or anti-CD22 antibodies); small molecule inhibitors, e.g., methotrexate and leflunomide; sirolimus (rapamycin) and analogs thereof, e.g., CCI-779; cox-2 and cPLA2 inhibitors; NSAIDs; p38 inhibitors, TPL-2, Mk-2 and NFκB inhibitors; RAGE or soluble RAGE; P-selectin or PSGL-1 inhibitors (e.g., small molecule inhibitors, antibodies to PSGL-1, antibodies to P-selectin); estrogen receptor beta (ERB) agonists or ERB-NFκB antagonists. Most preferred additional therapeutic agents that can be co-administered and / or co-formulated with one or more antibodies, e.g., bispecific antibodies, of the invention include one or more of: methotrexate, leflunomide, or a sirolimus (rapamycin) or an analog thereof, e.g., CCI-779.

[0195] Non-limiting examples of agents for treating or preventing an inflammatory disease or disorder, (e.g., IBD, CD, UC, IBS) with which an antibody, e.g., bispecific antigen binding protein, of the invention can be combined include the following: budenoside; epidermal growth factor; corticosteroids; cyclosporine; sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-1 monoclonal antibodies; anti-IL-6 monoclonal antibodies (e.g., anti-IL-6 receptor antibodies and anti-IL-6 antibodies); growth factors; elastase inhibitors; pyridinyl-imidazole compounds; IL-4, IL-10, IL-13 and / or TGF.beta. cytokines or agonists thereof (e.g., agonist antibodies); IL-11; glucuronide- or dextran-conjugated prodrugs of prednisolone, dexamethasone or budesonide; ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); slow-release mesalazine; methotrexate; antagonists of platelet activating factor (PAF); ciprofloxacin; and lignocaine.

[0196] Non-limiting examples of agents for treating or preventing multiple sclerosis with one or more antibodies, e.g., bispecific antibodies, of the invention can be combined include the following: interferons, e.g., interferon-α (e.g., AVONEX ®< , Biogen) and interferon-1b (BETASERON ®< , Chiron / Berlex); Copolymer 1 (Cop-1; COPAXONE ®< , Teva Pharmaceutical Industries, Inc.); dimethyl fumarate (e.g., BG-12; Biogen); hyperbaric oxygen; intravenous immunoglobulin; cladribine; corticosteroids; prednisolone; methylprednisolone; azathioprine; cyclophosphamide; cyclosporine: cyclosporine A, methotrexate; 4-aminopyridine; and tizanidine. Additional antagonists that can be used in combination with antibodies of the invention include antibodies to or antagonists of other human cytokines or growth factors, for example, LT, IL-1, IL-2, IL-6, EL-7, IL-8, IL-12 IL-15, IL-16, IL-18, EMAP-11, GM-CSF, FGF, and PDGF. Antibodies as described herein can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or their ligands. One or more antibodies, e.g., bispecific antibodies, of the invention may also be combined with agents, such as methotrexate, cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents which interfere with signaling by proinflammatory cytokines as described herein, IL-1b converting enzyme inhibitors (e.g., Vx740), anti-P7s, PSGL, TACE inhibitors, T-cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof, as described herein, and anti-inflammatory cytokines (e.g., IL-4, IL-10, IL-13 and TGF).

[0197] Preferred examples of therapeutic agents for multiple sclerosis with which the antibodies of the invention can be combined include dimethyl fumarate (e.g., BG-12; Biogen), interferon-beta, for example, IFN-β-1a and IFN-β-1b; COPAXONE ®< , corticosteroids, IL-1 inhibitors, antibodies to CD40 ligand and CD80, IL-12 antagonists.

[0198] Non-limiting examples of agents for treating or preventing psoriasis with which an antibody, e.g., bispecific antigen binding protein, of the invention can be combined include the following: corticosteroids; vitamin. D 3 and analogs thereof; retinoiods (e.g., soriatane); methotrexate; cyclosporine, 6-thioguanine; Accutane; hydrea; hydroxyurea; sulfasalazine; mycophenolate mofetil; azathioprine; tacrolimus; fumaric acid esters; biologics such as AMEVIVE ®< , Raptiva ustekinumab, and XP-828L; phototherapy; and photochemotherapy (e.g., psoralen and ultraviolet phototherapy combined).

[0199] Non-limiting examples of agents for treating or preventing inflammatory airway / respiratory disease (e.g., chronic obstructive pulmonary disorder, asthma) with which an antibody, e.g., bispecific antigen binding protein, of the invention can be combined include the following: beta2-adrenoceptor agonists (e.g., salbutamol (albuterol), levalbuterol, terbutaline, bitolterol); long-acting beta2-adrenoceptor agonists (e.g., salmeterol, formoterol, bambuterol); adrenergic agonists (e.g., inhaled epinephrine and ephedrine tablets); anticholinergic medications (e.g., ipratropium bromide); combinations of inhaled steroids and long-acting bronchodilators (e.g., fluticasone / salmeterol (ADVAIR ®< in the United States, and Seretide in the United Kingdom)) or. budesonide / formoterol (SYMBICORT ®< )); inhaled glucocorticoids (e.g., ciclesonide, beclomethasone, budesonide, flunisolide, fluticasone, mometasone, triamcinolone); leukotriene modifiers (e.g., montelukast, zafirlukast, pranlukast, and zileuton); mast cell stabilizers (e.g., cromoglicate (cromolyn), and nedocromil); antimuscarinics / anticholinergics (e.g., ipratropium, oxitropium, tiotropium); methylxanthines (e.g., theophylline, aminophylline); antihistamines; IgE blockers (e.g., omalizumab); M 3 muscarinic antagonists (anticholinergics) (e.g., ipratropium, tiotropium); cromones (e.g., chromoglicate, nedocromil); zanthines (e.g., theophylline); IL-17 inhibitors (e.g., brodalumab, secukinumab, ixekizumab), IL-4 inhibitors; and IL-13 inhibitors.

[0200] In one embodiment, an antibody, of the invention can be used in combination with one or more antibodies directed at other targets involved in regulating immune responses, e.g., transplant rejection.

[0201] Non-limiting examples of agents for treating or preventing immune responses with which an antibody, e.g., bispecific antigen binding protein, of the invention can be combined include the following: antibodies against other cell surface molecules, including but not limited to CD25 (interleukin-2 receptor-α), CD11a (LFA-1), CD54 (ICAM-1), CD4, CD45, CD28 / CTLA4 (CD80 (B7.1), e.g., CTLA4 Ig (abatacept , ORENCIA ®< ), ICOSL, ICOS and / or CD86 (B7.2). In yet another embodiment, an antibody of the invention is used in combination with one or more general immunosuppressive agents, such as cyclosporin A or FK506.

[0202] In other embodiments, antibodies are used as vaccine adjuvants against autoimmune disorders, inflammatory diseases, etc. The combination of adjuvants for treatment of these types of disorders are suitable for use in combination with a wide variety of antigens from targeted self-antigens, i.e., autoantigens, involved in autoimmunity, e.g., myelin basic protein; inflammatory self-antigens (e.g., amyloid peptide protein) or transplant antigens (e.g., alloantigens). The antigen may comprise peptides or polypeptides derived from proteins, as well as fragments of any of the following: saccharides, proteins, polynucleotides or oligonucleotides, autoantigens, amyloid peptide protein, transplant antigens, allergens, or other macromolecular components. In some instances, more than one antigen is included in the antigenic composition.

[0203] For example, desirable vaccines for moderating responses to allergens in a vertebrate host, which contain the adjuvant combinations of this invention, include those containing an allergen or fragment thereof. Examples of such allergens are described in U.S. Pat. No. 5,830,877 and PCT Publication No. WO 99 / 51259, and include pollen, insect venoms, animal dander, fungal spores and drugs (such as penicillin). The vaccines interfere with the production of IgE antibodies, a known cause of allergic reactions. In another example, desirable vaccines for preventing or treating disease characterized by amyloid deposition in a vertebrate host, which contain the adjuvant combinations of this invention, include those containing portions of amyloid peptide protein (APP). This disease is referred to variously as Alzheimer's disease, amyloidosis or amyloidogenic disease. Thus, the vaccines of this invention include the adjuvant combinations of this invention plus A.beta. peptide, as well as fragments of Aβ peptide and antibodies to Aβ peptide or fragments thereof.

[0204] In another embodiment, pharmaceutical compositions may be supplied as a kit comprising a container that comprises an antibody, bispecific antigen binding protein or antigen-binding fragment of the invention. Antibodies, e.g., bispecific antibodies, of the invention can be provided in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection. Alternatively, such a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of the antigen binding protein. Such a kit may further comprise written information on indications and usage of the pharmaceutical composition. Moreover, such information may include a statement that the antibody composition is contraindicated in patients with known hypersensitivity to TL1A and TNF-α .

[0205] In a further embodiment, the invention provides an article of manufacture, comprising: (a) a composition of matter comprising an antibody, bispecific antigen binding protein or antigen-binding fragment as described herein; (b) a container containing said composition; and (c) a label affixed to said container, or a package insert included in said container referring to the use of said antibody in the treatment of an immune related disease.

[0206] In another aspect, the composition comprises a further active ingredient, which may, for example, be a further antibody or an anti-inflammatory, cytotoxic or chemotherapeutic agent. Preferably, the composition is sterile.

[0207] The antibodies, as described herein are also useful to prepare medicines and medicaments for the treatment of immune-related and inflammatory diseases, including for example, IBS, IBD, CD, UC. In a specific aspect, such medicines and medicaments comprise a therapeutically effective amount of a bispecific antigen binding protein, antibody or antigen-binding fragment of the invention with a pharmaceutically acceptable carrier. In an embodiment, the admixture is sterile.

[0208] The bispecific antigen binding proteins which are not covered by the claims can be used for diagnostic purposes to detect, diagnose, or monitor diseases and / or conditions associated with TL1A and / or TNF-α. Also provided are methods for the detection of the presence of TL1A and / or TNF-α in a sample using classical immunohistological methods known to those of skill in the art. See, for example, Tijssen (1993), Practice and Theory of Enzyme Immunoassays, Vol 15 (Eds R.H. Burdon and P.H. van Knippenberg, Elsevier, Amsterdam); Zola (1987), Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc.); Jalkanen et al. (1985), J. Cell. Biol. 101:976-985; Jalkanen et al. (1987), J. Cell Biol. 105:3087-3096. The detection of either TL1A and / or TNF-α can be performed in vivo or in vitro.

[0209] Diagnostic applications provided herein include use of the antigen binding proteins to detect expression of TL1A and / or TNF-α and binding of these ligands to their receptors. Examples of methods useful in the detection of the presence of the ligand include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).

[0210] For diagnostic applications, the antigen binding protein typically will be labeled with a detectable labeling group. Suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3< H, 14< C, 15< N, 35< S, 90< Y, 99< Tc, 111< In, 125< I, 131< I), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labeling group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and may be used.

[0211] The bispecific antigen binding protein whice is not party of the claims, can be used to identify a cell or cells that express TL1A and / or TNF-α. In a specific embodiment, the antigen binding protein is labeled with a labeling group and the binding of the labeled antigen binding protein to TL1A and / or TNF-α is detected. In a further specific embodiment, the binding of the antigen binding protein to TL1A and / or TNF-α is detected in vivo. In a further specific embodiment, the bispecific antigen binding protein is isolated and measured using techniques known in the art. See, for example, Harlow and Lane, 1988, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor (ed. 1991 and periodic supplements); John E. Coligan, ed., 1993, Current Protocols In Immunology New York: John Wiley & Sons.WORKING EXAMPLES

[0212] The invention is further illuminated by the following working examples, which exemplify but do not limit the scope of the invention. The invention is defined in the appended claims.EXAMPLE 1 Preparation of XenoMouse ®< anti-TL1A monoclonal antibodiesMouse strains

[0213] Fully human antibodies to human TL1A were generated by immunizing XENOMOUSE ®< transgenic mice. U.S. Pat. Nos. 6,114,598; 6,162,963;6,833,268; 7,049,426; 7,064,244, which are incorporated herein by reference in their entirety; Green et al. (1994), Nature Genetics 7:13-21; Mendez et al. (1997), Nature Genetics 15:146-156; Green and Jakobovitis (1998), J. Ex. Med, 188:483-495; Kellerman and Green (2002), Current Opinion in Biotechnology 13, 593-597; each of which is incorporated by reference in its entirety. Animals from the XMG1-KL, XMG2-K, XMG2-K / Balbc, XMG2-KL, XMG4-K and XMG4-KL XENOMOUSE ®< strains were used for all immunizations.Generation of TL1A Immunogen

[0214] TL1A polypeptides containing the N-terminal His tag (H6) of which the first 22 amino acids are the VK1 signal peptide) were generated by transiently transfecting 293HEK cells with the corresponding cDNAs. The commonly used polyHis tag was employed to facilitate detection and subsequent purification.

[0215] 293-6E cells at 9.48x10 5< cells / ml were transfected with 0.5 mg / L DNA (0.1 mg / L His-TL1A in pTT5 vector with 0.4 mg / L empty pTT5 vector) (Durocher et al. (2002) NRCC, Nucleic Acids. Res. 30, e9) with 3 ml PEI / mg DNA in FreeStyle 293 media (Invitrogen). Tryptone N1 was added to cultures 1 hour after transfection. Cells were grown in suspension in FreeStyle 293 expression medium supplemented with 0.1% Pluronic F68 and 50 µg / ml Geneticin for 7 days and harvested for purification.EXAMPLE 2 Generation of TL1A

[0216] TL1A polypeptides containing the N-terminal His tag (H6) of which the first 22 amino acids are the VK1 signal peptide) were generated by transiently transfecting 293HEK cells with the corresponding cDNAs. The commonly used polyHis tag was employed to facilitate detection and subsequent purification.

[0217] 293-6E cells at 9.48x10 5< cells / ml were transfected with 0.5mg / L DNA (0.1mg / L His-TL1A in pTT5 vector with 0.4mg / L empty pTT5 vector) (Durocher et al. NRCC, Nucleic Acids. Res. (2002) 30, e9) with 3ml PEI / mg DNA in FreeStyle 293 media (Invitrogen). Tryptone N1 was added to cultures 1 hour after transfection. Cells were grown in suspension in FreeStyle 293 expression medium supplemented with 0.1% Pluronic F68 and 50 µg / ml Geneticin for 7 days and harvested for purification.Immunizations

[0218] Immunizations are conducted using one or more suitable forms of TL1A antigen, including recombinant human TL1A expressed on cells and recombinant human TL1A soluble protein or combinations thereof.

[0219] A suitable amount of immunogen (i.e., 10 µg of protein delivered by injection to the abdomen) is used for initial immunization in XenoMouse ®< . Following the initial immunization, subsequent boost immunizations of immunogen (i.e., 2 x 10 e6 cells or 5 µg of protein) are administered on a schedule and for the duration necessary to induce a suitable titer of anti-TL1A antibody in the mice. Titers are determined by any suitable method, for example, enzyme immunoassay or fluorescence activated cell sorting (FACS).

