Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
  • C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• LYMPHOTOXIN BETA RECEPTOR AGONIST BINDING PROTEINS [0001] The benefit under 35 U.S.C. ⁇ 119(e) of U.S. Provisional Application No.63/472,565, filed June 12, 2023, is hereby claimed, and the disclosure thereof is hereby incorporated by reference herein.
• FIELD [0002] The field of this invention relates to compositions and methods related to lymphotoxin beta receptor (“LT ⁇ R” or “LTBR”) binding proteins.
• LT ⁇ R lymphotoxin beta receptor
• BACKGROUND OF VARIOUS EMBODIMENTS [0003] Cancer immunotherapy enhances cancer patient survival by inducing or boosting an effective anti-tumor immune response in the patient. Immune checkpoint inhibition is one form of immunotherapy that has changed the treatment landscape for many tumors.
• This therapy works by blocking the immunosuppressive signals of immune checkpoint proteins, such as cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed cell death ligand 1 (PD-L1), that mediate tumor immune evasion.
• CTL-4 cytotoxic T lymphocyte antigen 4
• PD-1 programmed cell death protein 1
• PD-L1 programmed cell death ligand 1
• Blockade of this immunosuppressive signaling restores and/or enhances the body’s natural anti-tumor response to facilitate tumor eradication. While this form of immunotherapy has generated durable responses in many patients, it fails to generate therapeutic response in many others.
• patients with “cold tumors”, i.e., tumors characterized by a lack of infiltrating T cells and the presence of other immunosuppressive cells are typically resistant to immune checkpoint inhibitor therapy.
• a first aspect of the present disclosure is directed to an agonist lymphotoxin ⁇ receptor (LT ⁇ R) binding protein.
• the agonist LT ⁇ R binding protein of the disclosure binds one or more amino acid residues of human LT ⁇ R cysteine-rich domain 4 (CRD4) comprising amino acid residues 169-211 of SEQ ID NO: 1 and (a) does not inhibit LIGHT binding to LT ⁇ R or (b) does not inhibit LT ⁇ 1 ⁇ 2 binding to LT ⁇ R.
• Another aspect of the present disclosure is directed to a bispecific agonist LT ⁇ R binding protein.
• This bispecific binding protein comprises a LT ⁇ R binding domain, where the LT ⁇ R binding domain binds one or more amino acid residues of human LT ⁇ R CRD4 comprising amino acid residues 169- 211 of SEQ ID NO: 1; and a tumor-associated antigen binding domain.
• This bispecific binding protein agonizes LT ⁇ R activity, and (a) does not inhibit LIGHT binding to LT ⁇ R or (b) does not inhibit LT ⁇ 1 ⁇ 2 binding to LT ⁇ R.
• Other aspects of the present disclosure are directed to polynucleotides encoding the agonist LT ⁇ R binding proteins and bispecific LT ⁇ R binding proteins of the disclosure; vectors comprising these polynucleotides; and host cells comprising these vectors.
• Additional aspects of the present disclosure are directed to pharmaceutical compositions comprising an agonist LT ⁇ R binding protein, a bispecific agonist LT ⁇ R binding protein, or a polynucleotide or vector encoding the same as described herein.
• Another aspect of the disclosure is directed to methods of treating cancer in a subject that involve administering, to the subject having cancer, an agonist LT ⁇ R binding protein.
• the agonist LT ⁇ R binding protein used in these methods binds one or more amino acid residues of human LT ⁇ R cysteine-rich domain 4 (CRD4) comprising amino acid residues 169-211 of SEQ ID NO: 1 and (a) does not inhibit LIGHT binding to LT ⁇ R or (b) does not inhibit LT ⁇ 1 ⁇ 2 binding to LT ⁇ R [0009]
• Another aspect of the disclosure is directed to methods of treating cancer in a subject that involve administering, to the subject having cancer, a bispecific agonist LT ⁇ R binding protein.
• This bispecific agonist LT ⁇ R binding protein comprises a LT ⁇ R binding domain and a tumor-associated antigen binding domain.
• This LT ⁇ R binding domain binds one or more amino acid residues of human LT ⁇ R cysteine-rich domain (CRD4) comprising amino acid residues 169-211 of SEQ ID NO: 1.
• the bispecific LT ⁇ R binding protein further comprises a tumor-associated antigen binding domain.
• the bispecific LT ⁇ R binding protein agonizes LT ⁇ R activity and (a) does not inhibit LIGHT binding to LT ⁇ R or (b) does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R.
• LT ⁇ R agonism as an anti-tumor therapy was suggested more than 20 years ago (see e.g., Browning et al., “Signaling through the Lymphotoxin ⁇ Receptor Induces the Death of Some Adenocarcinoma Tumor Lines,” J. Exp. Med. 183:867 (1996)).
• Browning et al. “Signaling through the Lymphotoxin ⁇ Receptor Induces the Death of Some Adenocarcinoma Tumor Lines,” J. Exp. Med. 183:867 (1996).
• LT ⁇ R antibody has progressed to a Phase I clinical trial, and that trial ended prematurely.
• LT ⁇ R is ubiquitously expressed on various cells throughout the body and plays a critical role in the formation and maintenance of lymphoid organs and various immune cell populations.
• an LT ⁇ R antibody to be therapeutically effective and safe, it must selectively activate LT ⁇ R signaling in the tumor microenvironment without disrupting normal LT ⁇ R signaling outside of the tumor microenvironment.
• targeting agonism to the tumor environment can be achieved using a bispecific modality that provides cross-linking specific LT ⁇ R activation; if not properly designed, the cross-linking dependent agonist will act as an antagonist when bound to LT ⁇ R in a target-independent manner.
• antagonizing its activity presents serious safety concerns.
• the agonist LT ⁇ R binding proteins disclosed herein overcome this problem by exerting agonist activity in a cross-linking target-dependent manner without antagonizing normal LT ⁇ R signaling activity. This was achieved by specifically selecting for and engineering LT ⁇ R binding proteins that agonize LT ⁇ R in a cross-linking specific manner when bound to the cysteine rich domain 4 (CRD4) of LT ⁇ R without blocking endogenous LT ⁇ R ligand (i.e., LIGHT or LT ⁇ 1 ⁇ 2) binding and signaling activity.
• CCD4 cysteine rich domain 4
• TAA tumor associated antigen
• FIG.1A the total number of T cells (CD3 + T cells) infiltrating the tumor tissue increased at the 3 mg/kg dose of the human EpCAM x murine LT ⁇ R bispecific antibody relative to administration of an isotype control antibody.
• FIG.1B the total number of B cells (CD19 + B cells) infiltrating the tumor tissue increased with increasing doses (0.3, 3, and 30 mg/kg) of the huEpCAM x muLT ⁇ R BsAb relative to administration of an isotype control antibody.
• FIG. 2 shows tumor associated antigen (TAA)-targeted LT ⁇ R agonism induces HEV formation in the human EpCAM-expressing B16F10 melanoma tumor model.
• FIG. 2 shows the immunohistochemical analysis of HEV formation in tumor tissue following isotype control treatment (top image) and 3mg/kg of huEpCAM-muLT ⁇ R bispecific antibody treatment (bottom image) as described in Example 1 herein.
• FIG. 3 shows tumor associated antigen (TAA)-targeted LT ⁇ R agonism increased T cell infiltration in a human KPC pancreatic tumor model.
• FIG.3 shows T cells counts per gram of tumor tissue increased following administration of two different tool huLRRC15 x muLT ⁇ R BsAb to mice bearing a KPC M5 tumor.
• the huLRRC15 x muLT ⁇ R BsAb was administered twice a week for 3 doses before tumors were assessed with flow cytometry.
• One-way ANOVA with Dunnett’s multiple comparison test showed no significant difference against isotype, but treatments do show an upwards trend of T cell infiltration. Mean and SEM are represented in the graph.
• FIG. 4 shows tumor associated antigen (TAA)-targeted LT ⁇ R agonism inhibited tumor growth in a human KPC pancreatic tumor model.
• TAA tumor associated antigen
• the graph of FIG.4 shows mean tumor volume of KPC M5 pancreatic tumor model treated with PD-1 antibody, huLRRC15 x muLT ⁇ R BsAb, or the combination of these antibodies.
• huLRRC15 x muLT ⁇ R BsAb monotherapy ( ⁇ ) produced a greater effect than PD-1 monotherapy ( ⁇ ).
• the combination of huLRRC15 x muLT ⁇ R BsAb and PD-1 Ab did show greater TGI, but the difference between the two treatment groups was not statistically significant.