[0220] Multiple immunogens and routes of immunization were used to generate anti-human TL1A immune responses. For genetic immunizations, mice were immunized 12-16 times over 6-8 weeks using the Helios Gene Gun system according to the manufacturer's instructions (BioRad, Hercules, California). Briefly, expression vectors encoding wild type human or cynomolgus TL1A were coated onto gold beads (BioRad, Hercules, California) and delivered to the epidermis of a shaved mouse abdomen. For cell-based immunizations, mice were immunized with a suspension-adapted CHO-K1 cell line (Invitrogen, Carlsbad, California), stably transfected with an expression vector encoding human TL1A. Animals were immunized with cells mixed with Alum prepared from aluminum potassium sulfate (EMD Chemicals Inc., Gibbstown, NJ) and CpG-ODN (Eurofins MWG Operon LLC, Huntsville, AL) 10-12 times over 6-8 weeks using a protocol that alternated between subcutaneous and intraperitoneal injections. The initial boost was comprised of 4x10 6< cells while subsequent boosts contained 2x10 6< cells. For soluble, recombinant protein immunizations, mice were immunized with a 6x His-tagged, trimeric form of the human and cynomolgus TL1A extracellular domain (amino acids 72-251 of the TL1A sequences). Animals were immunized with recombinant protein mixed with Alum and CpG-ODN, 8-12 times over 4-8 weeks using sub-cutaneous injections. The initial boost was comprised of 10 µg while subsequent boosts contained 5 µg. Human TL1A-specific serum titers were monitored by live-cell FACS analysis on an Accuri or FacsCalibur (BD Biosciences) flow cytometer. Animals with the highest antigen-specific serum titers directed against human and cynomolgus TL1A were sacrificed and used for hybridoma generation (Kohler and Milstein, 1975).EXAMPLE 3 Preparation of Monoclonal AntibodiesHybridoma Generation

[0221] Animals exhibiting suitable titers are identified, and lymphocytes are obtained from draining lymph nodes and, if necessary, pooled for each cohort. Pooled lymphocytes (from each immunization cohort) were dissociated from lymphoid tissue by grinding in a suitable medium (for example, Dulbecco's Modified Eagle Medium (DMEM); Invitrogen, Carlsbad, CA).. B cells may be selected and / or expanded using a suitable method, and fused with suitable fusion partner; for example, non-secretory myeloma P3X63Ag8.653 cells (American Type Culture Collection CRL 1580; Kearney et al, J. Immunol. 123, 1979, 1548-1550), using techniques that are known in the art. B cells were selected and / or expanded using standard methods, and fused with a suitable fusion partner using techniques that were known in the art.

[0222] In one suitable fusion method, lymphocytes are mixed with fusion partner cells at a ratio of 1:4. The cell mixture is gently pelleted by centrifugation at 400 x g for 4 minutes, the supernatant decanted, and the cell mixture gently mixed (for example, by using a 1 ml pipette). Fusion is induced with PEG / DMSO (polyethylene glycol / dimethyl sulfoxide; obtainable from Sigma-Aldrich, St. Louis MO; 1 ml per million lymphocytes). PEG / DMSO is slowly added with gentle agitation over one minute followed, by one minute of mixing. IDMEM (DMEM without glutamine; 2 ml per million B cells), is then added over 2 minutes with gentle agitation, followed by additional IDMEM (8 ml per million B-cells) which is added over 3 minutes.

[0223] The fused cells are gently pelleted (400 x g, 6 minutes) and resuspended in 20 ml Selection media (for example, DMEM containing Azaserine and Hypoxanthine [HA] and other supplemental materials as necessary) per million B-cells. Cells are incubated for 20-30 minutes at 37 C and then resuspended in 200 ml Selection media and cultured for three to four days in T175 flasks prior to 96-well plating.

[0224] Cells are distributed into 96-well plates using standard techniques to maximize clonality of the resulting colonies. After several days of culture, supernatants are collected and subjected to screening assays as detailed in the examples below, including confirmation of binding to human TL1A, evaluation of cross-reactivity with other species' TL1A (for example, cynomologous monkey TL1A), and ability to inhibit the activity of TL1A. Positive cells are further selected and subjected to standard cloning and subcloning techniques. Clonal lines may be expanded in vitro, and the secreted human antibodies obtained for analysis.

[0225] In this manner, mice were immunized with recombinant human TL1A soluble protein for a total of 15 immunizations over a period of approximately 2 months; several hybridoma cell lines secreting TL1A-specific antibodies were obtained, and the antibodies were further characterized. The sequences thereof are presented in the Sequence Listing and in Tables A, C and D and results of various tests using these antibodies are shown herein.

[0226] Tables A to E herein show the sequences of anti-TL1A antibodies prepared in accordance with this working example.EXAMPLE 4 Antigen Enrichment of Hybridoma Pools

[0227] Fused hybridoma pools from select immune tissue harvest were used as a source of material for FACS-based enrichments. To enrich for hybridomas expressing antibodies specific to native (full length, on-cell) human TL1A membranes were prepared from 293T cells transiently expressing the TL1A cDNA construct. 24 hours after transfection using 293Fectin ™< (ThermoFisher Scientific Inc.) cells were biotinylated with E-Z link NHS-LC-LC- Biotin according to the manufacturer's recommendation (ThermoFisher Scientific Inc.). After biotinylation, cells were homogenized with a needle and syringe to form membrane fragments and referred to as "membrane preps". The biotinylated membrane preps were then used to detect hybridomas expressing surface antibodies specific to the target of interest via standard biotin-streptavidin chemistry.

[0228] To enrich hybridoma pools for the antigen of interest, they were first incubated with the membrane prep probe. Unbound probe was then washed away and the antigen-specific hybridomas were identified by simultaneous detection of surface IgG (with an Alexa 488 conjugated Gt anti-human Fc secondary antibody; Jackson ImmunoResearch) and the biotinylated membrane prep TL1A probe (Alexa Fluor 647 conjugated streptavidin; Jackson ImmunoResearch). Hybridomas expressing surface IgG and binding antigen were detected by FACS analysis on an Accuri flow cytometer. Dual positive events were sorted as single cells into 384-well plates on a FACS Aria cell sorter (BD Biosciences). After several days of culture, the hybridoma supernatants containing monoclonal antibodies were collected and used in the screening assays described in the examples below.EXAMPLE 5 Initial Selection of TL1A-Specific Binding Antibodies

[0229] Human TL1A was expressed on host Human Embryonic Kidney 293 cells by transfection using an expression vector expressing huTL1A cDNA, Gibco ™< Opti-MEM ®< media (Gibco, Cat. No. 31985088) and 293Fectin ™< reagent (Invitrogen, Cat. No. 12347019) following the protocol set out by the manufacturer. Hybridoma supernatants were screened for the presence of huTL1A-specific monoclonal antibodies using the FMAT 8200 Screening System (Molecular Devices) and the CellInsight ™< High Content Imaging Platform (ThermoFisher Scientific). The number of huTL1A positive wells (i.e., those that have signal over irrelevant hybridoma supernatant) is presented in Table 5.1. For CellInsight screens, 15 µl / well of hybridoma supernatant (and positive and negative controls) were added to black, 384-well, clear bottom plates (Corning Can. No. 3712) followed by the addition of 30 µl / well of a mixture of TL1A / 293T cells, nuclear Hoescht stain (Pierce, Cat. No. 62249) and Alexa 488-goat anti-human IgG (H+L) (Jackson, Cat. No. 109-545-088). After 3 hours of incubation at room temperature, plates were washed 2 times on an AquaMax 4000 plate washer (fitted with a 384-well cell wash head) and read on the CellInsight instrument according to the manufacturer's recommendations. For FMAT-based screens, 20 µl / well of hybridoma supernatant (and positive and negative controls) were added to black, 384-well, clear bottom plates followed by addition of 40 µl / well of a mixture of TL1A / 293T cells, 293T parental cells and Cy5-goat anti-human IgG (Fc) (Jackson, Cat. No. 109-075). After 3 hours of incubation at room temperature, plates were read on the FMAT 8200 system according to the manufacturer's recommendations. Table 5.1: TL1A-specific antibodies selectedHarvest # Platform Total # Positive 1FMAT 820014882FMAT 82009005Cellinsight18306FMAT 82004387&8Cellinsight1541 EXAMPLE 6 Identification of TL1A Receptor-Ligand Blocking Antibodies

[0230] Biotinylated huTL1A was prepared by reacting 100 µg / ml of NHS LC LC biotin (Pierce, Cat. No. 21338) and 100 µg huTL1A (prepared as described in Example 2) in 1 ml of PBS pH 8.5 for 1hr at room temperature. Un-reacted biotin was removed by ultra-filtration using a 5 kDa Amicon Ultra spin column (Millipore, Cat. No. UFC8 005). Hybridoma supernatants containing huTL1A-binding antibodies were assayed for their ability to block huTL1A binding to human Death Receptor 3 (huDR3) via an ELISA-based receptor-ligand assay. ELISA plates (Corning Cat. No. 3702) were coated with 40 µl / well of human DR3-Fc chimera (1 µg / ml) (R&D Systems, Cat. No. 943-D3) in coating buffer (1x PBS / 0.05% azide), then incubated overnight at 4°C. Plates were then washed with water 3 times and blocked with 90 µl diluent (1x PBS / 1% milk) for 30 min at room temperature. 15 µl of anti-TL1A hybridoma supernatant was preincubated with 45 µl of biotinylated TL1A (30 ng / ml final) in 96-well storage plates (Sigma, Cat. No. P6866) in assay diluent for 2 hr at room temperature prior to adding to the pre-blocked DR3 ELISA plates. Assay plates were then incubated for 1 hr at room temperature. Sample plates were subsequently washed 3 times followed by the addition of 40 µl / well of streptavidin-HRP (Pierce, Cat. No. 21126) and another 1 hr incubation at room temperature. Plates were washed an additional three times and 40 µl TMB (Neogen, Cat. No. 308177) was added. The TMB reaction was incubated for 30 min at room temperature and then quenched with 40 µl / well of 1 N hydrochloric acid. Finally, plates were read on an ELISA plate reader at a wavelength of 450 nm. Cutoffs were set at < 38% of signal of negative control, irrelevant ESNs (exhausted supernatants). The numbers of samples able to block the huTL1A-DR3 interaction (as defined by this cutoff) are indicated in Table 6.1. Table 6.1: Selected TL1A / DR3 blockersHarvest # Total #TL1A / OR3 blockers 1208239 EXAMPLE 7 TL1A functional blocking assays

[0231] In order to screen for hybridomas capable of blocking TL1A functional activity, IFNγ release from primary T cells or a NF-κB reporter assay in TF1 cells were employed. For the IFNγ release assay, purified primary human T cells (Biological Speciality Corp.,Cat. # 215-01-10) were stimulated with soluble human TL1A in the presence or absence of hybridoma supernatants specific to TL1A. 2 x 10 5< primary human T cells were stimulated with 8 - 16 ng / mL human TL1A (Amgen), 1 - 2 ng / mL IL-12 (Peprotech) and 0.5 - 1 ng / mL IL-18 (R&D Systems) in the presence of hybridoma supernatants containing anti-TL1A antibodies in 96-well round bottom plate at 37 °C for 72 hours. Culture supernatants were then tested for IFNγ level by ELISA according to the manufacturer's instructions (R&D Systems). For the TL1A responsive reporter assay, a TF-1 NF-κB reporter cell line (Amgen) was stimulated with soluble or membrane-bound human TL1A or soluble cynomolgus monkey TL1A. 0.2 - 3 nM or 2 - 20 nM of soluble human or cynomolgus monkey TL1A (respectively) was incubated with 10 4< - 10 5< TF-1 NF-κB reporter cells in the presence of serially diluted hybridoma supernatants (or controls) in 96 or 384-well plates at 37 °C overnight. For testing samples against membrane-bound TL1A, activity assays were performed by co-culturing the TF-1 NF-κB reporter cell line with human TL1A-expressing AM1D cells. 10 5< TF-1 NF-κB reporter cells and 10 3< AM1D cells were co-cultured in the presence of 5 ug / mL of anti-TL1A antibody (or controls) in a 384-well plate at 37 °C overnight. Reporter signal in each well was determine using the Steady-Glo Luciferase Assay System according to the manufacturer's recommendation (Promega).EXAMPLE 8 Molecular Rescue and Sequencing of TL1A Receptor-Ligand Blocking Antibodies

[0232] RNA (total or mRNA) was purified from wells containing the TL1A-neutralizing antibody-producing hybridoma cells using a Qiagen RNeasy mini or the Invitrogen mRNA catcher plus kit. Purified RNA was used to amplify the antibody heavy and light chain variable region (V) genes using cDNA synthesis via reverse transcription, followed by a polymerase chain reaction (RT-PCR). The fully human antibody gamma heavy chain was obtained using the Qiagen One Step Reverse Transcriptase PCR kit (Qiagen). This method was used to generate the first strand cDNA from the RNA template and then to amplify the variable region of the gamma heavy chain using multiplex PCR (see Table 8.1 for the complete primer list, SEQ ID NOS: 1191 to 1252, respectively). The 5' gamma chain-specific primer annealed to the signal sequence of the antibody heavy chain, while the 3' primer annealed to a region of the gamma constant domain. The fully human kappa light chain was obtained using the Qiagen One Step Reverse Transcriptase PCR kit (Qiagen). This method was used to generate the first strand cDNA from the RNA template and then to amplify the variable region of the kappa light chain using multiplex PCR. The 5' kappa light chain-specific primer annealed to the signal sequence of the antibody light chain while the 3' primer annealed to a region of the kappa constant domain. The fully human lambda light chain was obtained using the Qiagen One Step Reverse Transcriptase PCR kit (Qiagen). This method was used to generate the first strand cDNA from the RNA template and then to amplify the variable region of the lambda light chain using multiplex PCR. The 5' lambda light chain-specific primer annealed to the signal sequence of light chain while the 3' primer annealed to a region of the lambda constant domain.

[0233] The amplified cDNA was purified enzymatically using exonuclease I and alkaline phosphatase and the purified PCR product was sequenced directly. Amino acid sequences were deduced from the corresponding nucleic acid sequences bioinformatically. Two additional, independent RT-PCR amplification and sequencing cycles were completed for each hybridoma sample in order to confirm that any mutations observed were not a consequence of the PCR. The derived amino acid sequences were then analyzed to determine the germline sequence origin of the antibodies and to identify deviations from the germline sequence. The amino acid sequences corresponding to CDRs of the sequenced antibodies were aligned and these alignments were used to group the clones by similarity. Table 8.1: Multiplex primers used to amplify antibody V genes.Heavy chain Sequence 5' to 3' SEQ ID NO. C ACC ATG GAC TGG ACC TGG AGG ATC1191C ACC ATG GAC TGG ACC TGG AGC ATC1192C ACC ATG GAC TGC ACC TGG AGG ATC1193C ACC ATG GAC TGG ACC TGG AGA ATC1194C ACC ATG GAC TGG ACC TGG AGG G1195C ACC ATG GAC TGG ATT TGG AGG ATC C11965' primerC ACC ATG GAC ACA CTT TGC TCC ACG1197C ACC ATG GAC ACA CTT TGC TAC ACA CTC C1198C ACC ATG GAG TTT GGG CTG AGC TG1199C ACC ATG GAA TTG GGG CTG AGC TG1200C ACC ATG GAG TTG GGG CTG AGC TG1201C ACC ATG GAA CTG GGG CTC CGC1202C ACC ATG GAA TTT GGG CTG AGC TGG1203C ACC ATG GAG TTG GGG CTG TGC TG1204C ACC ATG GAG TTT GGG CTT AGC TGG1205C ACC ATG GAG TTT TGG CTG AGC TGG1206C ACC ATG AAA CAC CTG TGG TTC TTC CTC1207C ACC ATG AAG CAC CTG TGG TTC TTC C1208C ACC ATG AAA CAT CTG TGG TTC TTC CTT CTC1209C ACC ATG GGG TCA ACC GCC ATC C1210C ACC ATG TCT GTC TCC TTC CTC ATC TTC12113' primerGCTGAGGGAGTAGAGTCCTGAGGACTGT1212Kappa chain Sequence 5' to 3' SEQ ID NO. C ACC ATG GAC A TG AGG GTC CCC G1213C ACC ATG GAC ATG AGG GTC CCT GC1214C ACC ATG GAC ATG AGG GTC CTC GC1215C ACC ATG AGG CTC CCT GCT CAG C1216C ACC ATG AGG CTC CTT GCT CAG CTT C12175' primerC ACC ATG GAA ACC CCA GCG CAG C1218C ACC ATG GAA GCC CCA GCG CAG1219C ACC ATG GAA GCC CCA GCT CAG1220C ACC ATG GAA CCA TGG AAG CCC CAG1221C ACC ATG GTG TTG CAG ACC CAG GTC1222C ACC ATG GGG TCC CAG GTT CAC C1223C ACC ATG TTG CCA TCA CAA CTC ATT GGG1224C ACC ATG GTG TCC CCG TTG CAA TTC12253' primerACCCGATTGGAGGGCGTTATCCACC1226Lambda chaim Sequence 5' yo 3' SEQ ID NO. C ACC ATG GCC TGG TCC CCT CTC 1227C ACC ATG GCC TGG TCT CCT CTC C 1228C ACC ATG GCC AGC TTC CCT CTC C 1229C ACC ATG GCC GGC TTC CCT CTC 1230C ACC ATG ACC TGC TCC CCT CTC C 1231C ACC ATG GCC TGG GCT CTG CTC 1232C ACC ATG GCC TGG GCT CTG CTG 1233C ACC ATG GCA. TGG ATC CCT CTC TTC 1234C ACC ATG GCC TGG ACC GCT CTC 12355' primer C ACC ATG GCC TGG ACC CCT CTC 1236C ACC ATG GCC TGG ATC CCT CTC C 1237C ACC ATG GCC TGG ACC GTT CTC C 1238C ACC ATG GCA TGG GCC ACA CTC C 1239C ACC ATG GCC TGG ATC CCT CTA C 1240C ACC ATG GCC TGG GTC TCC TTC TAC 1241C ACC ATG GCC TGG ACC CAA CTC C 1242C ACC ATG GCT TGG ACC CCA CTC C 1243C ACC ATG GCC TGG ACT CCT CTC C 1244C ACC ATG GCC TGG ACT CCT CTT CTT C 1245C ACC ATG GCC TGG ACT CTT CTC CTT C 1246C ACC ATG GCC TGG GCT CCA CTA C 1247C ACC ATG GCC TGG ACT CCT CTC TTT C 1248C ACC ATG GCC TGG ATG ATG CTT CTC C 1249C ACC ATG GCC TGG GCT CCT CTG 1250C ACC ATG CCC TGG GCT CTG CTC 12513' primer GGA GGG TKT GGTGGTCTC CAC TCC C 1252 (where K = G + T) EXAMPLE 9 - Reference ExamplePreparation of hetero Ig constructs

[0234] Generation of a bispecific antibody through co-expression of two different antibodies leads to contaminants primarily consisting of mispaired heavy and light chains. The preferred bispecific, heterotetramer molecule with two different heavy chains associated with correctly paired light chains is only a minority of the total amount of combinations that can assemble. The contaminants occur mainly due to two different reasons. The first reason is that the heavy chain that comes together at the Fc region of the antibody can homodimerize, leading to conventional monospecific antibody, or heterodimerize, leading to a potential bispecific antibody. The second reason is that light chain is promiscuous and can pair with either of the heavy chains, leading to mispaired light-heavy chain Fab assembly that may not retain binding to the desired target. For these reasons, the bispecific engineering is a two-step process. The first goal is to prevent the homodimerization of the heavy chains and encourage heterodimerization. This can be achieved through engineering the Fc region of the antibodies, using, for example, the knobs-into-holes or charge pair mutations strategies. The second goal is to engineer the light-heavy chain interface in such a way that the light chain is specifically associated only with its cognate heavy chain.

[0235] The "Hetero-Ig" platform technology (see, e.g., WO2009089004 and WO2014081955,) takes advantage of the electrostatic steering mechanism to overcome the pairing problems mentioned above. Specifically, charged residues are introduced or exploited to drive heavy chain heterodimerization and correct light-heavy chain association. The charge pair mutations (CPMs) in the CH3 domain of the Fc region drive the heterodimerization of the two different heavy chains through opposite charges that cause electrostatic attraction (see, e.g., WO2009089004 and U.S. Patent No. 8,592,562); the two identical heavy chain combinations have identical juxtaposed charges and are therefore repelled.

[0236] The correct heavy chain-light chain pairing is facilitated by CPMs at the HC / LC binding interface or the HC1 / HC2 binding interface (see Figure 1). The correct heavy chain-light chain combinations will have opposite charges and therefore be attracted to each other, whereas the incorrect heavy chain-light chain combinations will have the same charges juxtaposed, resulting in repulsion. In Figure 1, correctly assembled hetero-Ig molecules have two or three HC1 / HC2 CPMs and two to four HC / LC CPMs that drive the assembly of the preferred heterotetramer comprising two different heavy chains and two different light chains so that the heterotetramer will be the majority component generated by the expression system. The DNAs encoding anti-TL1A / anti-TNF-α hetero Ig-s contain fragments coding for anti-TL1A (or anti-TNF-α) heavy chain and anti-TNF-α (or anti-TL1A) Fab. The DNAs were cloned into pTT5.1 vector. These expression vectors were then used to transfect and express anti-TL1A / anti-TNF-α bi-specifics in human 293 6E cells.

[0237] Anti-TNF-α antibodies 3.2, 234 and certolizumab were used with anti-TL1A antibodies 3B3, 2G11, 23B3 VH3, 23B3 VH4, and 3C6 to engineer hetero Ig molecules as described in Table J using high throughput cloning, expression and purification. Each of the bispecific hetero Ig molecules had one of the four formats shown in Figure 1 using the IgG1 effector functionless scaffold or an IgG2 scaffold. Preferred IgG molecules incorporate the charge mutations shown in Figure 1 (v2), which are shown in Table M. The IgG1 effector functionless scaffold comprises substitutions R292C and V302C and may also comprise substitution N297G (also known as the SEFL2 scaffold).EXAMPLE 10 - Reference exampleMethod for Expression and Purification of anti-TL1A / anti-TNFα Hetero-Ig Molecules

[0238] Hetero-Ig expression was performed via transient transfection of 293-6E cells. One day prior to transfection N-1 culture was set up in a 20 L Wave bag, at 36 °C-37 °C, 5% CO 2 , 0.2 LPM overlay with a total volume of 9 L of culture at 8.5 E5 vc / mL in Freestyle F-17 media (Thermo Fisher). The transfection complex was then prepared by mixing FreeStyle F-17 media, pre-warmed with 0.5 mg / L transfection DNA, with a 1:1:1:1 plasmid DNA chain ratio. Transfection complex (media+DNA+PEI reagent) volume was 10% of final culture volume. Four hours post-transfection , a feed of yeastolate and glucose was added. Culture was harvested on day six at 2.11 E6 cells / mL and 78.2% viability. Expression titer was measured using the ForteBio Octet Q System at 84.0 mg / L. The conditioned media was harvested by centrifugation and filtered using 0.2 µm cellulose acetate filter using a peristaltic pump.

[0239] For purification, the hetero-Ig molecules were affinity captured by MabSelect SuRe chromatography (GE Life Sciences, Piscataway, NJ), using Dulbecco's PBS without divalent cations (Invitrogen, Carlsbad, CA) as the wash buffer and 100 mM acetic acid, pH 3.6 as the elution buffer (Figure 3). All separations were carried out at ambient temperature. The elution peak was pooled based on the chromatogram, neutralized to pH 7.0 using 2 M tris base, diluted with 5-volumes water, and filtered through a 0.22 µm cellulose acetate filter. To remove half antibody species, the sample was then loaded on to an SP-HP sepharose column (GE Life Sciences, Piscataway, NJ) and washed with 8 column volumes of SP-Buffer A (20 mM sodium phosphate, pH 7.0) followed by elution using a 20 column volume gradient to 60% SP-Buffer B (20 mM sodium phosphate, 1 M NaCl, pH 7.0) (Figure 4). A pool was made based on the chromatogram and Caliper LabChip (Perkin Elmer, Waltham, MA) analysis of fractions. The pool was conditioned with an equal volume of 2X HIC Buffer (200 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0) and filtered through a 0.22 µm cellulose acetate filter. To remove mispaired species, the sample was loaded on to a Butyl-HP sepharose column (GE Life Sciences, Piscataway, NJ) and washed with 8 column volumes of Butyl-Buffer A (50 mM sodium phosphate, 0.75 M ammonium sulfate, pH 7.0) followed by elution using a 20 column volume gradient to 100% Butyl-Buffer B (50 mM sodium phosphate, pH 7.0) (Figure 5). A pool was made based on the chromatogram and Caliper LabChip (Perkin Elmer, Waltham, MA) analysis of fractions under non-reducing and reducing conditions. The pool was diafiltered against approximately 30 volumes of 10 mM sodium acetate, 9% sucrose, pH 5.2 using Slide-A-Lyzer dialysis cassettes with a 10 kDa cutoff membrane (Pierce, Rockford, IL) and further concentrated using a Vivaspin-20 centrifugal concentrator with a 10 kDa cutoff membrane (Sartorius Stedim Biotech, Goettingen, Germany). The concentrated material was then filtered through a 0.8 / 0.2 µm cellulose acetate filter and the concentration was determined by the absorbance at 280 nm using an extinction coefficient of approximately 212,000. Sample purity was determined by Caliper LabChip analysis under reducing (with 2% 2-mercaptoethanol) and non-reducing (with 25 mM iodoacetamide) conditions (Figure 6). Analytical SEC was carried out using a Zenix-C SEC-300 column (Sepax Technologies, Newark, DE) with an isocratic elution in 50 mM sodium phosphate, 250 mM NaCl, pH 6.9 over 18' (Figure 7).

[0240] In order to assess both hetero-Ig integrity and confirm heavy chain-light chain pairing, LC-MS was performed on non-reduced hetero-Ig, as well as hetero-Ig after limited lysyl endoproteinase C (Wako, Richmond, VA) digestion to produce the hetero-Ig Fab's (fragment antigen binding) regions of the hetero-Ig. Analysis of non-reduced hetero-Ig was performed by simple dilution of 30 µg native hetero-Ig sample, 1:1 in 0.1% TFA, and injecting 20 µg. Analysis of Fab's was achieved by incubation of hetero-Ig in the presence of lysyl endoproteinase C using a 1:400 enzyme / substrate ratio in the presence of 100 mM Tris, adjusted to pH8, for 30 minutes at 37 °C. The reaction was then stopped by dilution in an equal volume of 0.1% TFA. Initial cleavage of antibody by Lysyl Endoproteinase C is just above the hinge disulfides after the lysine in the sequence motif SCDK / THTCPPC yielding Fab and Fc fragments.

[0241] Mass analysis was performed using an Agilent 6230 ESI-TOF Mass Spectrometer and 1260 quaternary HPLC system equipped with a Zorbax 300SB-C8, 2.1 x 50 mm 3.5 µm column (Agilent, Santa Clara, CA). Mobile phase A consisted of 0.1% TFA, and mobile phase B consisted of 90% n-propanol, 0.1% TFA in water. Chromatographic gradient conditions for analysis of non-reduced hetero-Ig were as follows: 20% mobile phase B for 1 minute; 1-9 min, 20-70% B; 9-10 min, 70-100% B; 10-11 min, 100% B. Column temperature was kept at 75 °C and post column equilibration at 20% mobile phase B was performed for 7 minutes prior to injection of the next sample. The ESI-TOF settings were as follows: capillary voltage, 5900 V; gas temperature, 340 °C; dry gas, 13 L / min; nebulizer pressure, 25 psig; fragmentor voltage 460 V and skimmer voltage, 95 V (figure 8).

[0242] Analysis of hetero-Ig digested with lysl endoproteinase C was conducted by injecting 20µg onto the reverse-phase HPLC column and eluting using the following LC gradient: 2% mobile phase held for 2 minutes; 2-12 min, 2-45% B; 12-16 min, 45-90% B; 16-17 min 90% B. Column temperature was kept at 75 °C and post column equilibration at 20% mobile phase B was performed for 7 minutes prior to injection of the next sample (figure 9).EXAMPLE 11 - Reference examplePreparation of IgG-ScFv constructs

[0243] Each anti-TL1A / anti-TNF-α IgG-scFv antigen binding protein consists of two antigen binding domains, one directed against TL1A and the other against TNF-α. The DNAs encoding anti-TL1A / anti-TNF-α IgG-scFv contain fragments coding for anti-TL1A (or ant-TNF-α) heavy chain (HC) in which the C-terminus is fused to anti-TNF-α (or anti-TL1A) antibody single chain Fv (scFv) (see Figure 2) with or without cysteine clamp for the purpose of improving biophysical properties. In order to introduce the cysteine clamp, positions 44 (Kabat numbering) VH and 100 (Kabat numbering) in VL were mutated to cysteine. The DNAs were cloned into pTT5.1 vector. These expression vectors were then used to transfect and express anti-TL1A / anti-TNF-α bi-specifics in human 293-6E cells. Full sequences for anti-TL1A / anti-TNF-α IgG-scFv antigen binding proteins produced are shown in Tables L and M.EXAMPLE 12 -Reference exampleMethod for Purifying anti-TL1A / anti-TNFα IgG-scFv Molecules

[0244] IgG-ScFv expression was performed via transient 293 productions. One day prior to transfection, cultures were set up in eight 5 L Thompson Ultra Yield flasks, with a total volume of 2.250 L of culture at 8.5E5 vc / mL each in Freestyle F-17 media (Thermo Fisher). The cultures were kept at 36 °C-37 °C, 5% CO 2 at and shaking at 120 RPM. The transfection complex was prepared by mixing FreeStyle F-17 media with 0.5 mg / L DNA, using a 20% of coding plasmid and 80% empty pTT5 vector and PEI at 10% final culture volume Four hours later a yeast lysate and glucose feed was added to each flask. Six days post transfection the cells were harvested through centrifugation, pooled and filtered. Average titer was measured by Forte Bio Octet at 25 mg / L.

[0245] The IgG-scFv molecules were affinity captured by MabSelect SuRe chromatography (GE Life Sciences, Piscataway, NJ), using Dulbecco's PBS without divalent cations (Invitrogen, Carlsbad, CA) as the wash buffer and 100 mM acetic acid, pH 3.6 as the elution buffer (Figure 10). Affinity separations were carried out at ambient temperature. The elution peak was pooled based on the chromatogram, neutralized to pH 7.0 using 2 M tris base, diluted with one volume 10 mM citrate, 75 mM lysine, 4% trehalose, pH 7.0, and concentrated to approximately 20 mg / mL using a Vivacell-100 centrifugal concentrator with a 30 kDa cutoff membrane (Sartorius Stedim Biotech, Goettingen, Germany). To remove high molecular weight aggregates, the sample was filtered through a 0.8 / 0.2-µm cellulose acetate filter then loaded on to a Superdex 200 Prep Grade column (GE Life Sciences, Piscataway, NJ) equilibrated with 10 mM citrate, 75 mM lysine, 4% trehalose, pH 7.0 and eluted with an isocratic gradient with the same buffer (Figure 11). Gel filtration separations were carried out at approximately 7 °C. A pool was made based on the chromatogram and analytical SEC of the fractions using a Zenix-C SEC-300 column (Sepax Technologies, Newark, DE). The pool was further concentrated using a Vivaspin-20 centrifugal concentrator with a 30 kDa cutoff membrane (Sartorius Stedim Biotech, Goettingen, Germany) and filtered through a 0.8 / 0.2-µm cellulose acetate filter. The concentration was determined by the absorbance at 280 nm using an extinction coefficient of approximately 341,000. Sample purity was determined by Caliper LabChip analysis under reducing (with 2% 2-mercaptoethanol) and non-reducing (with 25 mM iodoacetamide) conditions (Figure 12). Analytical SEC was carried out using a Zenix-C SEC-300 column (Sepax Technologies, Newark, DE) with an isocratic elution in 50 mM sodium phosphate, 250 mM NaCl, pH 6.9 over 18' (Figure 13).

[0246] Due to the large size of the Ig-scFvs, 200 kDa or greater, accurate mass was not achievable by ESI-TOF mass analysis. In order to verify mass and assess the quality, Ig-scFv's were analyzed after digestion with IdeS protease (Promega, Madison WI), which cleaves just below the IgG hinge disulfides, between glycines in the sequence motif, CPPCPAPELLG / GP yielding (Fab) 2 and Fc with C-terminally fused scFv. The Ig-scFv's were incubated in the presence of IdeS protease using a 1:10 enzyme substrate ratio for 1 hour at 37 °C. The sample was then diluted 1:1 in 0.1% TFA, and 20 µg was injected onto LC-MS.