• Two-way ANOVA with Tukey’s multiple comparison test demonstrated significance against isotype for huLRRC15 x muLTbR BsAb and huLRRC15 x muLTbR BsAb + PD-1 Ab (p ⁇ 0.0001).
• FIG.5 contains Tables A and B summarizing characterization data of the LT ⁇ R antibodies identified in the first round of hybridoma screening.
• Table A contains functional potency values (EC 50 ) and binding data, i.e., LT ⁇ R antibody binding to TNRFSF1 and TNRFSF2, mouse LT ⁇ R, and cynomolgus LT ⁇ R. Data is presented as GeoMean fold over mock transfected cells.
• Table A also shows the percent inhibition of human LIGHT or human LT ⁇ 1 ⁇ 2 ligand binding to human LT ⁇ R in the presence of the indicated LT ⁇ R antibody as assessed using the single-point FACS bead-based receptor-ligand assay as described in Example 4.6.
• Table B shows LT ⁇ R antibody binding affinity measurements (ka, kd, and KD) to human and cynomolgus LT ⁇ R.
• FIG. 6 identifies the regions of the LT ⁇ R extracellular domain (ECD) (SEQ ID NO: 4) involved in the binding interaction with LT ⁇ R antibody, LIBC No.218990 (Bio. Reg.
• FIG. 7 identifies the regions of the LT ⁇ R ECD (SEQ ID NO: 4) involved in the binding interaction with LT ⁇ R antibody, LIBC No.218944 (Bio. Reg No.19321 / Well ID 31A3), as determined by HDX MS (see Example 6.1.1).
• FIG. 1 Shown are deuterium uptake graphs of peptides corresponding to residues 3–9, 20–29, and 38–47 of the LT ⁇ R ECD alone or in complex with the antibody. Protection from deuterium exchange in the complex derived peptides relative to non-complexed peptides indicates that one or more residues within these regions, i.e., residues 4-9, 21-29, and 39-47, of the LT ⁇ R ECD are involved in the binding interaction (note that the N-terminal residues of a peptide does not have an amide hydrogen for measure and is therefore excluded in reporting epitope regions). [0019] FIG.
• FIG. 9 identifies the regions of the LT ⁇ R ECD (SEQ ID NO: 4) involved in the binding interaction with LT ⁇ R antibody, LIBC No. 219081 (Bio Reg. No. 19325 / Well ID 41B2), as determined by HDX MS (see Example 6.1.1).
• FIG.10 identifies the regions of the LT ⁇ R ECD (SEQ ID NO: 4) involved in the binding interaction with LT ⁇ R antibody, LIBC No.219097 (Bio. Reg.
• LT ⁇ R ECD SEQ ID NO: 4
• LIBC No.218979 Bio. Reg. No.19319 / Well ID 23E9
• FIG.12 identifies the regions of the LT ⁇ R ECD (SEQ ID NO: 4) involved in the binding interaction with LT ⁇ R antibody, LIBC No.219051 (Bio. Reg. No.19323 / Well ID 35F5), as determined by HDX MS (see Example 6.1.2).
• FIG.13 identifies the variable heavy (VH) and variable light (VL) domain regions of LIBC No.
• FIGs.14A–14B identify the variable heavy (VH) and variable light (VL) domain regions of LIBC No. 218944 (19321/31A3) involved in the binding interaction with LT ⁇ R.
• FIG. 14A Shown are deuterium uptake graphs of VH (FIG. 14A) and VL (FIG. 14B) domain peptides alone or when bound to LT ⁇ R. Protection from deuterium exchange in the complexed peptides relative to non-complexed peptides indicates that one or more CDR residues within these regions of the antibody are involved in the binding interaction. The regions including these one or more residues are boxed in the shown VH (FIG.14A) and VL (FIG.14B) sequences and the three CDR regions of each variable domain are underlined. [0026] FIG.15 identifies the variable heavy (VH) and variable light (VL) domain regions of LIBC No.
• FIGs.16A–16B identify the variable heavy (VH) and variable light (VL) domain regions of LIBC No.
• VH VH
• VL VG. 16B domain peptides alone or when bound to LT ⁇ R. Protection from deuterium exchange in the complexed peptides relative to non-complexed peptides indicates that one or more CDR residues within these regions of the antibody are involved in the binding interaction. The regions including these one or more residues are boxed in the shown VH (FIG.16A) and VL (FIG.16B) sequences and the three CDR regions of each variable domain are underlined.
• FIGs.17A–17B identify the variable heavy (VH) and variable light (VL) domain regions of LIBC No. 219097 (19326/43D9) involved in the binding interaction with LT ⁇ R. Shown are deuterium uptake graphs of VH (FIG. 17A) and VL (FIG. 17B) domain peptides alone or when bound to LT ⁇ R. Protection from deuterium exchange in the complexed peptides relative to non-complexed peptides indicates that one or more CDR residues within these regions of the antibody are involved in the binding interaction.
• FIGs.18A–18B identify the variable heavy (VH) and variable light (VL) domain regions of LIBC No.218979 (Bio. Reg. No.19319 / Well ID 23E9) involved in the binding interaction with LT ⁇ R. Shown are deuterium uptake graphs of VH (FIG.18A) and VL (FIG.18B) domain peptides alone or when bound to LT ⁇ R.
• FIGs.19A–19B identify the variable heavy (VH) and variable light (VL) domain regions of LIBC No.219051 (Bio. Reg. No.19323 / Well ID 35F5) involved in the binding interaction with LT ⁇ R.
• FIG.19A Shown are deuterium uptake graphs of VH (FIG.19A) and VL (FIG.19B) domain peptides alone or when bound to LT ⁇ R. Protection from deuterium exchange in the complexed peptides relative to non-complexed peptides indicates that one or more CDR residues within these regions of the antibody are involved in the binding interaction. The regions including these one or more residues are boxed in the shown VH (FIG. 19A) and VL (FIG.19B) sequences and the three CDR regions of each variable domain are underlined. [0031] FIG.
• FIG. 20 is a graph showing potency of huLT ⁇ R x huCLDN6 bispecific antibodies (PUR149204-4, PUR149205-4, PUR149212-5, and PUR149213-4). Potency was measured by IL-8 release from LT ⁇ R expressing human melanoma cells co-cultured with CLDN6 expressing hamster ovarian cells (CHOs) in the presence of an increasing concentration of the indicated huLT ⁇ R x huCLDN6 bispecific antibody.
• the “signal” of the y-axis represents relative IL-8 release calculated by the ratio of signal at 665 nm to signal at 615 nm.
• FIG. 22 is a graph showing potency of huLT ⁇ R x huMUC17 bispecific antibodies (PUR149211-4, PUR149218-4, and PUR149219-4). Potency was as measured by IL-8 release from LT ⁇ R expressing human melanoma cells co-cultured with MUC17 expressing CHO cells in the presence of an increasing concentration of the indicated huLT ⁇ R x huMUC17 bispecific antibody.
• the “signal” of the y- axis represents relative IL-8 release calculated by the ratio of signal at 665 nm to signal at 615 nm.
• FIGs.24A-24C are graphs showing LIGHT (FIG.24A) and LT ⁇ 1 ⁇ 2 (FIG.24B) inhibition in the presence of increasing concentrations of various LT ⁇ R antibodies described herein.
• FIG. 24C is a graph showing anti-LT ⁇ R antibody binding to LT ⁇ R expressed by HEK293 cells to confirm that non-ligand blocking activity was not due to non-receptor binding by the tested antibody.
• FIGs.25A-25C are graphs showing LIGHT (FIG.25A) and LT ⁇ 1 ⁇ 2 (FIG.25B) inhibition in the presence of increasing concentrations of various LT ⁇ R-LRRC15 bispecific as described herein.
• FIG. 27 provides a table summarizing the additional agonist LT ⁇ R CRD4 binding, non- ligand blocking antibodies identified in the rescreening of XenoMouse ® hybridoma pools.
• Non-ligand blocking activity of the antibodies was assessed using the cell-based assay described in Example 9.3 and Caterra assay as described in Example 9.4.
• DETAILED DESCRIPTION OF THE VARIOUS EMBODIMENTS [0039]
• the present disclosure is directed to protein molecules that bind to and agonize the lymphotoxin-beta receptor (LT ⁇ R).