[0247] Mass analysis was performed using an Agilent 6230 ESI-TOF Mass Spectrometer and 1260 quaternary HPLC system equipped with a Zorbax 300SB-C8, 2.1 x 50 mm 3.5 µm column (Santa Clara, CA). Mobile phase A consisted of 0.1% TFA, and mobile phase B consisted of 90% n-propanol, 0.1% TFA in water. Analysis of Ig-scFv digested with IdeS Protease was conducted by injecting 20 µg and eluting using the following LC gradient: 2% mobile phase held for 2 minutes; 2-12 min, 2-45% B; 12-16 min, 45-90% B; 16-17 min 90% B. Column temperature was kept at 75 °C and post column equilibration at 20% mobile phase B was performed for 7 minutes prior to injection of the next sample (figure 14).EXAMPLE 13 TL1A binding assay

[0248] Goat anti-human Fc (Jackson Lab, Cat#109-005-098) was first immobilized on an SCM5 sensor chip using amine coupling. Anti-TL1A monoclonal antibodies, Fab fragment (using anti-huFab antibody, GE Healthcare cat # 28-9583-25) or anti-TL1A / anti-TNF-α bispecific molecules were injected to the chip at 10 µl / min for 1 minute. Various concentrations of human or cynomolgus TL1A protein from 0.78 to 25 nM in sample buffer (PBS, 0.005% P20, 0.1 mg / ml BSA) were injected at flow rate of 50 µl / min for 3 min association, 10 min dissociation. On rate, off rate and equilibrium dissociation constant were calculated using 1:1 binding model on BIAevaluation software.

[0249] Tables 13.1-13.3 show anti-TL1A binding data for anti-TL1A antibodies, hetero Ig bispecific antibodies, and IgG-scFv bispecific antibodies. Table 13.1: TL1A binding of anti-TL1A antibodiesAnti-TL1A mAb Protein ID SEQ ID NOS: human TL1A cyno TL1A ka (1 / Ms) kd (1 / s) : KD (M) ka(1 / Ms) kd (1 / s) KD (M) : 3C6PL-3911250, 52NA<5.0E-05<1.0E-105.7E+051.4E-032.4E-093B3 parentalPL-3911466, 684.0E+059.5E-052.4E-102.5E+051.4E-045.7E-105G4PL-39115455, 4575.0E+051.8E-043.6E-102.9E+051.9E-046.6E-1017E9PL-39113459, 4612.5E+055.3E-052.2E-107.3E+052.0E-032.8E-099C8PL-3911658, 601.6E+055.3E-053.4E-109.4E+046.9E-057.3E-1023B3PL-3654362, 644.7E+054.2E-048.9E-102.3E+053.2E-041.4E-092G11PL-3654454, 561.7E+051.8E-041.1E-096.6E+044.9E-057.3E-103B3 variantPL-36552130, 1344.3E+052.2E-04 :5.0E-102.8E+051.4E-045.0E-10 Table 13.2: TL1A binding of anti-TL1A Fab fragment Anti-TL1A Fab Protein ID human TL1A cyno TL1A ka (1 / Ms) kd (1 / s) KD (M) ka (1 / Ms) kd (1 / s) KD (M) 3C6PL-39193NA<5.0E-05<1.0E-10NB up to 100 nM3B3 parentalPL-391954.1E+051.6E-044.0E-103.5E+05 :1.8E-045.1E-105G4PL-391964.7E+053.3E-047.0E-104.4E+051.6E-043.7E-1017E9PL-39194NA<5.0E-05<1.0E-10NB up to 100 nM9C8PL-39197NA<5.0E-05<1.0E-10NA<5.0E-05<1.0E-1023B3PL-380247.6E+056.4E-048.5E-105.3E+051.6E-043.1E-102G11PL-38025NA<5.0E-05<3.0E-10NA<5.0E-05<1.0E-103B3 variantPL-380263.8E+054.1E-041.1E-094.5E+055.6E-051.3E-10 Table 13.3: TL1A binding of hetero Ig bispecific antibodies iPS anti-TNF anti-TL1A ka (1 / Ms) kd (1 / s) KD (M) 376541Certolizumab3B3 variant (322520)7.1E+052.1E-043.0E-10376542C2343B3 variant (322520)7.1E+052.1E-043.0E-10376543Certolizumab variant3B3 variant (322520)7.2E+052.3E-043.2E-10349461Certolizumab Variant2G11 Variant5.4E+051.3E-042.5E-10349463Certolizumab Variant23B3 VH46.3E+054.0E-046.4E-103712223.2 / CC3B3 variant (322520)6.6E+051.4E-042.1E-10 EXAMPLE 14 TL1A activity assay

[0250] TL1A activity assay was performed using TF-1 NF-κB reporter cell line. In brief, 30 ng / ml (EC90) of human or cynomolgus monkey TL1A was incubated with 10 4< TF-1 NF-κB reporter cells in the presence of serially diluted anti-TL1A antibodies or anti-TL1A / anti-TNF-α bispecific molecules in 96-well plate at 37 °C overnight. Each well was supplemented with 50 µl of Steady-glo Luciferase testing solution (Promega). Plate was covered and incubated while shaking for 10 minutes. Luciferase activity was analyzed by microbeta reader.

[0251] Tables 14.1-14.3 show anti-TL1A quality control (QC) and activity data for anti-TL1A antibodies, hetero Ig bispecific antibodies, IgG-scFv bispecific antibodies, and Fab's with both human and cynomolgus monkey TL1A. Table 14.1: anti-TL1A activity of anti-TL1A antibodies and Fab fragmentsmAb mAb / Fab huTL1A (0.2 nM) NF-kB IC50 nM cyno TL1A (2 nM) NF-kB IC50 nM 3C6mAb0.153>100Fab0.162>10017E9mAb0.168>100Fab70.83>1003B3S65AmAb0.0830.595Fab0.1780.4343B3 / 7B3mAb0.0370.390Fab0.1120.6475G4mAb0.0580.622Fab2.83810.062G11mAb0.2230.938Fab0.5511.6379C8mAb0.1310.721Fab2.4342.74823B3mAb0.0890.619Fab2.9922.629 Table 14.2: QC and activity of hetero Ig bispecific antibodies SE-HPLC Caliper Activity iPS No. TL1A TNF-α Final Conc. Final Yield HMW Main NR 1 NR2 HC1 H2 LC1 LC2 TL1A (pM) TNF-α (pM) 376541Certolizumab (parent)3B3 (variant)12.052950.799.372284258514918751349461Certolizumab (variant)2G11 (variant)10.491880.799.3455510005149128460349463Certolizumab (variant)23B3 (VH4)10.614500.699.4633750503367346464376542C2343B3 (variant)9.761740.0100.0712944565050147544376543Certolizumab (variant)3B3 (variant)13.603200.499.6752543575149158107 Table 14.3: QC and activity of IgG-scFv bispecific antibodies SE-HPLC Caliper Activity iPS IgG scFv Final Conc. Final Yield HMW Main Shoulder NR 1 NR 2 HC1 LC1 TL1A (pM) TNF-α (pM) 371213TNF-α (adalimumab)TL1A (3B3 Var2)12.40310.0598.31.4118981196164371217TNF-α (C234)TL1A (3B3 Var2 / CC)13.062900.498.51.2396181186940369989TNF-α (adalimumab)TL1A (9C8)13.831990.398.11.12773761814059369995TNF-α (3.2)TL1A (9C8)15.812171.097.91.6100801719953370001TNF-α (C234)TL1A (9C8)12.412890.298.21.14258841611952369992TNF-α (adalimumab)TL1A (9C8 / CC)11.481690.39821.61684841615172370004TNF-α (C234)TL1A (9C8 / CC)11.341110.398.21.54555841616370370013TL1A (9C8)TNF-α (3.2)16.534640.398.31.54159791817125 371219TL1A (3B3 Var2)TNF-α (3.2)10.91970.498.11.410070205322371222TL1A (3B3 Var2)TNF-α (3.2 / CC)20.071570.298.31.510078188626370018TL1A (23B3)TNF-α (adalimumab)11.591122.296.51.5100861415725370021TL1A (23B3)TNF-α (adalimumab / CC)15.293210.0100.01.3793861413924 EXAMPLE 15 TNF-α binding assay

[0252] Goat anti-human Fc was first immobilized on SCM5 sensor chip using amine coupling. Anti-TNF monoclonal antibodies, Fab fragment or TL1A / TNF bispecifics were injected to the chip at 10 µl / min for 1 min. Various concentration of human or cynomologus TNF protein from 0.78 to 25 nM in sample buffer (PBS, 0.005% P20, 0.1 mg / ml BSA) were injected at flow rate of 50 µl / min for 3 min association, 10 min dissociation. On rate, off rate and equilibrium dissociation constant were calculated using 1:1 binding model on BIAevaluation software.

[0253] Tables 15.1-15.3 show TNF-α binding activity data for anti-TNF-α antibodies, hetero Ig bispecific antibodies, IgG-scFv bispecific antibodies, and IgG-Fab bispecific antibodies. Table 15.1: TNF-α binding activity of anti-TNF-α antibodies and Fab fragmentHuman TNF-α Cyno TNF-α k a (1 / Ms) k d (1 / s) KD (M) k a (1 / Ms) k d (1 / s) KD(M) 3.2mAb1.5E+067.3E-054.8E-119.0E+059.9E-051.1E-10Fab<5.0E-5<5.0E-54.14mAb5.3E+056.1E-051.2E-103.5E+051.0E-042.9E-10Fab<5.0E-5<5.0E-5234mAb1.5E+067.9E-055.2E-111.2E+061.0E-048.7E-11Fab<5.0E-5<5.0E-5AdalimumabmAb7.1E+056.1E-058.6E-116.4E+055.5E-058.7E-11Fab4.5E+051.4E-043.2E-103.6E+058.1E-052.3E-10Certolizumab pegolPeg-Fab3.9E+066.2E-051.6E-116.4E+062.2E-033.5E-10 Table 15.2: TNF-α binding activity of hetero Ig bispecific antibodies iPS Lot anti-TNF-α anti-TL1A ka (1 / Ms) kd (1 / s) KD (M) 376541PL-42786Certolizumab parent3B3 variant (322520)3.2E+064.4E-05*1.4E-11376542PL-42789C2343B3 variant (322520)2.3E+061.1E-044.8E-11376543PL-42790Certolizumab variant3B3 variant (322520)3.6E+065.5E-051.6E-11349461PL-42787Certolizumab variant2G11 Variant3.4E+066.4E-051.9E-11349463PL-42788Certolizumab variant23B3 VH43.4E+065.5E-051.6E-11 Table 15.3: TNF-α binding activity of IgG-scFv bispecific antibodies iPS Lot IgG scFV ka (1 / Ms) kd (1 / s) KD (M) 369989PL-42765TNF-α (Adalimumab)TL1A (9C8)1.2E+067.6E-056.6E-11369992PL-42770TNF-α (Adalimumab)TL1A (9C8 / CC)1.2E+068.4E-057.3E-11369995PL-42779TNF-α (3.2)TL1A (9C8)1.7E+067.7E-054.4E-11370001PL-42767TNF-α (C234)TL1A (9C8)1.7E+069.9E-055.9E-11370004PL-42785TNF-α (C234)TL1A (9C8 / CC)1.7E+061.0E-045.9E-11370013PL-42781TL1A (9C8)TNF-α (3.2)1.3E+068.8E-056.5E-11370018PL-42763TL1A (23B3)TNF-α (Adalimumab)1.1E+065.3E-055.0E-11370021PL-42778TL1A (23B3)TNF-α (Adalimumab / CC)1.1E+064.9E-05*4.5E-11371213PL-42771TNF-α (Adalimumab)TL1A (3B3.322520 / CC)1.3E+068.2E-056.5E-11371217PL-42782TNF-α (C234)TL1A (3B3.322520 / CC)1.9E+068.8E-054.8E-11371219PL-42774TL1A (3B3.322520)TNF-α (3.2)1.3E+065.5E-054.2E-11371222PL-42783TL1A (3B3.322520)TNF-α (3.2 / CC)1.3E+066.3E-054.7E-11 EXAMPLE 16 TNF-α activity assays

[0254] TNF-α activity assay was performed using TF-1 NF-kB reporter cell line. In brief, 1 ng / ml (EC90) of human or cynomolgus monkey TNF-α was incubated with 10 4< TF-1 NF-κB reporter cells in the presence of a serially diluted anti-TNF-α antibodies or TL1A / TNF-α bispecific molecules in 96-well plate at 37 °C overnight. 50 µl of Steady-glo Luciferase testing solution (Promega) was added to each well. Plate was covered and incubated while shaking for 10 minutes. Luciferase activity was analyzed by microbeta reader.

[0255] Table 16.1 shows anti-TNF-α QC and activity data for anti-TNF antibodies. Tables 14.2 and 14.3 show anti-TNF-α binding activity for hetero Ig bispecific antigen binding proteins, and IgG-scFv bispecific antigen binding proteins, respectively. Table 16.1: Anti-TNF-α activity of TNF-α antibodies and Fab fragmentsAntibody designation Modality Human TNF (20 pM) IC50 (pM) NFkB Cyno TNF (20 pM) IC50 (pM) NFkB 3.2mAb75334Fab36729504.14mAb127739Fab5263105234mAb74138Fab8252027adalimumabmAb119180Fab6701800certolizumab pegolPeg-Fab911.38 µM EXAMPLE 17 Bispecific molecule activity

[0256] The human and cynomolgus monkey TL1A and TNF-α binding activities of bispecific antigen binding proteins were determined as described in Examples 13 and 15. Data are shown in Table 17.1 Table 17.1: human and cyno TL1A and TNF binding activitiesleads TL1A warhead TNF-α warhead iPS no. TL1A binding (Kd pM) TNF binding (Kd pM) Hu TL1ACyno TL1AHu TNFCyno TNFHetero-Ig3B3 V2certolizumab376543112612210IgG-scFv9C8 / CCadalimumab3815051201502133IgG-scFv9C8 / CCC234381489160941019IgG-scFv3B3 V2 / CCadalimumab3815131.86.61923

[0257] The human and cynomolgus monkey TL1A and TNF-α blocking activities of bispecific antigen binding proteins were determined as described in Examples 14 and 16. Data are shown in Table 17.2. Table 17.2: human and cyno TL1A and TNF-α blocking activitesleads Anti-TL1A warhead Anti-TNF-α warhead iPS No. TL1A assay IC50 pM TNF assay IC50 pM soluble humem husoluble cynosoluble humem husoluble cynoHetero-Ig3B3 V2certolizumab37654345.550.149.7101.1942.60.9uMIgG-scFv9C8 / CCadalimumab38150552.246.545.372.9719.596.4IgG-scFv9C8 / CCC23438148966.746.362.695.8639.2157.2IgG-scFv3B3 V2 / CCadalimumab38151335.619.223.769.7906.391.3 EXAMPLE 18 SNP genotyping

[0258] To evaluate potential TL1A genotype association with expression, genomic DNA (gDNA) was isolated from healthy PBMC donors using Gentra Puregene Tissue kit from Qiagen. Genomic DNA were genotyped using TaqMan SNP genotyping assays for rs7848647, rs6478109, rs6478108, and rs3810936 assays from LifeTech and standard protocols on the Bio-Rad droplet digital PCR platform. Briefly, 10 ng of gDNA from each donor was mixed with ddPCR ™< Supermix for Probes (Bio-Rad) and TaqMan SNP genotyping assay (LifeTech) for each SNP of interest. Droplets were generated for each reaction using the QX100 ™< droplet generator (Bio-Rad) and subjected to thermal cycling on a C1000 Touch ™< thermal cycler (Bio-Rad). Following amplification, droplet fluorescence was read on a QX100 ™< droplet reader (Bio-Rad) and data were analyzed using QuantaSoft software (Bio-Rad). Donors were considered homozygous risk haplotype if only risk alleles were present at all 4 genotyped SNPs (rs7848647, rs6478109, rs6478108, and rs3810936). Donors were considered homozygous non-risk haplotype if only non-risk alleles were present at all 4 genotyped SNPs. Donors were considered heterozygous haplotype if both risk and non-risk alleles were present at all 4 genotyped SNPs. Donors that were considered "recombinant" had only homozygous risk alleles at rs7848647, rs6478109, and rs6478108, but were heterozygous (risk and non-risk alleles present) at rs3810936.

[0259] The presence of synonymous SNP (rs3810936) in the exon 4 of the TL1A gene enabled us to track risk allele vs non-risk allele expression by droplet digit PCR (ddPCR) using allelic specific fluorescent probes in the allelic expression imbalance (AEI) study. The frequency of risk vs non-risk allele usage in heterozygous PBMC at basal level or after immune complex stimulation was evaluated at various time points. In brief, PBMCs heterozygous for TL1A genotype were treated with immune complex for various lengths of time. RNA was then isolated from PBMC using an RNeasy mini kit with on-column DNase I digestion from Qiagen. RNA from each sample was converted to cDNA using the High Capacity cDNA Reverse Transcription Kit according to manufacturer's instructions (LifeTech). Allele-specific expression at rs3810936 was determined using TaqMan SNP genotyping assay specific for rs3810936 (LifeTech) and standard protocols on the Bio-Rad droplet digital PCR platform. In brief, the amount of input cDNA was first optimized for TNFSF15 expression at each time point in order to increase precision by maximizing the number of positive droplets counted for each sample by QuantaSoft software. The optimal amount of cDNA per sample was mixed with ddPCR ™< Supermix for Probes (Bio-Rad) and TaqMan SNP genotyping assay (LifeTech) for rs3810936. Droplets were generated for each reaction using the QX100 ™< droplet generator (Bio-Rad) and subjected to thermal cycling on a C1000 Touch ™< thermal cycler (Bio-Rad). Following amplification, droplet fluorescence was read on a QX100 ™< droplet reader (Bio-Rad). Data were analyzed using QuantaSoft software (Bio-Rad) and copies / ml of each allele at rs3810936 was determined. The allelic expression ratio was calculated by dividing the copies / ml of the risk allele by the copies / ml of the non-risk allele. Total copy number of each allele was normalized by the input amount of cDNA (copies / ng), and the fold-induction for each allele at each time point was calculated by dividing the copies / ng at the time point of interest by the copies / ng at baseline (0 hr). Higher induction of TL1A risk allele was observed in PBMC after immune complex treatment compared to non-risk allele in heterozygous PBMC in allelic expression imbalance study.

[0260] The inventor(s) identified a small number of donors homozygous for risk alleles at rs7848647, rs6478109, and rs6478108, but heterozygous at rs3810936, likely due to recombination between rs6478109 and synonymous SNP rs3810936. Since these donors are heterozygous at synonymous SNP rs381093, the inventor(s) could track the risk allele and non-risk allele usage and evaluate if the allelic expression imbalance was still retained in these donors. Interestingly, compared to heterozygous donors, no allelic expression imbalance was detected in these recombinant individuals before or after immune complex stimulation. This demonstrates that synonymous SNP rs381093 itself is not responsible for allelic imbalance regulation. The regulatory SNPs likely derive from rs7848647, rs6478109, rs6478108 and / or other SNPs 5' to the synonymous SNP rs3810936.

[0261] To evaluate if TL1A allelic expression imbalance data correlate with expression quantitative trait loci (eQTL), PBMC donors with homozygous TL1A risk allele, homozygous non-risk alleles or heterozygous alleles were treated with immune complex. RNA from each sample was isolated and then converted to cDNA as described previously. Total expression of TL1A (TNFSF15) in each donor was determined by digital PCR. In brief, cDNA from each sample was mixed with ddPCR ™< Supermix for Probes (Bio-Rad) and PrimeTime ®< qPCR assay ID Hs.PT.56a.41003970. Droplets were generated for each reaction using the QX100 ™< droplet generator (Bio-Rad) and subjected to thermal cycling on a C1000 Touch ™< thermal cycler (Bio-Rad). Following amplification, droplet fluorescence was read on a QX100 ™< droplet reader (Bio-Rad). Data were analyzed using QuantaSoft software (Bio-Rad) and copies / µl of TL1A was determined and normalized for the amount of input cDNA (copies / ng). P values for differences in TL1A expression levels between different TNFSF15 haplotypes were determined using the student t test. TL1A is expressed at a very low basal level in PBMCs, but strongly induced after immune complex stimulation. At basal level, PBMCs from donors homozygous for TL1A risk SNPs have lower TL1A mRNA compared to non-risk homozygous donors, albeit at very low copy number, consistent with the allelic expression imbalance study which demonstrated lower ratio of risk allele versus non-risk allele without stimulation. However, after immune complex stimulation, PBMCs from donors homozygous for TL1A risk SNPs have higher TL1A expression compared to non-risk homozygous donors. These data, together, demonstrated TL1A risk SNPs regulate higher TL1A induction. The inventor(s) speculate that in inflammatory conditions such as IBD, donors carrying TL1A risk SNPs likely encounter higher TLIA induction upon encounter of stimuli such as cytokines, opsonized or non-opsonized microbes. Therefore, TL1A risk SNPs could be used for patient stratification for TL1A inhibitor or anti-TL1A / anti-TNF-α bispecific inhibitors.

[0262] To evaluate if TL1A genotype-mediated expression regulation is tissue-specific, HUVEC cells from various donors were genotyped as described previously. Genotyped HUVEC cells from various donors were treated with IL-1 at various time points. Total copy number of each allele was measured as described previously. The allelic expression ratio was calculated by dividing the copies / ml of the risk allele by the copies / ml of the non-risk allele. No allelic expression imbalance was observed in HUVEC cells with or without IL-1 treatment.EXAMPLE 19 Mouse IBD models

[0263] To evaluate role of TL1A in IBD, a series of preclinical experiments were performed in mouse IBD models. The effect of TL1A inhibition was evaluated in mouse mdrla- / - spontaneous colitis model. Mdrla gene encodes multiple drug resistance gene for P-glycoprotein 170. Mice with knockout of mdr1a gene are prone to develop spontaneous colitis from 12 weeks of age due to weakened intestinal barrier. Disease course is affected by gut microbes. In our experiment, female Mdr1a - / -< or wild type FVB controls were obtained from Taconic at 4-6 weeks of age. Animal weight and clinical disease activity was monitored regularly. The clinical disease activity score was arrived at using a summation of the scores obtained evaluating anal inflammation (0=none, 1=mild, 2=moderate, 3=severe, 4=rectal prolapse) and stool consistency (0=normal, 1=moist / sticky, 2=soft, 3=diarrhea, 4=bloody). Mice (n=10, 6-7 weeks age) were randomized to different groups based upon baseline clinical disease activity measurements, and treated intra-peritoneally once a week with 500 µg of anti-mouse TL1A antibody, anti-IL23p19 antibody, anti-mouse IL17RA antibody, or mouse isotype control or no treatment once per week for 8 weeks. Mice were then sacrificed, sections of the intestine were taken and processed for H&E staining prior to scoring for disease by a pathologist. The following score was assigned based upon the histopathology: 0 = normal; 1 = minimal, mononuclear infiltrate and / or epithelial hypertrophy / hyperplasia; 2 = mild, mononuclear infiltrate and / or epithelial hypertrophy / hyperplasia; 3 = moderate, mononuclear infiltrate and / or epithelial hypertrophy / hyperplasia with rare crypt abscesses; 4 = marked, same as for 3 plus abundant crypt abscesses and crypt dropout and / or focal ulceration; 5 = severe, same as for 4 but with large areas of crypt dropout and / or extensive areas of ulceration. Statistical analysis was performed using one-way ANOVA with Dunett's compared to mIgG1 group. In conclusion, prophylactic treatment with anti-TL1A mAb inhibited spontaneous colitis development in mdr1a - / -< Mice.

[0264] The inventor(s) also evaluated if challenge with TL1A protein will exacerbate colitis development in mdr1a - / -< mice, which are prone to develop spontaneous colitis from 12 weeks of age due to a weakened intestinal barrier. Mdr1a - / -< mice or wild-type control mice at 6-8 weeks of age were treated intra-peritoneally once a week with 150 µg of recombinant mFc-TL1A fusion protein or isotype control three times each week for 4 weeks. Clinical disease activity was monitored by evaluating anal inflammation and stool consistency as described previously. After 4 weeks of treatment, mice were sacrificed, sections of the intestine were taken and processed for H&E staining prior to scoring for disease as described previously. Statistical analysis was performed using one-way ANOVA with Dunett's compared to mIgG1 group. Challenge of TL1A protein severely exacerbated colitis development in mdr1a - / -< mice but not in wild-type mice.

[0265] The inventor(s) further examined the impact of TL1A challenge in lamina propria lymphocytes in mdr1a - / -< mice versus wild-type mice. In brief, lamina propria lymphocytes were isolated as previously described (D'Souza WN et al., JI, V168:5566-5572, 2002). In brief, intestines were isolated, cut open longitudinally, rinsed with buffer and cut into 0.5 cm pieces. They were then washed twice in EDTA and the supernatants were discarded. The pieces were washed with RPMI and incubated in collagenase / DNase for 30 minutes. The isolated cells were run through a percoll gradient and the cells collected at the interphase were stained for surface antigens and analyzed by FACS. TL1A challenge in mdr1a - / -< mice resulted in increased inflammatory cells in lamina propria.EXAMPLE 20 Pharmacodynamics studies

[0266] To evaluate if TL1A and TNF challenges result in similar or different pharmacodynamics effects, C57Bl / 6 mice (8 week, female) were first intraperitonially injected with 500 µg / mice of anti-mouse TL1A, anti-mouse TNF-α or PBS. After 4 hours, the mice were then challenged with 100 µg / mice of TL1A or 10 µg / mice of TNF-α or without challenging. The sera were collected after 24 hours. The cytokines were measured by MSD (IL-22 was measured by ELISA). Challenge with TL1A or TNF protein in mice resulted in distinct cytokine induction. TL1A challenge mainly induced IFN-y and IL-5, whereas TNF challenge induced IL-6, IL-8 and IL-10.

[0267] The inventor(s) evaluated cytokine induction by TL1A or TNF treatment in human PBMC. In brief, PBMC freshly isolated from human blood were cultured in media (RPMI1640 supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 5 X 10 -5< M 2-ME, and antibiotics) in the presence of 100 ng / ml of human TL1A or TNF-α. Supematant was collected after 72 hours. The cytokines in the supernatant were measured by MSD (IL-22 was measured by ELISA). Similar to the mice challenge experiment, treatment of TL1A with human PBMC resulted in induction of IFN-y, IL-5 and IL-22, whereas TNF treatment resulted in increase of IL-8, IL-10 and MCP-1. Therefore, TL1A and TNF induce distinct cytokine profiles in human PBMC.EXAMPLE 21 - Reference exampleIgG-Fab molecules

[0268] Bispecific antigen binding proteins were prepared with a subset of the anti-TNFα and anti-TL1A antibodies. In some embodiments of this IgG-Fab format, a polypeptide comprising a VH-CH1 domain from a second antibody is fused through a peptide linker to the carboxyl- terminus of the heavy chain of a first antibody to form a modified heavy chain. A polypeptide comprising the remaining domains of the Fab fragment from the first antibody (i.e. a VL-CL domain) is co-expressed with the light chain of the first antibody and the modified heavy chain to produce the complete molecule. Assembly of the full molecule creates a tetravalent binding protein having two antigen binding domains against a first antigen located on the amino terminal side of a dimerized immunoglobulin Fc region and two antigen binding domains against a second antigen located on the carboxyl terminal side of the dimerized Fc region.

[0269] The TNFα / TL1A IgG-Fab consists of two antigen binding domains, one directed against TNFα and the other against TL1A. The DNA molecules encoding TNFα / TL1A IgG-Fab molecules contain fragments encoding an anti- TNFα (or anti- TL1A ) antibody light chain, an anti- TNFα (or anti-TL1A ) antibody heavy chain in which the C-terminus is fused to (i) an anti-TL1A (or anti- TNFα ) antibody light chain or (ii) an anti-TL1A (or anti- TNFα ) Fd (VH-CH1), and a third polypeptide comprising the other half of the Fab fragment to complete the carboxy-terminal binding domain; for example, (i) an anti-TL1A (or anti- TNFα) Fd or (ii) an anti-TL1A (or anti- TNFα) antibody light chain. The IgG-Fab bispecific molecules contain charge pair mutations introduced into CH1 and CL domains of each Fab region (Fab 1 and Fab 2 as illustrated in Figure 3). The charge pairs are designed to allow preferential assembly of anti-TNFAR light chain / VHCH1(Fd) pair and anti-TL1A light chain / VHCH1 (Fd) pair. As an additional approach to promote correct pairing of the light chain / VHCH1 (Fd) pair, for a subset of the IgG-Fab molecules generated, the CL and CH1 regions in the carboxyl-terminal Fab (i.e. Fab 2) were swapped such that the polypeptide fused to the carboxyl-terminal region of the heavy chain of the second antibody comprised VL and CH1 regions from the first antibody and the second polypeptide comprised VH and CL regions from the first antibody. See molecules listed in Tables 21.1 and 21.3, with the VL and VH CDRs listed in Table 21.2A and 21.2B and purity listed in Table 21.4. The DNA molecules were generated by synthesized gBlocks and cloned into the pTT5.1 vector. These expression vectors were used to transfect and express the TNFα / TL1A bispecific molecules in human 293-6E cells. 144 different IgG-Fab bispecific molecules were generated. The full sequences for each molecule are set forth in Table 21.1 and the Sequence Listing.

[0270] The IgG-Fab molecules were purified using affinity captured by MabSelect SuRe chromatography (GE Life Sciences, Piscataway, NJ) using a Large Format Autosampler (LFAS, Amgen, Inc., Thousand Oaks, CA). Clarified, conditioned media was loaded onto a 1 mL HiTrap MabSelect SuRe column (GE Life Sciences, Piscataway, NJ) equilibrated with Dulbecco's phosphate buffered saline without divalent cations (D-PBS, Life Technologies, Grand Island, NY). MabSelect columns were washed with 8 column volumes of D-PBS and eluted with 100 mM acetic acid, pH 3.6. When protein A eluates had an absorbance above 5 mAU at 280 nm, the eluent was directly loaded onto a HiTrap Desalting column (GE Life Sciences, Piscataway, NJ) and developed with 1.2 column volumes of 10 mM sodium acetate, 150 mM NaCl, pH 5.0. When desalting eluates had an absorbance above 3 mAU at 280 nm, sample collection was triggered and fractions were collected in 96-well deepwell blocks to a maximum of 2 mL each. Sample purity was determined by Caliper LabChip analysis under reducing (with 2% 2-mercaptoethanol) and non-reducing (with 25 mM iodoacetamide) conditions. Analytical SEC was carried out using a Zenix-C SEC-300 column (Sepax Technologies, Newark, DE) with an isocratic elution in 50 mM sodium phosphate, 250 mM NaCl, pH 6.9 over 8'.

[0271] The IgG-Fab molecules were tested for their expressability (titer and recovery) and activity. The results are shown in Figures 27 to 33 and in Table 21.3. In Table 21.3 and throughout, "ada" refers to adalimumab.

[0272] TL1A activity assay was performed using TF-1 NF-κB reporter cell line. In brief, 30 ng / ml (EC90) of human or cynomolgus monkey TL1A was incubated with 10 4< TF-1 NF-κB reporter cells in the presence of serially diluted anti-TL1A antibodies or TL1A / TNF-α bispecific molecules in 96-well plate at 37 °C overnight. Each well was supplemented with 50 µl of Steady-glo Luciferase testing solution (Promega). Plate was covered and incubated while shaking for 10 minutes. Luciferase activity was analyzed by microbeta reader.