• the present disclosure provides agonist LT ⁇ R binding proteins that bind one or more amino acid residues of human LT ⁇ R cysteine-rich domain 4 (CRD4) comprising amino acid residues 169-211 of SEQ ID NO: 1 and (a) do not inhibit LIGHT binding to LT ⁇ R or (b) do not inhibit LT ⁇ 1 ⁇ 2 binding to LT ⁇ R.
• the present disclosure also provides bispecific agonist LT ⁇ R binding proteins comprising an LT ⁇ R binding domain that binds one or more amino acid residues of human LT ⁇ R CRD4 and (a) does not inhibit LIGHT binding to LT ⁇ R or (b) does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R, and a tumor-associated antigen binding domain.
• nucleic acid molecules comprising a sequence of polynucleotides that encode all or a portion of the agonist LT ⁇ R binding proteins disclosed herein.
• the present disclosure further provides vectors and plasmids comprising such nucleic acids, and cells or cell lines comprising such nucleic acids and/or vectors and plasmids.
• the provided methods further include, for example, methods of making, identifying, and isolating agonist LT ⁇ R binding proteins, and methods for administering, to a human subject, an agonist LT ⁇ R binding protein of the disclosure.
• sequence identity refers to the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. The identity between two sequences is preferably defined by assessing their identity across the whole length of the sequence as identified herein.
• the percentage of sequence identity between a first sequence and a second sequence may be calculated using methods known by a person skilled in the art, e.g., by dividing the number of residues in the first sequence that are identical to the residues at the corresponding positions in the second sequence by the total number of residues in the first sequence and multiplying by 100% or by using a known computer algorithm for sequence alignment such as NCBI Blast, e.g., BLASTN and BLASTP (Altschul, S. F. et al, J. Mol. Biol.
• Binding affinity generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a LT ⁇ R binding protein as describe herein) and its binding partner (e.g., human LT ⁇ R).
• binding affinity refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., LT ⁇ R binding protein and LT ⁇ R).
• the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD).
• KD dissociation constant
• Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (K D ) and equilibrium association constant (K A ).
• K D equilibrium dissociation constant
• K A equilibrium association constant
• Binding affinity can be determined using a variety of techniques as described herein and as known in the art, for example but not limited to, equilibrium methods, e.g., enzyme-linked immunosorbent assay (ELISA), KinExA (see, e.g., Rathanaswami et al.
• ELISA enzyme-linked immunosorbent assay
• KinExA see, e.g., Rathanaswami et al.
• Affinity can also be determined using other methods such as indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
• binding protein refers to any one of many forms of binding proteins known in the art.
• binding proteins of the present disclosure encompass (i) full-length immunoglobulin molecules, i.e., antibodies, (ii) epitope binding fragments of antibodies, and (iii) antibody derivatives, e.g. multi-specific antibodies.
• an antibody refers to a protein having a conventional immunoglobulin format, comprising heavy and light chains, and comprising variable and constant regions.
• an antibody may be an IgG which is a “Y-shaped” structure of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa).
• An antibody has a variable region and a constant region.
• the variable region is generally about 100-110 or more amino acids, comprises three complementarity determining regions (CDRs) and is primarily responsible for antigen recognition.
• CDRs of antibodies have been described in the art. Briefly, in an antibody scaffold, the CDRs are embedded within a framework in the heavy and light chain variable region where they constitute the regions largely responsible for antigen binding and recognition.
• a variable region typically comprises at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol.
• Antibodies can comprise any constant region known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. In the present invention, antibodies are of the IgG isotype.
• IgG has several subclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4.
• the light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a human kappa- or lambda-type light chain constant region [0057]
• An “epitope binding fragment” of an antibody encompasses any polypeptide fragment, region, portion, or domain of a full-length antibody that exhibits the same or substantially similar binding properties of the full-length antibody and can be obtained, for example, by protease cleavage of an intact parental antibody.
• an “antibody derivative” is a protein or polypeptide that contains at least one epitope binding domain of an antibody and is typically formed using recombinant techniques or via chemical modification of a parent antibody or portion thereof.
• An antibody derivative comprises an amino acid sequence that is substantially similar to the amino acid sequence of one or more parental antibodies or relevant portions thereof.
• Exemplary antibody derivatives of the present disclosure include multispecific binding proteins, e.g., a bispecific binding protein.
• epitope refers to the site or portion of a protein to which an antibody binds.
• An epitope can be composed of either or both contiguous amino acid residues or discontiguous amino acid residues that form a conformational spatial unit.
• paratope refers to the portion of a binding protein, e.g., an antibody, that binds to the epitope of a protein target.
• a paratope can be linear in nature or can be discontinuous, formed by a spatial relationship between non-contiguous amino acid residues of an antibody rather than a linear series of amino acids.
• light chain paratope amino acid residues refer to antibody light chain and/or heavy chain residues involved in the binding interaction with the protein target.
• the paratope may comprise amino acid residues of a single heavy chain CDR, e.g., heavy chain CDR3, or a combination of amino acid residues from the heavy chain CDRs (HCDR1, CDR2, HCDR3) and light chain CDRs (LCDR1, LCDR2, and LCDR3).
• HCDR1, CDR2, HCDR3 a combination of amino acid residues from the heavy chain CDRs
• LCDR1, LCDR2, and LCDR3 light chain CDRs
• LT ⁇ R binding proteins agonist lymphotoxin beta receptor (LT ⁇ R) binding proteins.
• LT ⁇ R also known as tumor necrosis factor (TNF) receptor superfamily member 3 and tumor necrosis factor receptor type III (TNFR-III)
• TNF tumor necrosis factor
• TNFR-III tumor necrosis factor receptor type III
• LT ⁇ R is a member of the TNF family that is expressed on most cell types, including fibroblasts, epithelial cells, monocytes, dendritic cells, and cells of the myeloid lineage.
• LT ⁇ R is not expressed by T or B lymphocytes.
• LT ⁇ R is a receptor for two TNF family cytokine ligands.
• the first of these LT ⁇ R ligands is a heterotrimer of lymphotoxin ⁇ and lymphotoxin ⁇ subunits known as LT ⁇ 1 ⁇ 2.
• the second ligand of LT ⁇ R is a homotrimer of LIGHT (Tumor necrosis factor ligand superfamily member 14 (TNFSF14)).
• LIGHT Tumor necrosis factor ligand superfamily member 14
• LT ⁇ 1 ⁇ 2 and LIGHT binding to LT ⁇ R initiates a signal transduction pathway that involves primarily the NF ⁇ B pathway.
• LT ⁇ R signaling is involved in lymph node (LN) organogenesis during development and the maintenance of lymphoid organs and various immune cells, including neutrophils, natural killer (NK) cells and invariant natural killer T (iNKT) cells in adulthood.
• LN lymph node
• NK natural killer
• iNKT invariant natural killer T
• LT ⁇ R also plays a critical role in the formation of LN-like cell clusters known as tertiary lymphoid structures (TLS) in non-lymph organs and the induction of genes critical for T cell migration across the endothelium, both of which are key to facilitating an effective anti-tumor response.
• TLS tertiary lymphoid structures
• Human LT ⁇ R is a 61 kDa transmembrane protein having an amino acid sequence of SEQ ID NO: 1 (isoform 1) or SEQ ID NO: 2 (isoform 2) as shown below.
• the LT ⁇ R binding proteins as described herein bind to human LT ⁇ R.
• the LT ⁇ R binding proteins of the present disclosure bind to one or more epitopes within the extracellular domain of human LT ⁇ R (comprising amino acid residues 31-227 of SEQ ID NO: 1), which contains four cysteine-rich domains (i.e., CRD1–CRD4).
• the LT ⁇ R binding proteins described herein also bind to cynomolgus (cyno) LT ⁇ R.
• the amino acid sequence of cyno LT ⁇ R is provided below (SEQ ID NO: 3).
• the LT ⁇ R binding proteins described herein do not bind to murine LT ⁇ R.
• the LT ⁇ R binding proteins described herein bind to human LT ⁇ R and cynomolgus LT ⁇ R but do not bind to murine LT ⁇ R.
• the LT ⁇ R binding proteins are agonist LT ⁇ R binding proteins, for example, an agonist LT ⁇ R antibody, an agonist LT ⁇ R epitope binding fragment of an antibody, or an agonist LT ⁇ R antibody derivative (e.g., an agonist LT ⁇ R bispecific binding protein).