[0273] TNFα activity assay was performed using TF-1 NF-κB reporter cell line. In brief, 1 ng / ml (EC90) of human or cynomolgus monkey TNF-α was incubated with 10 4< TF-1 NF-κB reporter cells in the presence of a serially diluted anti-TNF-α antibodies or TL1A / TNFα bispecific molecules in 96-well plate at 37 °C overnight. 50 µl of Steady-glo Luciferase testing solution (Promega) was added to each well. Plate was covered and incubated while shaking for 10 minutes. Luciferase activity was analyzed by microbeta reader. Table 21.1: anti-TL1A anti-TNF-α IgG-Fab molecule amino acid sequencesMolecule Designation (iPS no.) IgG source, Fab source, aa substitutions Light chain 1 SEQ ID NO Light chain 2 SEQ ID NO Heavy chain SEQ ID NO 376597001_Adalimumab_IgG.001_3B3v2_VH_CH1 (S183E)125412561258376601002_Adalimumab_IgG.001_9C8_VH_CH1(S 183E)126012621264376605003_Adalimumab_IgG.001_23B3_VH4_VH_ CH1(S183E)126612681270376609004_3.2_IgG.001_3B3v2_VH_CH1(S183E)127212741276376613005_3.2_IgG.001_9C8_VH_CH1(S183E)127812801282376617006_3.2_IgG.001_23B3_VH4_VH_CH1(S18 3E)128412861288376621007_3B3v2_IgG.001_Adalimumab_VH_CH1 (S183E)129012921294376625008_3B3v2_IgG.001_3.2_VH_CH1(S183E)129612981300376629009_9C8_IgG.001_Adalimumab_VH_CH1(S 183E)130213041306376633010_9C8_IgG.001_3.2_VH_CH1(S183E)130813101312376637011_23B3_VH4_IgG.001_Adalimumab_VH_ CH1(S183E)131413161318376641012_23B3_VH4_IgG.001_3.2_VH_CH1(S18 3E)132013221324376645013_Adalimumab_CK(S176K)_Fc_3B3v2_V H_CH1(S183E)132613281330376651014_Adalimumab_CK(S176K)_Fc_9C8_VH_ CH1(S183E)133213341336376655015_Adalimunmb_CK(S176K)_Fc_23B3_VH 4_VH_CH1(S183E)133813401342376659016_3.2_CL(S176K)_Fc_3B3v2_VH_CH1(S 183E)134413461348376665017_3.2_CL(S176K)_Fc_9C8_VH_CH1(S18 3E)135013521354376669018_3.2_CL(S176K)_Fc_23B3_VH4_VH_C H1(S183E)135613581360376673019_3B3v2_CK(S176K)_Fc_Adalimumab_V H_CH1(S183E)136213641366376679020_3B3v2_CK(S176K)_Fc_3.2_VH_CH1(S 183E)136813701372376683021_9C8_CK(S176K)_Fc_Adalimumab_VH CH1(S183E)137413761378376689022_9C8_CK(S176K)_Fc_3.2_VH_CH1(S18 3E)138013821384376693023_23B3_VH4_CK(S176K)_Fc_Adalimuma b_VH_CH1(S183E)138613881390376699024_23B3_VH4_CK(S176K)_Fc_3.2_VH_C H1(S183E)139213941396376703025_Adalimumab_IgG.001_3B3v2_VH_CK( S176E) NA139814001402376709026_Adalimumab_IgG.001_9C8_VH_CK(S1 76E)140414061408376715027_Adalimumab_IgG.001_23B3_VH4_VH_ CK(S176E)141014121414376721028_3.2_IgG.001_3B3v2_VH_CK(S176E)141614181420376725029_3.2_IgG.001_9C8_VH_CK(S176E)142214241426376729030_3.2_IgG.001_23B3_VH4_VH_CK(S176 E)142814301432376733031_3B3v2_IgG.001_Adalimumab_VH_CK( S176E)143414361438376739032_3B3v2_IgG.001_3.2_VH_CL(S176E)144014421444376745033_9C8_IgG.001_Adalimumab_VH_CK(S1 76E)144614481450376750034_9C8_IgG.001_3.2_VH_CL(S176E)145214541456376755035_23B3_VH4_IgG.001_Adalimumab_VH_ CK(S176E)145814601462376760036_23B3_VH4_IgG.001_3.2_VH_CL(S176 E)146414661468376765037_Adalimumab_CK(S176K)_Fc_3B3v2_V H_CK(S176E)147014721474376770038_Adalimumab_CK(S176K)_Fc_9C8_VH CK(S176E)147614781480376775039_Adalimumab_CK(S176K)_Fc_23B3_VH 4_VH_CK(S176E)148214841486376779040_3.2_CL(S176K)_Fc_3B3v2_VH_CK(S1 76E)148814901492376784041_3.2_CL(S176K)_Fc_9C8_VH_CK(S176 E)149414961498376789042_3.2_CL(S176K)_Fc_23B3_VH4_VH_C K(S176E)150015021504376793043_3B3v2_CK(S176K)_Fc_Adalimumab_V H_CK(S176E)150615081510376797044_3B3v2_CK(S176K)_Fc_3.2_VH_CL(S1 76E)151215141516376801045_9C8_CK(S176K)_Fc_Adalimumab_VH CK(S176E)151815201522376806046_9C8_CK(S176K)_Fc_3.2_VH_CL(S176 E)152415261528376811047_23B3_VH4_CK(S176K)_Fc_Adalimuma b_VH_CK(S176E)153015321534376816048_23B3_VH4_CK(S176K)_Fc_3.2_VH_C L(S176E)153615381540376821059_Adalimumab_IgG.002_3B3v2_VH_CH1 (S183E).001154215441546376826060_Adalimumab_IgG.002_9C8_VH_CH1(S 183E).001154815501552376830061_Adalimumab_IgG.002_23B3_VH4_VH_ CH1(S183E).001155415561558376834062_3.2_IgG.002_3B3v2_VH_CH1(S183E).0 01156015621564376868063_3.2_IgG.002_9C8_VH_CH1(S183E).001156615681570376842064_3.2_IgG.002_23B3_VH4_VH_CH1(S18 3E).001157215741576376846065_3B3v2_IgG.002_Adalimumab_VH_CH1 (S183E).001157815801582376850066_3B3v2_IgG.002_3.2_VH_CH1(S183E).0 01158415861588376854067_9C8_IgG.002_Adalimumab_VH_CH1(S 183E).001159015921594376858068_9C8_IgG.002_3.2_VH_CH1(S183E).001159615981600376862069_23B3_VH4_IgG.002_Adalimumab_VH_ CH1(S183E).001160216041606376867070_23B3_VH4_IgG.002_3.2_VH_CH1(S18 3E).00 1160816101612376872071_Adalimumab_CK(S176K)_Fc.001_3B3v 2_VH_CH1(S183E).001161416161618376879072_Adalimumab_CK(S176K)_Fc.001_9C8 VH_CH1(S183E).001162016221624376884073_Adalimumab _CK(S176K)_Fc.001_23B3 _VH4_VH_CH1(S183E).001162616281630376889074_3.2_CL(S176K)_Fc.001_3B3v2_VH_C H1(S183E).001163216341636376896075_3.2_CL(S176K)_Fc.001_9C8_VH_CH1( S183E).001163816401642376901076_3.2_CL(S176K)_Fc.001_23B3_VH4_V H_CH1(S183E).001164416461648376906077_3B3v2_CK(S176K)_Fc.001_Adalimuma b_VH_CH1(S183E).001165016521654376913078_3B3v2_CK(S176K)_Fc.001_3.2_VH_C H1(S183E).001165616581660376918079_9C8_CK(S176K)_Fc.001_Adalimumab VH_CH1(S183E).001166216641666376925080_9C8_CK(S176K)_Fc.001_3.2_VH_CH1( S183E).001166816701672376930081_23B3_VH4_CK(S176K)_Fc.001_Adalim unmb_VH_CH1(S183E).001167416761678376937082_23B3_VH4_CK(S176K)_Fc.001_3.2_V H_CH1(S183E).001168016821684376941083_Adalimumab_IgG.002_3B3v2_VH_CK( S176E).001168616881690376948084_Adalimumab_IgG.002_9C8_VH_CK(S1 76E).00 1169216941696376955085_Adalimumab _IgG.002_23B3_VH4_VH_ CK(S176E).001169817001702376962086_3.2_IgG.002_3B3v2_VH_CK(S176E).00 1170417061708376967087_3.2_IgG.002_9C8_VH_CK(S176E).001171017121714376972088_3.2_IgG.002_23B3_VH4_VH_CK(S176 E).001171617181720376976089_3B3v2_IgG.002_Adalimumab_VH_CK( S176E).001172217241726376983090_3B3v2_IgG.002_3.2_VH_CL(S176E).00 1172817301732376990091_9C8_IgG.002_Adalimumab_VH_CK(S1 76E).001173417361738376995092_9C8_IgG.002_3.2_VH_CL(S176E).001174017421744377000093_23B3_VH4_IgG.002_Adalimumab_VH_ CK(S176E).001174617481750377005094_23B3_VH4_IgG.002_3.2_VH_CL(S176 E).001175217541756377010095_Adalimumab _CK(S176K)_Fc.001_3B3v 2_VH_CK(S176E).001175817601762377015096_Adalimumab_CK(S176K)_Fc.001_9C8 VH_CK(S176E).001176417661768377020097_Adalimumab_CK(S176K)_Fc.001_23B3 VH4_VH_CK(S176E).001177017721774377025098_3.2_CL(S176K)_Fc.001_3B3v2_VH_C K(S176E).001177617781780377030099_3.2_CL(S176K)_Fc.001_9C8_VH_CK(S 176E).001178217841786377035100_3.2_CL(S176K)_Fc.001_23B3_VH4_V H_CK(S176E).001178817901792377040101_3B3v2_CK(S176K)_Fc.001_Adalimuma b_VH_CK(S176E).001179417961798377045102_3B3v2_CK(S176K)_Fc.001_3.2_VH_C L(S176E).001180018021804377050103_9C8_CK(S176K)_Fc.001_Adalimumab VH_CK(S176E).001180618081810377055104_9C8_CK(S176K)_Fc.001_3.2_VH_CL(S 176E).001181218141816377060105_23B3_VH4_CK(S176K)_Fc.001_Adalim unmb_VH_CK(S176E).001181818201822377064106_23B3_VH4_CK(S176K)_Fc.001_3.2_V H_CL(S176E).001182418261828377068117_Adalimumab_IgG.003_3B3v2_VH_CH1 (S183E).002183018321834377072118_Adalimumab_IgG.003_9C8_VH_CH1(S 183E).002183618381840377077119_Adalimumab_IgG.003_23B3_VH4_VH CH1(S183E).002184218441846377082120_3.2_IgG.003_3B3v2_VH_CH1(S183E).0 02184818501852377087121_3.2_IgG.003_9C8_VH_CH1(S183E).002185418561858377092122_3.2_IgG.003_23B3_VH4_VH_CH1(S18 3E).002186018621864377097123_3B3v2_IgG.003_Adalimumab_VH_CH1 (S183E).002186618681870377102124_3B3v2_IgG.003_3.2_VH_CH1(S183E).0 02187218741876377107125_9C8_IgG.003_Adalimumab_VH_CH1(S 183E).0021878188018823771112126_9C8_IgG.003_3.2_VH_CH1(S183E).002188418861888377116127_23B3_VH4_IgG.003_Adalimumab_VH_ CH1(S183E).002189018921894377121128_23B3_VH4_IgG.003_3.2_VH_CH1(S18 3E).002189618981900377126129_Adalimumab_CK(S176K)_Fc.002_3B3v 2_VH_CH1(S183E).002190219041906377133130_Adalimumab_CK(S176K)_Fc.002_9C8_ VH_CH1(S183E).002190819101912377138131_Adalimumab_CK(S176K)_Fc.002_23B3 _VH4_VH_CH1(S183E).002191419161918377142132_3.2_CL(S176K)_Fc.002_3B3v2_VH_C H1(S183E).002192019221924377148133_3.2_CL(S176K)_Fc.002_9C8_VH_CH1( S183E).002192619281930377152134_3.2_CL(S176K)_Fc.002_23B3_VH4_V H_CH1(S183E).002193219341936377156135_3B3v2_CK(S176K)_Fc.002_Adalimuma b_VH_CH1(S183E).002193819401942377162136_3B3v2_CK(S176K)_Fc.002_3.2_VH_C H1(S183E).002194419461948377166137_9C8_CK(S176K)_Fc.002_Adalimumab VH_CH1(S183E).002195019521954377172138_9C8_CK(S176K)_Fc.002_3.2_VH_CH1( S183E).002195619581960377176139_23B3_VH4_CK(S176K)_Fc.002_Adalim umab_VH_CH1(S183E).002196219641966377182140_23B3_VH4_CK(S176K)_Fc.002_3.2_V H_CH1(S183E).002196819701972377186141_Adalimumab_IgG.003_3B3v2_VH_CK( S176E).002197419761978377193142_Adalimumab_IgG.003_9C8_VH_CK(S1 76E).002198019821984377200143_Adalimumab_IgG.003_23B3_VH4_VH_ CK(S176E).002198619881990377207144_3.2_IgG.003_3B3v2_VH_CK(S176E).00 2199219941996377212145_3.2_IgG.003_9C8_VH_CK(S176E).002199820002002377217146_3.2_IgG.003_23B3_VH4_VH_CK(S176 E).002200420062008377222147_3B3v2_IgG.003_Adalimumab_VH_CK( S176E).002201020122014377229148_3B3v2_IgG.003_3.2_VH_CL(S176E).00 2201620182020377236149_9C8_IgG.003_Adalimumab_VH_CK(S1 76E).002202220242026377241150_9C8_IgG.003_3.2_VH_CL(S176E).002202820302032377246151_23B3_VH4_IgG.003_Adalimumab_VH_ CK(S176E).002203420362038377251152_23B3_VH4_IgG.003_3.2_VH_CL(S176 E).002204020422044377256153_Adalimumab_CK(S176K)_Fc.002_3B3v 2_VH_CK(S176E).002204620482050377261154_Adalimumab_CK(S176K)_Fc.002_9C8_ VH_CK(S176E).002205220542056377266155_Adalimumab_CK(S176K)_Fc.002_23B3 _VH4 VH_CK(S176E).002205820602062377271156_3.2_CL(S176K)_Fc.002_3B3v2_VH_C K(S176E).002206420662068377276157_3.2 CL(S176K)_Fc.002_9C8_VH_CK(S 176E).002207020722074377281158_3.2_CL(S176K)_Fc.002_23B3_VH4_V H_CK(S176E).002207620782080377286159_3B3v2_CK(S176K)_Fc.002_Adalimuma b_VH_CK(S176E).002208220842086377290160_3B3v2_CK(S176K)_Fc.002_3.2_VH_C L(S176E).002208820902092377295161_9C8_CK(S176K)_Fc.002_Adalimumab VH_CK(S176E).002209420962098377300162_9C8_CK(S176K)_Fc.002_3.2_VH_CL(S 176E).002210021022104377305163_23B3_VH4_CK(S176K)_Fc.002_Adalim umab_VH_CK(S176E).002210621082110377310164_23B3_VH4_CK(S176K)_Fc.002_3.2_V H_CL(S176E).002211221142116

[0274] The foregoing amino acid sequences are encoded by the nucleic acid sequences immediately preceding them in the Sequence Listing.