• An “agonist LT ⁇ R binding protein” as used herein is an LT ⁇ R binding molecule that induces LT ⁇ R-mediated signaling either directly, by binding to the receptor, or indirectly, via binding to the receptor and inducing its higher order clustering at the cell surface, e.g., by use of cross-linking antibodies. Binding of a LT ⁇ R binding protein of the disclosure to LT ⁇ R induces NF ⁇ B signaling, gene expression associated with T cell migration across the endothelium, and/or TLS cluster formation. Assays for measuring LT ⁇ R activation and/or the induction of LT ⁇ R signaling by a binding protein of the present disclosure are known in the art and are described herein.
• LT ⁇ R agonism is measured by assaying interleukin-8 (IL8) release from LT ⁇ R expressing cells following incubation with an agonist LT ⁇ R binding protein as described in Example 7 herein.
• An increase in the level of cellular IL8 release in the presence of an LT ⁇ R binding protein of the present disclosure as compared to its absence is an indication that the LT ⁇ R binding protein functions as an agonist LT ⁇ R binding protein.
• LT ⁇ R agonism is measured by assaying transactivation of endothelial cells.
• LT ⁇ R-mediated endothelial cell transactivation can be measured by assaying the endothelial cell expression of adhesion molecules, such as vascular cell adhesion protein 1 (VCAM1; also known as CD106) and/or intracellular adhesion molecule 1 (ICAM; also known as CD54), and/or the expression or secretion of inflammatory chemokines, such as, e.g., CCL5, CCL2, and CXCL10.
• VCAM1 vascular cell adhesion protein 1
• IAM intracellular adhesion molecule 1
• An increase in endothelial cell VCAM or ICAM expression in the presence of an agonist LT ⁇ R binding protein as compared to in its absence is an indication that the LT ⁇ R binding protein functions as an agonist LT ⁇ R binding protein.
• an increase in endothelial cell expression or secretion of CCL5, CCL2, and/or CXCL10 in the presence of an LT ⁇ R binding protein as compared to in its absence is an indication that the LT ⁇ R binding protein functions as an agonist LT ⁇ R binding protein.
• the agonist activity of an LT ⁇ R binding protein described herein induces at least a 10% increase in LT ⁇ R signaling activity, as measured by, e.g., IL8 release or endothelial cell transactivation, relative to LT ⁇ R signaling activity in the absence of the agonist LT ⁇ R binding protein.
• the LT ⁇ R binding protein described herein increases LT ⁇ R signaling activity by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% relative to the level of LT ⁇ R signaling in the absence of the agonist LT ⁇ R binding protein.
• the LT ⁇ R binding protein described herein increases LT ⁇ R signaling activity by at least 100% relative to the level of LT ⁇ R signaling in the absence of the agonist LT ⁇ R binding protein.
• the agonist activity of the LT ⁇ R binding proteins described herein is measured using one or more of the in vitro functional endpoints as described above and the activity is expressed as a half maximal effective concentration (EC 50 ).
• the EC 50 of the LT ⁇ R binding proteins described herein is within about 1 pM to about 500 pM, about 1 pM to about 250 pM, about 1 pM to about 200 pM, about 1 pM to about 150 pM, about 1 pM to about 125 pM, about 1 to about 100 pM, about 1 pM to about 90 pM about 1 to about 80 pM about 1 pM to about 70 pM, about 1 pM to about 60 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 to about 30 pM, about 1 to 200 pM, about 10 to about 200 pM, about 20 pM to about 200 pM, about
• the EC 50 of agonist LT ⁇ R binding protein mediated IL-8 release as described herein is about 10 pM to about 200 pM, about 20 pM to about 150 pM, or about 30 pM to about 140 pM.
• the LT ⁇ R binding proteins as described herein bind to and agonize human LT ⁇ R while allowing endogenous LT ⁇ R ligand binding to the receptor and resulting activity to occur. As described supra, LT ⁇ R binds at least two different endogenous ligands, i.e., LIGHT and LT ⁇ 1 ⁇ 2.
• an LT ⁇ R binding protein of the present disclosure When an LT ⁇ R binding protein of the present disclosure is bound to its corresponding epitope of LT ⁇ R, at least one of LIGHT or LT ⁇ 1 ⁇ 2 can also bind to LT ⁇ R to initiate endogenous ligand-mediated signaling activity.
• binding of an LT ⁇ R binding protein described herein does not inhibit, prevent, or preclude LT ⁇ 1 ⁇ 2 binding to LT ⁇ R.
• binding of an LT ⁇ R binding protein described herein does not inhibit, prevent, or preclude LIGHT binding to LT ⁇ R.
• binding of an LT ⁇ R binding protein described herein does not inhibit, prevent, or preclude either LIGHT binding to LT ⁇ R or LT ⁇ 1 ⁇ 2 binding to LT ⁇ R.
• non- ligand blocking activity is a beneficial feature of the agonist LT ⁇ R binding proteins of the present disclosure because it maximizes safety upon administration of the binding protein to, e.g., a patient.
• the LT ⁇ R binding proteins described herein preferentially agonize LT ⁇ R in a cross-linking dependent manner, i.e., the binding molecule must bind to LT ⁇ R and another protein (e.g., an Fc receptor or a targeting protein) to induce LT ⁇ R activation.
• non-ligand blocking activity of an agonist LT ⁇ R binding protein as described herein is characterized by the percent inhibition that its binding has on LIGHT and LT ⁇ 1 ⁇ 2 binding to LT ⁇ R.
• LT ⁇ R binding by a binding protein of the present disclosure results in negligible inhibition or blocking of LIGHT binding ( ⁇ 20% inhibition) to LT ⁇ R or LT ⁇ 1 ⁇ 2 binding ( ⁇ 20% inhibition) to LT ⁇ R as measured by the cell based receptor-ligand assay described infra (see also Example 8).
• LT ⁇ R binding by a binding protein as described herein inhibits ⁇ 20% of LIGHT binding to LT ⁇ R, ⁇ 20% LT ⁇ 1 ⁇ 2 binding to LT ⁇ R, or ⁇ 20% of LIGHT and LT ⁇ 1 ⁇ 2 binding to LT ⁇ R as measured by the cell-based receptor-ligand assay described herein.
• the LT ⁇ R binding protein as described herein inhibits ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% of LIGHT binding to LT ⁇ R or LT ⁇ 1 ⁇ 2 binding to LT ⁇ R.
• LT ⁇ R binding by a binding protein as described herein inhibits ⁇ 15% of LIGHT binding to LT ⁇ R or inhibits ⁇ 15% of LT ⁇ 1 ⁇ 2 binding to LT ⁇ R as measured by the cell-based receptor ligand assay described herein and exemplified in Example 8.2.
• LT ⁇ R binding by a binding protein as described herein inhibits ⁇ 15% of LIGHT binding to LT ⁇ R and inhibits ⁇ 15% of LT ⁇ 1 ⁇ 2 binding to LT ⁇ R as measured by the cell-based receptor ligand assay described herein and exemplified in Example 8.2. In one embodiment, LT ⁇ R binding by a binding protein described herein inhibits ⁇ 10% of LIGHT binding to LT ⁇ R or inhibits ⁇ 10% LT ⁇ 1 ⁇ 2 binding to LT ⁇ R as measured by the cell-based receptor ligand assay described herein.
• LT ⁇ R binding by a binding protein described herein inhibits ⁇ 10% of LIGHT binding to LT ⁇ R and inhibits ⁇ 10% LT ⁇ 1 ⁇ 2 binding to LT ⁇ R as measured by the cell-based receptor ligand assay described herein. In one embodiment, LT ⁇ R binding by a binding protein described herein inhibits ⁇ 5% of LIGHT binding to LT ⁇ R or inhibits ⁇ 5% LT ⁇ 1 ⁇ 2 binding to LT ⁇ R as measured by the cell-based receptor ligand assay described herein.
• LT ⁇ R binding by a binding protein described herein inhibits ⁇ 5% of LIGHT binding to LT ⁇ R and inhibits ⁇ 5% LT ⁇ 1 ⁇ 2 binding to LT ⁇ R as measured by the cell-based receptor ligand assay described herein.
• LT ⁇ R binding by a binding protein described herein inhibits 0% of LIGHT binding to LT ⁇ R or inhibits 0% of LT ⁇ 1 ⁇ 2 binding to LT ⁇ R as measured by the cell- based receptor ligand assay described herein.
• LT ⁇ R binding by a binding protein described herein inhibits 0% of LIGHT binding to LT ⁇ R and inhibits 0% of LT ⁇ 1 ⁇ 2 binding to LT ⁇ R as measured by the cell-based receptor ligand assay described herein.
• Exemplary LT ⁇ R binding proteins described herein that do not inhibit LIGHT binding to LT ⁇ R and do not inhibit LT ⁇ 1 ⁇ 2 binding to LT ⁇ R include, without limitation, the agonist LT ⁇ R binding proteins that bind to the CRD4 domain of LT ⁇ R.
• non-ligand blocking activity of an agonist LT ⁇ R binding protein as described herein is characterized by the percentage of endogenous LT ⁇ R ligand binding that occurs in the presence of bound agonist LT ⁇ R binding protein.
• LT ⁇ R binding by a binding protein as described herein allows at least 80% of endogenous LT ⁇ R ligand (i.e., LIGHT or LT ⁇ 1 ⁇ 2) binding to occur as measured by the cell-based receptor-ligand assay described herein (see Example 8.2).
• LT ⁇ R binding by a binding protein as described herein allows at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of LIGHT binding to LT ⁇ R or LT ⁇ 1 ⁇ 2 binding to LT ⁇ R to occur.
• LT ⁇ R binding by a binding protein as described herein allows at least 90% of LIGHT binding to LT ⁇ R to occur or at least 90% of LT ⁇ 1 ⁇ 2 ligand binding to LT ⁇ R to occur as measured by the cell based receptor-ligand assay described herein and exemplified in Example 8.
• LT ⁇ R binding by a binding protein as described herein allows at least 90% of LIGHT binding to LT ⁇ R to occur and at least 90% of LT ⁇ 1 ⁇ 2 ligand binding to LT ⁇ R to occur as measured by the cell based receptor-ligand assay described herein and exemplified in Example 8. In one embodiment, LT ⁇ R binding by a binding protein as described herein allows at least 95% of LIGHT binding to LT ⁇ R to occur or at least 95% of LT ⁇ 1 ⁇ 2 ligand binding to LT ⁇ R to occur as measured by the cell based receptor-ligand assay described herein and exemplified in Example 8.
• LT ⁇ R binding by a binding protein as described herein allows at least 95% of LIGHT binding to LT ⁇ R to occur and at least 95% of LT ⁇ 1 ⁇ 2 ligand binding to LT ⁇ R to occur as measured by the cell based receptor-ligand assay described herein and exemplified in Example 8.
• LT ⁇ R binding by a binding protein as described herein allows 100% of LIGHT binding to LT ⁇ R occur or 100% of LT ⁇ 1 ⁇ 2 ligand binding to LT ⁇ R to occur as measured by the cell based receptor-ligand based assay described in Example 8.
• LT ⁇ R binding by a binding protein as described herein allows 100% of LIGHT binding to LT ⁇ R occur and 100% of LT ⁇ 1 ⁇ 2 ligand binding to LT ⁇ R to occur as measured by the cell based receptor-ligand based assay described in Example 8.
• Exemplary agonist LT ⁇ R binding proteins described herein that allow 100% of LIGHT binding to LT ⁇ R and 100% LT ⁇ 1 ⁇ 2 binding to LT ⁇ R are the agonist LT ⁇ R binding proteins that bind to the CRD4 domain of LT ⁇ R.
• Non-ligand blocking activity of the agonist LT ⁇ R binding proteins described herein is measured and quantified using a cell-based receptor-ligand binding assay.
• This assay measures the percent inhibition of LIGHT and/or LT ⁇ 1 ⁇ 2 binding to LT ⁇ R in the presence of the agonist LT ⁇ R binding protein of the disclosure (see Example 8).
• This cell-based receptor-ligand binding assay involves incubating LT ⁇ R expressing cells with cell culture media containing the agonist LT ⁇ R binding protein for 1 hour at 4°C to allow the agonist LT ⁇ R binding protein to bind LT ⁇ R expressed on the cells.
• Suitable LT ⁇ R expressing cells include any cell type that naturally expresses LT ⁇ R, e.g., endothelial cells, macrophages, natural killer cells, etc., as well as cells engineered to express LT ⁇ R, e.g., a cell line (such as CHO cells, HEK 293 cells, melanoma cells, etc.) transfected with a LT ⁇ R expression vector.
• a cell line such as CHO cells, HEK 293 cells, melanoma cells, etc.
• the concentration of agonist LT ⁇ R binding protein in the cell culture media is at a concentration effective to saturate binding of all LT ⁇ R expressed by the LT ⁇ R expressing cells.
• the concentration of agonist LT ⁇ R binding protein in the cell culture media is at a concentration of 5-30nM to saturate binding of all LT ⁇ R expressed by the LT ⁇ R expressing cells.
• the assay further involves interacting the LT ⁇ R expressing cells, after incubating with the agonist LT ⁇ R binding protein, with a detectable LIGHT ligand, a detectable LT ⁇ 1 ⁇ 2 ligand, or both (i.e., detectable LIGHT and LT ⁇ 1 ⁇ 2 ligands) under conditions effective for the ligands to bind to LT ⁇ R.
• LIGHT and LT ⁇ 1 ⁇ 2 ligands are rendered detectable by directly coupling them to a detectable label, e.g., a fluorescent label or tag.
• Binding of the detectable LIGHT and/or LT ⁇ 1 ⁇ 2 ligands to the LT ⁇ R expressing cells in the presence of the agonist LT ⁇ R binding protein is detected using suitable means (e.g., Fluorescence-activated cell sorting).
• suitable means e.g., Fluorescence-activated cell sorting.
• a quantitative assessment of ligand binding inhibition is determined by comparing the level of LIGHT and/or LT ⁇ 1 ⁇ binding to LT ⁇ R in the presence of an agonist LT ⁇ R binding protein to the corresponding level of LIGHT and/or LT ⁇ 1 ⁇ binding to LT ⁇ R in the absence of the agonist LT ⁇ R binding protein.
• Agonist LT ⁇ R binding proteins that minimally inhibit ( ⁇ 20% inhibition) or do not inhibit (0% inhibition) LIGHT or LT ⁇ 1 ⁇ 2 ligand binding to LT ⁇ R are identified as suitable for use in the methods described herein.
• the binding of the detectable LIGHT and/or LT ⁇ 1 ⁇ 2 ligands to LT ⁇ R expressing cells in the presence or absence of the LT ⁇ R binding protein is detected via FACs on a flow cytometry machine.
• Exemplary agonist LT ⁇ R binding proteins described herein exhibit the desired functional properties (e.g., cyno and human LT ⁇ R binding and non-ligand blocking activity) as a result, in part, of the epitope of LT ⁇ R bound by the agonist LT ⁇ R binding protein.
• the LT ⁇ R binding proteins as disclosed herein bind to an epitope of LT ⁇ R comprising one or more segments of a cysteine rich domain (CRD) of the extracellular domain, i.e., one or more residues of CRD1, CRD2, CRD3, or CRD4.
• CRD cysteine rich domain
• the human LT ⁇ R extracellular domain is provided below as SEQ ID NO: 4.
• the agonist LT ⁇ R binding protein of the present disclosure binds to an epitope comprising residues of CRD1 of LT ⁇ R.
• CRD1 of LT ⁇ R comprises residues 12–51 of SEQ ID NO: 4, which corresponds to residues 42-81 of the full-length LT ⁇ R of SEQ ID NO: 1.
• the LT ⁇ R binding protein of the present disclosure binds to an epitope comprising residues of LT ⁇ R CRD2.
• LT ⁇ R CRD2 comprises residues 52–94 of SEQ ID NO: 4, which corresponds to residues 82-124 of the full-length LT ⁇ R of SEQ ID NO: 1.
• the LT ⁇ R binding protein of the present disclosure binds to an epitope comprising residues of LT ⁇ R CRD3.
• LT ⁇ R CRD3 comprises residues 95–138 of SEQ ID NO: 4, which corresponds to residues 125-168 of the full-length LT ⁇ R of SEQ ID NO: 1.
• the LT ⁇ R binding protein of the present disclosure binds to an epitope comprising residues of LT ⁇ R CRD4.
• LT ⁇ R CRD4 comprises residues 139–181 of SEQ ID NO: 4, which corresponds to residues 168–211 of the full-length LT ⁇ R amino acid sequence of SEQ ID NO: 1.
• an agonist LT ⁇ R binding protein of the present disclosure binds to an epitope that comprises residues of the extracellular CRD2 and CRD3 of human LT ⁇ R.
• the agonist LT ⁇ R binding protein binds to an epitope within CRD2 comprising one or more residues at positions 56–64 of SEQ ID NO: 4 and binds to an epitope spanning CRD2 and CRD3 comprising one or more residues at positions 81–101 of SEQ ID NO: 4 as determined by hydrogen/deuterium exchange (HDX) mass spectrometry (MS).