[0275] The CDR sequences of the VL and VH domains of the foregoing are as shown in Tables 21.2A and 21.2B below. Table 21.2A: anti-TL1A / anti-TNF-α IgG-Fab VL CDR sequencesAb Type CDRL1 CDRL2 CDRL3 Anti-TNF-alpha (Adalimumab) NASEQ ID NO: 957SEQ ID NO: 959SEQ ID NO: 960AARASQGIRNYLAAASTLQSQRYNRAPYTSEQ ID NO: 92SEQ ID NO: 242SEQ ID NO: 96Anti-TNF-alpha (3.2) NASEQ ID NO: 962SEQ ID NO: 965SEQ ID NO: 1189AATGSSSNIGAGYDVHGNSNRPSQSYDSSLSGSVSEQ ID NO: 146SEQ ID NO: 148SEQ ID NO: 150Anti-TL1A (3B3v2) NASEQ ID NO: 94SEQ ID NO: 467SEQ ID NO: 104AARASQSVRSSYLAGASSRATQQYGSSPTSEQ ID NO: 122SEQ ID NO: 124SEQ ID NO: 126Anti-TL1A (9C8) NASEQ ID NO: 263SEQ ID NO: 2117SEQ ID NO: 2118AARASQSINNYLNAASSLQSQQSYSTPRTSEQ ID NO: 110SEQ ID NO: 112SEQ ID NO: 108Anti-TL1A (23B3) NASEQ ID NO: 2119SEQ ID NO: 2120SEQ ID NO: 2121AARSSQSVLYSSNNKNYLVWASTRESQQYYKTPLTSEQ ID NO: 128SEQ ID NO: 118SEQ ID NO: 120 Table 21.2B: anti-TL1A / anti-TNF-α IgG-Fab VH CDR sequences Ab Type CDRH1 CDRH2 CDRH3 Anti-TNF-alpha (Adalimumab) NAGATTATGCGATGCATSEQ ID NO: 2122SEQ ID NO: 2123SEQ ID NO: 2124AADYAMHAITWNSGHIDYADSVEGVSYLSTASSLDYSEQ ID NO: 158SEQ ID NO: 160SEQ ID NO: 162Anti-TNF-alpha (3.2) NAAGCTACTGGATCGGCAGTAACTGGGGTCTTGACTACSEQ ID NO: 2125SEQ ID NO: 2126SEQ ID NO: 2127AASYWIGIIYLGDSDTRYSPSFQGSNWGLDYSEQ ID NO: 212SEQ ID NO: 214SEQ ID NO: 216Anti-TL1A (3B3v2) NAGGTTACTACTGGAACSEQ ID NO: 2128SEQ ID NO: 2129SEQ ID NO: 2130AAGYYWNEINHAGNTNYNPSLKSGYCRSTTCYFDYSEQ ID NO: 188SEQ ID NO: 190SEQ ID NO: 192Anti-TL1A (9C8) NAAGTTACTTCTGGAGCSEQ ID NO: 2131SEQ ID NO: 2132SEQ ID NO: 2133AASYFWSYIYYSGQTKYNPSLKSETGSYYGFDYSEQ ID NO: 170SEQ ID NO: 633SEQ ID NO: 180Anti-TL1A (23B3) NASEQ ID NO: 2134SEQ ID NO: 2135SEQ ID NO: 2136AATNSVAWNRTYYRSKWYNDYAVSLKSEDGDSYYRYGMDVSEQ ID NO: 182SEQ ID NO: 196SEQ ID NO: 186 Table 21.3: Titer, Recovery, and Activity of IgG-Fabs iPS No. IgG Fab CL / CH1 Swap CPM Titer (mg / L) Recovery (mg / L) hu TL1A (pM) hu TL1A Fold Change hu TNFa (pM) hu TNFa Fold Change 376597TNFa (ada)TL1A (3B3v2)No swapv163.556.26698197.33.65370487.50.603448376601TNFa (ada)TL1A (9C8)No swapv149.444.46469136.50.90397496.50.665517376605TNFa (ada)TL1A (23B3)No swapv14637.35632689.65.56129941.76.494483376609TNFa (3.2)TL1A (3B3v2)No swapv128.937.88288440881.6296394.21.345714376613TNFa (3.2)TL1A (9C8)No swapv134.541.14599260.31.72384175.31.075714376617TNFa (3.2)TL1A (23B3)No swapv130.337.1352910258.26612979.61.137143376621TL1A (3B3v2)TNFa (ada)No swapv153.155.2317166.31.22777833.30.229655376625TL1A (3B3v2)TNFa (3.2)No swapv151.262.1359263.81.18148137.30.532857376629TL1A (9C8)TNFa (ada)No swapv146.143.10706142.10.9410622.40.154483376633TL1A (9C8)TNFa (3.2)No swapv144.353.37693215.71.42847744.60.637143376637TL1A (23B3)TNFa (ada)No swapv145.943.74483348.82.81290327.50.189655376641TL1A (23B3)TNFa (3.2)No swapv149.756.93208.81.68387143.90.627143376645TNFa (ada)TL1A (3B3v2)N-term swapv15.824.959868139.12.5759261310.903448376651TNFa (ada)TL1A (9C8)N-term swapv15.737.33602337.82.237086157.51.086207376655TNFa (ada)TL1A (23B3)N-term swapv16.546.945786200.21.614516198.51.368966376659TNFa (3.2)TL1A (3B3v2)N-term swapv17.337.57669273.61.36296392.41.32376665TNFa (3.2)TL1A (9C8)N-term swapv118.320.86174910.60264941.70.595714376669TNFa (3.2)TL1A (23B3)N-term swapv121.821.9279377.60.62580673.51.05376673TL1A (3B3v2)TNFa (ada)N-term swapv127.327.46576482589.3518541.70.287586376679TL1A (3B3v2)TNFa (3.2)N-term swapv128.840.933076250115.740739.60.565714376683TL1A (9C8)TNFa (ada)N-term swapv133.428.837164142.74172235.20242759376689TL1A (9C8)TNFa (3.2)N-term swapv132.439.77615787.15.212583450.642857376693TL1A (23B3)TNFa (ada)N-term swapv125.419.9532229.50.203448376699TL1A (23B3)TNFa (3.2)N-term swapv122.925.8504340.70.581429376703TNFa (ada)TL1A (3B3v2)C-term swapv139.230.58153150.42.78518578.60.542069376709TNFa (ada)TL1A (9C8)C-term swapv15041.99633199.51.32119280.40.554483376715TNFa (ada)TL1A (23B3)C-term swapv140.729.97569640.45.16451664.20.442759376721TNFa (3.2)TL1A (3B3v2)C-term swapv123.224.91097213.23.94814892.81.325714376725TNFa (3.2)TL1A (9C8)C-term swapv126.231.24415185.81.23046448.30.69376729TNFa (3.2)TL1A (23B3)C-term swapv131.728.69498330.82.667742791.128571376733TL1A (3B3v2)TNFa (ada)C-term swapv136.931.9554866.11.2240742631.813793376739TL1A (3B3v2)TNFa (3.2)C-term swapv12.951.921946376745TL1A (9C8)TNFa (ada)C-term swapv124.922.1839108.70.719868135.30.933103376750TL1A (9C8)TNFa (3.2)C-term swapv17.718.2236972231.47682171.41.02376755TL1A (23B3)TNFa (ada)C-term swapv19.117.361222271.52.189516433.32.988276376760TL1A (23B3)TNFa (3.2)C-term swapv17.175.960717305.62.464516129.81.854286376765TNFa (ada)TL1A (3B3v2)Both swapv13.664.304958343.66.3629631821.255172376770TNFa (ada)TL1A (9C8)Both swapv12.681.7125810376775TNFa (ada)TL1A (23B3)Both swapv14.992.967115389.73.14274290.40.623448376779TNFa (3.2)TL1A (3B3v2)Both swapv14.923.717088707.113.09444226.83.24376785TNFa (3.2)TL1A (9C8)Both swapv19.369.546385149.50.990066420.6376789TNFa (3.2)TL1A (23B3)Both swapv112.211.32345554.24.469355741.057143376793TL1A (3B3v2)TNFa (ada)Both swapv119.315.350211937.535.87963347.92.39931376797TL1A (3B3v2)TNFa (3.2)Both swapv110376801TL1A (9C8)TNFa (ada)Both swapv113.611.14213286.81.8993383102.137931376806TL1A (9C8)TNFa (3.2)Both swapv110.961822376811TL1A (23B3)TNFa (ada)Both swapv13.221.671524376817TL1A (23B3)TNFa (3.2)Both swapv110376822TNFa (ada)TL1A (3B3v2)No swapv253.643.05822128.52.3796384.70.584138376826TNFa (ada)TL1A (9C8)No swapv227.422.923981060.70198780.80.557241376830TNFa (ada)TL1A (23B3)No swapv234.225.04152622.11290373.30.505517376834TNFa (3.2)TL1A (3B3v2)No swapv21817.62724246.84.57037971.385714376838TNFa (3.2)TL1A (9C8)No swapv217.317.06775548.83.63443731.60.451429376842TNFa (3.2)TL1A (23B3)No swapv214.912.78592469.853.78911382.91.184286376846TL1A (3B3v2)TNFa (ada)No swapv246.745.5738271.81.3296331.50.217241376850TL1A (3B3v2)TNFa (3.2)No swapv224.425.4700983.31.54259384.81.211429376854TL1A (9C8)TNFa (ada)No swapv239.536.01855229.31.51854330.80.212414376858TL1A (9C8)TNFa (3.2)No swapv217.517.37068190.81.263576350.5376862TL1A (23B3)TNFa (ada)No swapv223.519.86708436.33.51854834.40.237241376867TL1A (23B3)TNFa (3.2)No swapv211.310.117781090.879032290.414286376872TNFa (ada)TL1A (3B3v2)N-term swapv25.265.36285527.7250.513426519.53.582759376879TNFa (ada)TL1A (9C8)N-term swapv24.242.812933253.21.676821173.21.194483376885TNFa (ada)TL1A (23B3)N-term swapv23.012.592265310.32.502419940.86.488276376889TNFa (3.2)TL1A (3B3v2)N-term swapv210.817101376897TNFa (3.2)TL1A (9C8)N-term swapv22.765.4946267605.033113472.96.755714376902TNFa (3.2)TL1A (23B3)N-term swapv210.985458376907TL1A (3B3v2)TNFa (ada)N-term swapv236.729.31227321359.535.80.246897376913TL1A (3B3v2)TNFa (3.2)N-term swapv219.425.2242681315.0555652.550.750714376918TL1A (9C8)TNFa (ada)N-term swapv239.631.18441203.61.34834417.8550.123138376925TL1A (9C8)TNFa (3.2)N-term swapv222.427.25854189.651.2559629.350.419286376931TL1A (23B3)TNFa (ada)N-term swapv222.920.43729719558.0241926.770.184621376937TL1A (23B3)TNFa (3.2)N-term swapv212.29.722544176.952.527857376941TNFa (ada)TL1A (3B3v2)C-term swapv240.631.7986622.730.42092650.40.347586376949TNFa (ada)TL1A (9C8)C-term swapv238.735.04348.1650.31897449.0950.338586376955TNFa (ada)TL1A (23B3)C-term swapv23024.1244333.952.69314557.150.394138376962TNFa (3.2)TL1A (3B3v2)C-term swapv29.9613.7926854010195727.95714376967TNFa (3.2)TL1A (9C8)C-term swapv28.7312.01796376972TNFa (3.2)TL1A (23B3)C-term swapv22.584.41917512490100.72587870112.4286376976TL1A (3B3v2)TNFa (ada)C-term swapv226.316.3204931.310.579815172.21.187586376983TL1A (3B3v2)TNFa (3.2)C-term swapv210.993437376991TL1A (9C8)TNFa (ada)C-term swapv240.538.4284959.90.3966891481.02069376995TL1A (9C8)TNFa (3.2)C-term swapv210.339488377001TL1A (23B3)TNFa (ada)C-term swapv225.825.4905841.6750.336089158.451.092759377006TL1A (23B3)TNFa (3.2)C-term swapv210.124147377011TNFa (ada)TL1A (3B3v2)Both swapv213.586728270250.037043065.521.14138377015TNFa (ada)TL1A (9C8)Both swapv23.23.312442141050972.7586377020TNFa (ada)TL1A (23B3)Both swapv211.15137377025TNFa (3.2)TL1A (3B3v2)Both swapv210.778064377031TNFa (3.2)TL1A (9C8)Both swapv212.94023917640116.821212965185.2143377036TNFa (3.2)TL1A (23B3)Both swapv210.595615377041TL1A (3B3v2)TNFa (ada)Both swapv210.810.829891689.531.287042014.513.8931377045TL1A (3B3v2)TNFa (3.2)Both swapv210377050TL1A (9C8)TNFa (ada)Both swapv222.116.88003269.051.78178867.20.463448377056TL1A (9C8)TNFa (3.2)Both swapv210377060TL1A (23B3)TNFa (ada)Both swapv213.78306237119.12097171.51.182759377064TL1A (23B3)TNFa (3.2)Both swapv210377068TNFa (ada)TL1A (3B3v2)No swapv369.261.3541423.50.43518556.10.386897377073TNFa (ada)TL1A (9C8)No swapv364.957.5309947.080.31178836.7950.253759377077TNFa (ada)TL1A (23B3)No swapv36050.4456867.90.54758149.660.342483377082TNFa (3.2)TL1A (3B3v2)No swapv335.650.01275194.93.6092591213.517.33571377087TNFa (3.2)TL1A (9C8)No swapv344.733.18984148.80.9854341.5550.593643377092TNFa (3.2)TL1A (23B3)No swapv33543.58861598.54.82661338.8450.554929377097TL1A (3B3v2)TNFa (ada)No swapv370.574.2530337.290.69055620.850.143793377102TL1A (3B3v2)TNFa (3.2)No swapv368.184.2387231.5650.58453729.980.428286377107TL1A (9C8)TNFa (ada)No swapv35755.2256878.350.51887416.7750.11569377112TL1A (9C8)TNFa (3.2)No swapv337.945.7669253.50.35430526.3150.375929377116TL1A (23B3)TNFa (ada)No swapv344.344.0240573.90.59596815.230.105034377121TL1A (23B3)TNFa (3.2)No swapv340.728.5981262.550.50443522.850.326429377126TNFa (ada)TL1A (3B3v2)N-term swapv311.981845377133TNFa (ada)TL1A (9C8)N-term swapv32.652.948434806.55.341065523.806897377138TNFa (ada)TL1A (23B3)N-term swapv311.33942377142TNFa (3.2)TL1A (3B3v2)N-term swapv310.211.7593432.780.60703745.340.647714377148TNFa (3.2)TL1A (9C8)N-term swapv311.113.0763599.650.65993448.4950.692786377152TNFa (3.2)TL1A (23B3)N-term swapv31312.6972546.510.37508139.740.567714377156TL1A (3B3v2)TNFa (ada)N-term swapv330.834.834781584.529.3425919.90.137241377162TL1A (3B3v2)TNFa (3.2)N-term swapv326.234.40556250446.3703724.8750.355357377166TL1A (9C8)TNFa (ada)N-term swapv331.926.76967830.54966916.9550.116931377172TL1A (9C8)TNFa (3.2)N-term swapv331.338.7044181.50.53973527.260.389429377176TL1A (23B3)TNFa (ada)N-term swapv311.311.48623530542.7822628.730.198138377182TL1A (23B3)TNFa (3.2)N-term swapv31415.541828185227.298448.170.688143377186TNFa (ada)TL1A (3B3v2)C-term swapv346.436.675337.570.69574135.750.246552377193TNFa (ada)TL1A (9C8)C-term swapv33228.52527128.050.84801330.930.21331377200TNFa (ada)TL1A (23B3)C-term swapv356.643.6029217.351.75282338.4250.265377208TNFa (3.2)TL1A (3B3v2)C-term swapv322.126.10432212.353.932407170.152.430714377212TNFa (3.2)TL1A (9C8)C-term swapv317.618.673368745.788079306.84.382857377217TNFa (3.2)TL1A (23B3)C-term swapv327.425.63219271.42.18871114.81.64377223TL1A (3B3v2)TNFa (ada)C-term swapv340.536.0718230.1750.5587961671.151724377229TL1A (3B3v2)TNFa (3.2)C-term swapv33.053.03109379.51.4722221711.524.45377236TL1A (9C8)TNFa (ada)C-term swapv348.343.31554.250.35927261.70.425517377241TL1A (9C8)TNFa (3.2)C-term swapv38.958.4908456064.013245268.43.834286377246TL1A (23B3)TNFa (ada)C-term swapv322.420.5244568.70.554032118.50.817241377252TL1A (23B3)TNFa (3.2)C-term swapv35.055.690246219.81.772581138119.72857377256TNFa (ada)TL1A (3B3v2)Both swapv313.203042377261TNFa (ada)TL1A (9C8)Both swapv313.032322377266TNFa (ada)TL1A (23B3)Both swapv32.573.395799377272TNFa (3.2)TL1A (3B3v2)Both swapv313.07164713300246.2963377276TNFa (3.2)TL1A (9C8)Both swapv31416.058461237.58.195364362.45.177143377281TNFa (3.2)TL1A (23B3)Both swapv312.613.705761937.515.625282.354.033571377286TL1A (3B3v2)TNFa (ada)Both swapv325.529.40929486.79.012963521.53.596552377290TL1A (3B3v2)TNFa (3.2)Both swapv310.304074377295TL1A (9C8)TNFa (ada)Both swapv326.329.74724177.551.175828132.350.912759377300TL1A (9C8)TNFa (3.2)Both swapv33.584.35596817575116.390769750996.4286377305TL1A (23B3)TNFa (ada)Both swapv31113.60209595.54.106897377310TL1A (23B3)TNFa (3.2)Both swapv310.64267 Table 21.4: anti-TL1A / anti-TNF-α IgG-Fab purity CaliperNon-reducedReducedPS Nopre-MP HMWMPpost-MPMP 1MP 2MP 3LMWHC 1HC 2HC 3LC 1R LC 23765973.832961010.2068970.7931033766012.894971010.353160.646843766053.36296.63410113766091.24199100.1538460.84615413766131.838981010.2986580.7013423766171.1629910113766211.82798.172101013766252.55297100.1365030.8634970.5114940.4885063766291.868981010.5214290.4785713766335.854941010.4542250.5457753766371.09998.9011010.6833330.3166673766413.25596.7451010.448980.551023766458.84463.7541010.3360660.66393437665139.874600.460.54010.3649290.6350713766558.19191.80910.3350520.6649483766593.51596.4850.910.090.074380.925620.5145990.4854013766652.981971010.4981680.5018323766692.21397.78710113766734.63560.8420.950.050.1101150.88988513766791.77498.2260.70.30.1234940.2030080.67349 913766835.787940.530.220.2510.3301890.6698113766893.89696.1040.70.310.2093860.7906143766932.3296.1311.5480.80.1113766991.27990.7150.285113767037.4693100.1647560.8352440.4370860.5629143767098.439921010.439850.560153767152.451980.40.6010.2851240.7148763767215.54494.4560.850.150.1975950.8024050.3277510.6722493767257.76671.4451010.4133330.5866673767294.728951010.4143430.5856573767339.277911010.4743590.52564137673911.35870.08310.7194810.28051937674518.482821010.4796750.5203253767505.3694.64100.1535090.84649113767554.451961010.5092250.4907753767606.60993.39110113767654.75157.8681010.3051360.6948643767707.17650.33113767753663.70510.5270270.4729733767795.2594.751010.4161070.58389337678519.12480.8761010.4433330.5566673767892.556971010.3852940.6147063767939.16179.135100.1201120.879888137679737680116.03783.96310113768063.9119637681135.5741.522113768173768223.67961010.3046880.6953133768261.52198.479100.0977920.9022080.4877380.5122623768303.37714.01510113768343.39796.613100.4956670.5043330.2298580.7701423768382.64497.3561010.254630.745373768422.839971010.1498770.8501233768462.2897.7210113768502.2897.72100.3321920.6678080.7355770.2644233768540.96399.0371010.5339510.4660493768582.90397.0971010.5138890.4861113768622.56797.4331010.6666670.3333333768672.06297.9381010.4957020.50429837687250.1925010.1712540.82874637687910.70565.81210.3032070.6967933768851.20277.07210.2326280.7673723768899.57983.9820.5460.4543768976.19793.80310.5401460.4598543769027.84192.125 9113769072.50897.4920.810.190.2139460.7860540.1761520.8238483769131.5998.410.637630.36210.5890910.4109090.1425390.8574613769182.689970.6070.39310.3100780.6899223769252.70197.479110.2452830.7547173769313.8251723.9710.8450.15113769376.425736.77710113769416.0993.910.99500.1044520.8955480.4567310.5432693769494.91595.0850.1360.8050.05910.4321610.5678393769553.58996.4110.1970.2870.51010.2228410.7771593769625.59486.487.9260.6360740.6360740.36392 610.3216780.6783223769676.54793.4531010.4868690.5131313769723.03196.96910.425490.574513769763.38796.6131010.4879650.51203537698334.082663769919.46790.5331010.5012190.49878137699503770012.54597.4551010.5192880.480712377006066.13633.8643770112.172790.5449440.4550560.2473120.7526883770159.233571103770207.1278551.5513770258.1328515.9483770313.924910.4731490.5268510.5774190.4225813770363.7889637704116.236 183.982100.3158710.684129137704537705011.1647710.49610113770563770607.0757733.064113770643770683.292971010.2991450.7008553770731.92798.07410.3769470.6230533770772.5839710.1107490.8892513770821.13599100.2250860.7749140.0714290.9285713770872.00697.9291010.2749330.7250673770921.99498.0061010.0943950.9056053770971.26198.73910113771022.1197.767100.1642510.8357490.5013190.4986813771071.501981010.4858930.5141073771123.322971010.4686570.5313433771162.65971010.5570030.4429973771213.284971010.4597010.54029937712612.40 466113771336.5535710.4365080.5634923771383.9887310.4009320.5990683771422.077981010.528610.471393771483.772961010.5297450.4702553771521.436991010.502890.497113771562.61477100.1187340.88654413771621.39498.5360.640.330.0654660.1456630.4850.1722370.8277633771665.575942.3130.2950.450.25410.3591160.6408843771723.898960.2610.3250.41410.3493330.65066737717619.39 977101137718232.58 8501113771866.81393100.1719750.8280250.4540540.5459463771931.39498.6051010.4461080.5538923772005.103950.150.1650.688010.3344480.6655523772083.092970.840.160.2297870.7702130.3389830.66101737721220.00 5800.8670.13310.4128880.5871123772175.089951010.447090.552913772237.4593100.0655740.9344260.4974230.50257737722920.13 8621137723618.44821010.4884390.51156137724110.02 29011377246792.6941010.5198780.4801223772521683.8046.111113772560100137726101001013772660100113772728.6758410.3878410.61215937727611.00 688.6261010.4864860.5135143772814.72295.2791010.4854880.5145123772865.49764100.1343280.8656721377290010037729513.80 710.89910113773006.9328710.5665530.43344737730511.80 883100.3314180.6685820.81250.18753773100100