• HDX hydrogen/deuterium exchange
• the agonist LT ⁇ R binding protein binds to the aforementioned regions of CRD2 and CRD3 and does not bind to a region of LT ⁇ R outside of CRD2 and CRD3.
• the agonist LT ⁇ R binding protein binds to regions of LT ⁇ R consisting of one or more residues within CRD2 and CRD3. Binding of an LT ⁇ R binding protein to the identified regions of LT ⁇ R agonizes receptor signaling while allowing endogenous LT ⁇ R ligand binding. Agonist LT ⁇ R binding proteins that bind this epitope also bind cyno LT ⁇ R.
• an agonist LT ⁇ R binding protein of the present disclosure binds to an epitope that comprises residues of the extracellular CRD1 of human LT ⁇ R.
• the agonist LT ⁇ R binding protein described herein binds to an epitope within CRD1 comprising one or more residues at positions 3–9, 20–29, and/or 38–47 of SEQ ID NO: 4 as determined by HDX MS.
• the agonist LT ⁇ R binding protein binds to the aforementioned regions of CRD1 and does not bind to a region of LT ⁇ R outside of CRD1.
• the agonist LT ⁇ R binding protein binds to a region of LT ⁇ R consisting of residues within CRD1. Binding of an LT ⁇ R binding protein to this epitope of LT ⁇ R agonizes receptor signaling while allowing endogenous LIGHT ligand binding to LT ⁇ R and minimally inhibiting LT ⁇ 1 ⁇ 2 ligand binding ( ⁇ 20%). Agonist LT ⁇ R binding proteins that bind this epitope also bind cyno LT ⁇ R. An exemplary agonist LT ⁇ R binding protein having these properties is identified herein as LIBC218994 (19321).
• an agonist LT ⁇ R binding protein of the present disclosure binds to an epitope that comprises residues of the extracellular CRD1 of human LT ⁇ R.
• the agonist LT ⁇ R binding protein described herein binds to an epitope within CRD1 comprising one or more residues at positions 1–19 of SEQ ID NO: 4 as determined by HDX MS.
• the agonist LT ⁇ R binding protein binds to the aforementioned regions of CRD1 and does not bind to a region of LT ⁇ R outside of CRD1.
• the agonist LT ⁇ R binding protein binds to a region of LT ⁇ R consisting of residues within CRD1.
• an agonist LT ⁇ R binding protein of the present disclosure binds to an epitope that comprises residues of the extracellular CRD4 of human LT ⁇ R.
• the agonist LT ⁇ R binding protein described herein binds to an epitope within CRD4 comprising one or more residues at positions 168–179 of the extracellular domain of LT ⁇ R (SEQ ID NO: 4) (residues 197-209 of SEQ ID NO: 1) as determined by HDX MS.
• the agonist LT ⁇ R binding protein binds to the aforementioned region of CRD4 and does not bind to a region of LT ⁇ R outside of CRD4.
• the agonist LT ⁇ R binding protein binds to a region of LT ⁇ R consisting of residues within CRD4.
• Agonist LT ⁇ R binding proteins that bind this epitope also bind cyno LT ⁇ R.
• Exemplary agonist LT ⁇ R binding protein having these properties are identified herein as LIBC219081 (19325) and LIBC218979 (19319).
• LT ⁇ R binding proteins that have an identical or overlapping epitope with the exemplary agonist LT ⁇ R binding proteins disclosed herein will compete with each other for binding to LT ⁇ R.
• an exemplary agonist LT ⁇ R binding protein of the disclosure competes for binding to CRD4 with LIBC219081 (19325) and/or LIBC218979 (19319).
• To “compete” or “be in competition with” means the LT ⁇ R binding protein competes for the same epitope or binding site on a target.
• Such competition can be determined by an assay in which a reference agonist LT ⁇ R binding protein, such as agonist LT ⁇ R binding proteins LIBC219081 (19325) and LIBC218979 (19319), prevents or inhibits specific binding of a test LT ⁇ R binding protein.
• Example 9.2 An exemplary competitive binding assay that can be utilized to identify agonist LT ⁇ R binding proteins encompassed by the present disclosure that compete for binding to CRD4 of LT ⁇ R is described herein in Example 9.2.
• a competing LT ⁇ R binding protein when it is present in excess, it will inhibit binding of a reference agonist LT ⁇ R binding protein to a common epitope by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.
• binding of a reference agonist LT ⁇ R binding protein to LT ⁇ R is inhibited by at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more.
• An agonist LT ⁇ R binding protein of the present disclosure is a protein that binds to and agonizes LT ⁇ R without blocking or inhibiting one or both endogenous LT ⁇ R ligands from binding LT ⁇ R.
• the LT ⁇ R binding proteins described herein comprise one or more amino acid binding domains that alone, or in combination, bind a region of LT ⁇ R and activate signaling activity of the receptor.
• the agonist LT ⁇ R binding proteins further comprise a scaffold or framework portion that allows the one or more binding domains to adopt a conformation that promotes a binding interaction between the binding domain(s) and LT ⁇ R.
• LT ⁇ R binding proteins include, without limitation, LT ⁇ R antibodies (i.e., immunoglobulins), epitope binding fragments of an LT ⁇ R antibody, and LT ⁇ R antibody derivatives, each of which is described in more detail herein.
• An agonist LT ⁇ R binding protein as disclosed herein binds to its corresponding epitope within the extracellular domain of LT ⁇ R.
• an agonist LT ⁇ R binding protein of the present disclosure binds epitope residues within CRD4 of the extracellular domain of LT ⁇ R (i.e., residues 139–181 of SEQ ID NO: 4 (ECD); residues 169–211 of SEQ ID NO: 1 (full-length LT ⁇ R)) more frequently, more rapidly, with greater duration and/or with greater affinity or avidity than an alternative epitope.
• the agonist LT ⁇ R binding proteins described herein bind to any 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues of the extracellular CRD4 domain of LT ⁇ R, in particular one or more residues of 168-179 of SEQ ID NO: 4 or one or more residues of 168–178 of SEQ ID NO: 4.
• an agonist LT ⁇ R binding protein binds to LT ⁇ R with a tight binding affinity as determined by an equilibrium dissociation constant (K D ) value of 10 -7 M or less.
• an agonist LT ⁇ R binding protein of the present disclosure binds human LT ⁇ R with an equilibrium dissociation constant or K D (k off /k on ) of less than 10 -7 M, less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, or less than 10 -11 M less than 10 -12 M, or less than 10 -13 M (lower values indicating tighter binding affinity).
• agonist LT ⁇ R binding proteins of the disclosure bind human LT ⁇ R with an equilibrium dissociation constant or K D (k off /k on ) of between about 10 -7 M and about 10 -11 M, between about 10 -7 M and about 10 -10 M, between about 10 -7 M and about 10 -9 M, between about 10 -7 M and about 10 -8 M, between about 10 -8 M and about 10 -9 M, between about 10 -9 M and about 10 -10 M, between about 10 -10 M and about 10 -11 M, between about 10 -11 M and about 10 -12 M, between about 10 -12 M and about 10 -13 M.
• K D equilibrium dissociation constant
• agonist LT ⁇ R binding proteins of the disclosure specifically bind human LT ⁇ R with a K D of between about 10 -8 M and about 10 -11 M or between about 10 -9 M and about 10 -10 M.
• agonist LT ⁇ R binding proteins of the disclosure e.g., antibodies, epitope-binding fragments, and antibody derivatives
• K D k off /k on
• K D k off /k on
• agonist LT ⁇ R binding proteins of the disclosure specifically bind human LT ⁇ R with an equilibrium dissociation constant or K D (k off /k on ) of between about 0.01 mM and about 10 nM, between about 0.01 nM and about 9 nM, between about 0.01 nM and about 8 nM, between about 0.01 nM and about 7 nM, between about 0.01 nM and about 6 nM, between about 0.01 nM and about 5 nM, between about 0.01 nM and about 4 nM, between about 0.01 nM and
• the agonist LT ⁇ R binding proteins of the disclosure that bind to human LT ⁇ R also bind to cynomolgus monkey (cyno) LT ⁇ R with the same or similar affinities.
• the amino acid sequence of cyno LT ⁇ R is provided herein as SEQ ID NO: 3.
• the agonist LT ⁇ R binding proteins described herein bind to cynomolgus monkey LT ⁇ R with a K D of between about 10 -7 M and about 10 -8 M, between about 10 -8 M and about 10 -9 M, between about 10 -9 M and about 10 -10 M, between about 10 -10 M and about 10 -11 M, between about 10 -11 M and about 10 -12 M.
• LT ⁇ R binding proteins of the disclosure specifically bind cyno LT ⁇ R with a K D of between about 10 -8 M and about 10 -10 M.
• agonist LT ⁇ R binding proteins of the disclosure bind cynomolgus monkey (cyno) LT ⁇ R with an equilibrium dissociation constant or K D (k off /k on ) of between about 0.1 nM and about 30 nM, between about 0.1 nM and about 20 nM, between about 0.1 nM and about 10 nM, between about 0.1 nM and about 9 nM, between about 0.1 nM and about 8 nM, between about 0.1 nM and about 7 nM, between about 0.1 nM and about 6 nM, between about 0.1 nM and about 5 nM, between about 0.1 nM and about 4 nM, between about 0.1 nM and about 30 nM, between about 0.1 nM and about 30 nM, between about 0.1 n
• the agonist LT ⁇ R binding proteins of the present disclosure bind with high affinity to both human LT ⁇ R and cyno LT ⁇ R.
• the K D of the agonist LT ⁇ R binding protein for human LT ⁇ R is within about 100-fold, about 50-fold, about 25-fold, about 10-fold, about 5-fold, or about 2-fold or less of the KD of the agonist LT ⁇ R binding protein for cyno LT ⁇ R.
• the KD of the agonist LT ⁇ R binding protein for human LT ⁇ R is about 10-fold or less of the K D of the agonist LT ⁇ R binding protein for cyno LT ⁇ R.
• the agonist LT ⁇ R binding proteins as disclosed herein bind to the LT ⁇ R extracellular domain with an affinity corresponding to a K D that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its affinity for binding to a non-specific antigen (e.g., TNFR-I or TNFR-II, etc.).
• a non-specific antigen e.g., TNFR-I or TNFR-II, etc.
• the amount with which the affinity is lower is dependent on the K D of the binding protein to LT ⁇ R, so that when the K D of the agonist LT ⁇ R binding protein is very low (that is, the binding protein is highly specific), then the amount with which the affinity for LT ⁇ R is lower than the affinity for a non-specific antigen may be at least 10,000-fold.
• the agonist LT ⁇ R binding protein of the present disclosure is an LT ⁇ R antibody , an epitope-binding fragment of an LT ⁇ R antibody (e.g., an LT ⁇ R binding Fab, Fab′, F(ab′) 2 ), or an LT ⁇ R antibody derivative (e.g., LT ⁇ R scFv, minibody, diabody, or multi-specific binding protein) as described herein.
• an agonist LT ⁇ R binding protein of the present disclosure is an antibody as defined herein.
• antibody also includes antibodies comprising 1, 2, 3, 4, or 5 amino acid residue insertions or deletions at the N-terminus and/or C-terminus of the heavy and/or light chain that retain the same or similar binding and/or function as the antibodies comprising two heavy chains and two light chains not comprising these amino acid residue substitutions, insertions, or deletions.
• the heavy and light chain variable regions (V H and V L , respectively) of an antibody and other binding molecules described herein are responsible for LT ⁇ R recognition and binding, while the heavy and light chain constant regions mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
• variable (V) and constant regions (C) 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 Ch.7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989), which is hereby incorporated by reference in its entirety).
• the variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites.
• VH and VL regions of an LT ⁇ R antibody and other LT ⁇ R binding proteins as described herein are further subdivided into regions of hypervariability, termed “complementarity determining regions,” or “CDRs,” that are interspersed within regions of more conserved sequence, termed “framework regions” (FR).
• CDRs complementarity determining regions
• FR framework regions
• One or more residues within one or more variable region CDRs of the heavy and light chains form a binding domain that interacts with an antigen, e.g., LT ⁇ R.
• Exemplary V H and V L domain sequences and V H and V L CDR sequences of LT ⁇ R antibodies and binding proteins of the present disclosure are described in more detail infra.
• an agonist LT ⁇ R binding protein of the present disclosure is an epitope- binding fragment of a LT ⁇ R antibody.
• fragment region
• portion domain
• Fragments of antibodies e.g., Fab and (Fab′) 2 fragments
• the epitope binding fragment of the LT ⁇ R antibody is an amino acid sequence that comprises a portion of the amino acid sequence of such parental antibody.
• Exemplary LT ⁇ R-binding fragments encompassed by the present disclosure include, without limitation, (i) Fab' or Fab fragments, which are monovalent fragments containing the VL, VH, CL and CH1 domains as described supra; (ii) F(ab') 2 fragments, which are bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting essentially of the VH and CH1 domains; (iv) Fv fragments consisting essentially of a VL and VH domain; (v) single domain antibodies or dAb fragments (Ward et al.
• An agonist LT ⁇ R epitope-binding fragment of the present disclosure may contain 1, 2, 3, 4, 5 or all 6 of the CDR domains of an LT ⁇ R antibody as disclosed herein.
• an epitope- binding fragment of an LT ⁇ R antibody comprises, essentially consists of, or consists of 30 to 100 amino acid residues, or 50 to 150 amino acid residues, or 70 to 200 amino acid residues of the parental LT ⁇ R antibody.
• the length of an epitope-binding fragment of an LT ⁇ R antibody is at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the length of the LT ⁇ R antibody.
• an LT ⁇ R epitope-binding fragment of the present disclosure elicits the same or similar activity of the LT ⁇ R antibody from which the fragment is derived from.
• agonist LT ⁇ R epitope-binding fragments and LT ⁇ R antibodies as described herein elicit detectable LT ⁇ R binding activity and induce LT ⁇ R signaling as disclosed herein.
• Epitope-binding fragments of an LT ⁇ R antibody may be obtained using conventional techniques known to those of skill in the art.
• agonist LT ⁇ R F(ab') 2 fragments may be generated by treating an agonist LT ⁇ R antibody with pepsin.
• the resulting LT ⁇ R F(ab') 2 fragment may be treated to reduce disulfide bridges to produce LT ⁇ R Fab' fragments.
• Agonist LT ⁇ R Fab fragments may be obtained by treating an agonist LT ⁇ R antibody with papain, and LT ⁇ R Fab' fragments may be obtained with pepsin digestion of an agonist LT ⁇ R antibody.
• An agonist LT ⁇ R Fab' fragment may be obtained by treating an agonist LT ⁇ R F(ab') 2 fragment with a reducing agent, such as dithiothreitol.
• LT ⁇ R-binding fragments may also be generated by expression of nucleic acids encoding such fragments in recombinant cells (see e.g., Evans et al. “Rapid Expression Of An Anti-Human C5 Chimeric Fab Utilizing A Vector That Replicates In COS And 293 Cells,” J. Immunol. Meth.184:123-38 (1995), which is hereby incorporated by reference in its entirety).
• a chimeric gene encoding a portion of a LT ⁇ R F(ab') 2 fragment could include DNA sequences encoding the CH1 domain and hinge region of the heavy chain, followed by a translational stop codon to yield such a truncated antibody fragment molecule.
• Such gene sequences or their encoding cDNA are joined, using recombinant methods, by a flexible linker (typically of about 10, 12, 15 or more amino acid residues) that enables them to be made as a single protein chain in which the V L and V H regions associate to form monovalent epitope-binding proteins (see e.g., Bird et al. “Single-Chain Antigen-Binding Proteins,” Science 242:423-426 (1988); and Huston et al. “Protein Engineering Of Antibody Binding Sites: Recovery Of Specific Activity In An Anti-Digoxin Single-Chain Fv Analogue Produced In Escherichia coli,” Proc. Natl. Acad. Sci.
• a flexible linker typically of about 10, 12, 15 or more amino acid residues
• Another exemplary agonist LT ⁇ R antibody derivative of the present disclosure is a bispecific scFv.
• a bispecific LT ⁇ R scFv can be formed by employing a flexible linker that enables the V L and V H regions of different single polypeptide chains (each having different epitope binding specificities) to associate together.
• each scFv in the bivalent tandem scFv or diabody can be the same, i.e., recognize the same target epitope of LT ⁇ R or can be different, i.e., recognize and bind different target epitopes.
• the agonist LT ⁇ R antibody derivative is a triabody, i.e., a trivalent single chain variable fragment, engineered by linking three scFvs together, either in tandem or in a trimer formation to form a triabody.
• each scFv of the triabody can be the same, i.e., recognize the same target epitope, or can be different, i.e., recognize and bind different target epitopes.