[0276] It is understood that the disclosed invention is not limited to the particular methodology, protocols and materials described as these can vary. It is also understood that the terminology used herein is for the purposes of describing particular embodiments only and is not intended to limit the scope of the appended claims.

[0277] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.ABBREVIATIONS

[0278] Abbreviated terms used throughout this specification are defined as follows. aa, AAamino acid AEIallelic expression imbalance ANOVAanalysis of variance BSAbovine serum albumin CDRcomplementarity determining regions CHOChinese hamster ovary cells CPMcharge pair mutation DMEMDulbecco's Modified Eagle Medium DMSOdimethyl sulfoxide ELISAenzyme-linked immunosorbent assay eQTLexpression quantitative trait loci ESI-TOFelectrospray ionization time of flight ESNexhausted supernatant FACSfluorescence-activated cell sorting FBSfetal bovine serum FPLCfast protein liquid chromatography FVBa strain of mice inbred for the Friend leukemia virus 1b (Fvlb) allele H&EHematoxylin and eosin HAhypoxanthine HIChydrophobic interaction chromatography HPLChigh performance liquid chromatography HRPhorse radish peroxidase HUVEChuman umbilical vein epithelial cell IBDinflammatory bowel disease IDMEMDMEM without glutamine IFNinterferon ILinterleukin MCPmonocyte chemotactic protein MSDmacromolecular structure database NAnucleic acid PBMCperipheral blood mononuclear cell PBSphosphate-buffered saline PCRpolymerase chain reaction PEGpolyethylene glycol PEIpolyethylenimine QTLquantitative trait loci RPMImedia developed at Roswell Park Memorial Institute RT-PCRpolymerase chain reaction at room temperature SNPsingle nucleotide polymorphism TFAtrifluoroacetic acid TL1ATNF-like ligand 1A (TNFSF15) TMBtetramethylbenzene TNFtumor necrosis factor-α

Claims

1. An antigen binding protein specific for TL1A, wherein the antigen binding protein comprises a combination of sequences for LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 selected from the following Table A, wherein the combination of sequences for LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 is selected from the rows shown in Table A: LCDR1 (SEQ ID NO)LCDR2 (SEQ ID NO)LCDR3 (SEQ ID NO)HCDR1 (SEQ ID NO)HCDR2 (SEQ ID NO)HCDR3 (SEQ ID NO)DASSLQS (100)SYGMH (164)ATSSLQS (106)SYFWS (170)AASSLQS (112)SYFWS (170)WASTRES (118)TNSVAWN (182)WASTRES (118)TNSVAWN (182)WASTRES (118)TNSVAWN (182)GASSRAT (124)QQYGSSPT (126)GYYWN (188)GASSRAT (124)QQYGSSPT (126)GYYWN (188)AASSLQS (112)SYGMH (164)LGSSRAS (685)TYYMS (777)WASTRES (118)TYGMH (783)WASTRES (118)SYGMH (164)GNNNRPS (699)SYVMS (792)GNSHRPS (705)NYAMS (798)GNSHRPS (705)NYAMN (804)AASSLQS (112)SSSATWN (809)TASSLQS (721)SNSATWN (815)SNNKRPS (725)GFYMH (819)EGIAVALTY (823)VASSLQS (731)GYYWS (265)AASGLQG (737)SYGMH (164)AASSLQS (112)QQSYSSIT (745)SYAMS (836)EMAGAFDI (840)TASSLQS (749)GYYWS (265)KVSNWDS (755)AYYMH (847)AASRLQS (761)AYYMH (847)AASSLQS (112)SYAMS (836)GASRLQS (769)GYYMH (857)AASSLQS (112)SYAMS (836)2. The antigen binding protein of claim 1, wherein the combination of sequences of LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 is selected from: a. antibody 9C8, SEQ ID NOS. 110, 112, 108, 170, 178 and 180; and b. antibody 383, SEQ ID NOS. 122, 124, 126, 188, 190, and 192.

3. The antigen binding protein of claim 1 comprising light and heavy chain variable domain sequence combinations having at least 90% sequence identity with antigen binding proteins selected from Table D, SEQ ID NOS: a. 6 and 8; b. 10 and 12; c. 14 and 16; d. 18 and 20; e. 22 and 24; f. 26 and 28; g. 30 and 32; h. 491 and 495; i. 493 and 497; j. 866 and 868; k. 870 and 872; l. 874 and 876; m. 878 and 880; n. 882 and 884; o. 890 and 892; p. 894 and 896; q. 898 and 900; r. 902 and 904; s. 906 and 908; t. 910 and 912; u. 914 and 916; v. 918 and 920; w. 922 and 920; x. 924 and 926; y. 928 and 930; and z. 932 and 934.

4. The antigen binding protein of claim 3 comprising light and heavy chain variable domain sequence combinations of antigen binding proteins selected from: a. antibody 9C8, SEQ ID NOS: 14 and 16; and b. antibody 383, SEQ ID NOS: 22 and 24.

5. The antigen binding protein of claim 1 comprising a combination of light and heavy chain sequences having at least 90% sequence identity with a combination of light and heavy chain sequences selected from Table E, SEQ ID NOS: a. 50 and 52, b. 54 and 56, c. 58 and 60, d. 62 and 64, e. 66 and 68, f. 70 and 72, g. 74 and 76, h. 455 and 457, i. 459 and 461 j. 1116 and 1118, k. 1120 and 1122, I. 1124 and 1126, m. 1128 and 1130, n. 1132 and 1134, o. 1136 and 1138, p. 1140 and 1142, q. 1144 and 1146, r. 1148 and 1150, s. 1152 and 1154, t. 1156 and 1158, u. 1160 and 1162, v. 1164 and 1166, w. 1168 and 1170, x. 1172 and 1174, y. 1176 and 1178, z. 1180 and 1182, aa. 1184 and 1186, and bb. 1188 and 1190.

6. The antigen binding protein of claim 5 wherein the protein comprises a combination of light and heavy chain sequences selected from: a. SEQ ID NOS. 54 and 56; and b. SEQ ID NOS. 58 and 60.

7. The antigen binding protein of claims 1-6 wherein the antigen binding protein is an antibody.

8. The antigen binding protein of claims 1-6 wherein the antigen binding protein comprises an antibody fragment.

9. One or more isolated nucleic acids encoding the antigen binding protein of any one of claims 1 to 8.

10. One or more expression vectors comprising the nucleic acid or acids of claim 9.

11. A host cell comprising the one or more expression vectors of claim 10.

12. A method for the preparation of an antigen binding protein, comprising: a. culturing the host cell of claim 11 under conditions that allow expression of the antigen binding protein; and b. recovering the antigen binding protein from the culture.

13. A pharmaceutical composition comprising the antigen binding protein of any one of claims 1 to 11 and a pharmaceutically acceptable diluent, excipient or carrier.

14. The antigen binding protein of any one of claims 1 to 8 or the pharmaceutical composition of claim 13 for use in a method of treating a disease by therapy.

15. The antigen binding protein of any one of claims 1 to 8 or the pharmaceutical composition of claim 13 for use in a method of treating an inflammatory disease characterized by the presence of elevated levels of TL1A in a patient in need thereof, wherein the method comprises administering to the patient an effective amount of the antigen binding protein or the pharmaceutical composition.

16. The antigen binding protein or the pharmaceutical composition for use according to claim 15, wherein the inflammatory disease characterized by the presence of elevated levels of TL1A is inflammatory bowel disease (IBD), Crohn's disease (CD), or ulcerative colitis (UC).

17. The antigen binding protein of any one of claims 1 to 8 or the pharmaceutical composition of claim 13 for use in a method of treating a disease in a patient in need thereof, wherein the method comprises administering to the patient an effective amount of the antigen binding protein or the pharmaceutical composition and wherein the disease is selected from the group consisting of inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome (IBS), bladder syndrome / intersticial cystitis, urinary bowel disfunction, sepsis, uveitis, encephalomyelitis, myasthenia gravis, Sjogren's syndrome (SS), scleroderma, multiple sclerosis (MS), cystic fibrosis (CF), inflammation in chronic kidney disease (CKD), psoriasis (Pso), psoriatic arthritis (PsA), ankylosing spondylitis (AS), rheumatoid arthritis (RA), juvenile rheumatoid arthritis (JRA), osteoarthritis (OA), spondyloarthropathy, primary sclerosing cholangitis, primary biliary cirrhosis, atherosclerosis, splenomegaly, inflammation in chronic kidney disease (CKD), atopic dermatitis (AD), eczematous dermatitis, contact dermatitis systemic sclerosis, systemic lupus erythematosus (SLE), lupus nephritis (LN), cutaneous lupus erythematosus, autoimmune thyroiditis, IgA nephropathy, diabetic kidney disease, antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV), minimal change disease (lipoid nephrosis), focal segmental glomerulosclerosis (FSGS), nephrogenic systemic fibrosis (NSF), nephrogenic fibrosing dermopathy, fibrosing cholestatic hepatitis, eosinophilic fasciitis (Shulman's syndrome), scleromyxedema (popular mucinosis), scleroderma, lichen sclerosusetatrophicus, inflammatory lung injury, idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease (COPD), airway hyper-responsiveness, chronic bronchitis, allergic asthma, eczema, Helicobacter pylori infection, intraabdominal adhesions and / or abscesses as results of peritoneal inflammation, nephrotic syndrome, idiopathic demyelinating polyneuropathy, Guillain-Barre syndrome, transplant rejection, organ allograft rejection, graft vs. host disease (GVHD), IgA nephropathy, diabetic kidney disease, diabetes mellitus, minimal change disease (lipoid nephrosis), nephrogenic systemic fibrosis (NSF), nephrogenic fibrosing dermopathy, fibrosing cholestatic hepatitis, eosinophilic fasciitis, (Shulman's syndrome), scleromyxedema (popular mucinosis), scleroderma, lichen sclerosusetatrophicus, Takatsuki disease (PEP syndrome), nephrotic syndrome, POEMs syndrome, Crow-Fukase syndrome, nephrotic syndrome, antineutrophil cytoplasmic antibodies, vasculitis, giant cell arteritis and multiple-myeloma-induced lytic bone disease, streptococcal cell wall (SCW)-induced arthritis, gingivitis / periodontitis, herpetic stromal keratitis, gluten-sensitive enteropathy restenosis, Kawasaki's disease, immune-mediated renal diseases, cancer, and angiogenesis.