• the agonist LT ⁇ R antibody derivative is a tetrabody of four single chain variable fragments, where each of the scFv recognize the same or different target epitopes.
• the agonist LT ⁇ R antibody derivative is a “linear antibody” which is an antibody comprising a pair of tandem Fd segments (VH-CH1- VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (see Zapata et al.
• the agonist LT ⁇ R antibody derivative is a minibody, comprising single-chain Fv regions coupled to the CH3 region (i.e., scFv-CH3).
• the agonist LT ⁇ R antibody derivative is a modified domain antibody, e.g., a modified single VL or VH domain or two or more VH domains joined by a peptide linker (see, e.g., Ward et al., Nature, 341:544-546 (1989), which is hereby incorporated by reference in its entirety); a maxibody, i.e., two scFvs fused to Fc region (see, e.g., Fredericks et al., Protein Engineering, Design & Selection, 17:95-106 (2004) and Powers et al., J. Immunol.
• a modified domain antibody e.g., a modified single VL or VH domain or two or more VH domains joined by a peptide linker (see, e.g., Ward et al., Nature, 341:544-546 (1989), which is hereby incorporated by reference in its entirety); a maxibody, i.e., two s
• the agonist LT ⁇ R antibody derivative is an immunoglobulin fusion protein, which comprises an agonist LT ⁇ R binding domain (i.e., polypeptide comprising one or more heavy chain and/or light chain CDRs as described herein) coupled to a hinge region polypeptide and one or more immunoglobulin constant regions (see e.g., U.S. Patent Appl. Pub. No.
• immunoglobulin fusion protein formats include, without limitation, an IgG-scFv, an IgG-Fab, 2scFv-IgG, 4scFv-IgG, VH-IgG, IgG-VH, and Fab-scFv-Fc.
• Another exemplary agonist LT ⁇ R antibody derivative of the present disclosure is a multi- specific binding antibody, e.g., a bispecific or tri-specific binding protein.
• Multi-specific agonist LT ⁇ R binding proteins of the present disclosure comprise a first binding domain that binds to LT ⁇ R and is coupled to one or more additional binding domains, where each additional binding domain binds a target moiety that is not LT ⁇ R.
• An exemplary agonist LT ⁇ R multi-specific binding protein of the disclosure is a bispecific agonist LT ⁇ R binding protein that comprises an LT ⁇ R binding domain and a tumor-associated antigen binding domain.
• Exemplary LT ⁇ R binding domains of a bispecific agonist LT ⁇ R binding protein include the binding domains from any one of the LT ⁇ R antibodies disclosed herein.
• the bispecific agonist LT ⁇ R binding protein comprises an LT ⁇ R binding domain that binds one or more amino acid residues of human LT ⁇ R cysteine-rich domain 4 (CRD4) comprising amino acid residues 169- 211 of SEQ ID NO: 1.
• This bispecific agonist binding protein does not inhibit LIGHT binding to LT ⁇ R and does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R.
• the agonist LT ⁇ R binding protein is a human agonist LT ⁇ R binding protein.
• a human agonist LT ⁇ R binding protein comprises variable and constant regions or domains which correspond substantially to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (1991).
• the human agonist LT ⁇ R binding protein disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and, in particular, CDR3.
• the human agonist LT ⁇ R binding proteins of the present disclosure may have at least one, two, three, four, five, or more positions replaced with an amino acid residue that is not encoded by the human germline immunoglobulin sequence.
• human agonist binding proteins as used herein also contemplates fully human antibodies, which include non-artificially and/or non-genetically altered human sequences of antibodies as those can be derived by using technologies or systems known in the art, such as, for example, phage display technology or transgenic mouse technology, including but not limited to the XenoMouse ® , which is a transgenic mouse capable of producing human antibodies.
• the agonist LT ⁇ R binding protein of the present disclosure is an IgG isotype. The choice of isotype typically will be guided by the desired effector functions, such as antibody- dependent cellular cytotoxicity (ADCC) induction.
• ADCC antibody- dependent cellular cytotoxicity
• Exemplary IgG isotypes are IgG1, IgG2, IgG3, and IgG4.
• the agonist LT ⁇ R binding protein of the present disclosure is an IgG1 isotype.
• the agonist LT ⁇ R binding protein of the present disclosure is an IgG2 isotype.
• the binding domain of an exemplary agonist LT ⁇ R binding protein disclosed herein comprises one or more VH and VL CDRs that can be delineated using any one of the standard methods known in the art, including and without limitation, the Kabat numbering scheme (Kabat et al., Sequences of Proteins of Immunological Interest, 5 th ed., U.S. Dept.
• an agonist LT ⁇ R binding protein of the present disclosure comprises a HCDR1, HCDR2, and HCDR3 of any one of the heavy chain variable region sequences as provided in Tables 4, 8, and 12, and a LCDR1, LCDR2, and LCDR3 of the corresponding light chain variable region as also provided in Tables 4, 8, and 12, where the CDRs are defined by the Kabat numbering scheme.
• an agonist LT ⁇ R binding protein of the present disclosure comprises a HCDR1, HCDR2, and HCDR3 of any one of the heavy chain variable region sequences as provided in Tables 4, 8, and 12, and a LCDR1, LCDR2, and LCDR3 of the corresponding light chain variable region as also provided in Tables 4, 8, and 12, where the CDRs are defined by the Chothia numbering scheme.
• an agonist LT ⁇ R binding protein of the present disclosure comprises a HCDR1, HCDR2, and HCDR3 of any one of the heavy chain variable region sequences as provided in Tables 4, 8, and 12, and a LCDR1, LCDR2, and LCDR3 of the corresponding light chain variable region as provided in Tables 4, 8, and 12, where the CDRs are defined by the AbM numbering scheme.
• an agonist LT ⁇ R binding protein of the present disclosure comprises a HCDR1, HCDR2, and HCDR3 of any one of the heavy chain variable region sequences as provided in Tables 4, 8, and 12, and a LCDR1, LCDR2, and LCDR3 of the corresponding light chain variable region as also provided in Tables 4, 8, and 12, where the CDRs are defined by the IMGT numbering scheme.
• an agonist LT ⁇ R binding protein of the present disclosure comprises a HCDR1, HCDR2, and HCDR3 of any one of the heavy chain variable region sequences as provided in Tables 4, 8, and 12, and a LCDR1, LCDR2, and LCDR3 of the corresponding light chain variable region as also provided in Tables 4, 8, and 12, where the CDRs are defined by the Contact numbering scheme.
• the agonist LT ⁇ R binding protein is an antibody.
• the agonist LT ⁇ R binding protein is a bispecific antibody.
• agonist LT ⁇ R binding proteins of the present disclosure bind to an epitope comprising residues of CRD4 of LT ⁇ R.
• CRD4 of LT ⁇ R span residues 139–181 of SEQ ID NO: 4 (LT ⁇ R ECD), which corresponds to residues 169–211 of SEQ ID NO: 1 (full-length LT ⁇ R).
• the agonist LT ⁇ R binding protein described herein binds to an epitope comprising one or more CRD4 residues at positions 168–179 of the extracellular domain of LT ⁇ R (SEQ ID NO: 4) (residues 197- 209 of SEQ ID NO: 1) as determined by HDX MS.
• the agonist LT ⁇ R binding proteins of the present disclosure bind to an epitope comprising residues of LT ⁇ R CRD4 and also binds to one or more residues outside of the CRD4.
• the agonist LT ⁇ R binding protein binds to an epitope comprising residues of CRD4 and residues of the adjacent CRD3 of LT ⁇ R.
• CRD3 of LT ⁇ R comprises residues 95–138 of SEQ ID NO: 4, which corresponds to residues 125-168 of the full- length LT ⁇ R of SEQ ID NO: 1.
• the agonist LT ⁇ R binding proteins of the present disclosure bind to an epitope of LT ⁇ R within CRD4 of LT ⁇ R and do not bind to a region of LT ⁇ R outside of the CRD4 as defined herein.
• the agonist LT ⁇ R binding protein binds to an epitope consisting of residues within CRD4 of LT ⁇ R.
• the agonist LT ⁇ R binding protein having these properties is an antibody.
• Exemplary agonist LT ⁇ R antibodies having these functional features are disclosed herein (see Tables 2–4 and 6–8).
• the agonist LT ⁇ R binding protein having these properties is a bispecific binding protein.
• Exemplary agonist LT ⁇ R bispecific binding proteins having these functional features comprise a LT ⁇ R binding domain from the exemplary agonist LT ⁇ R antibodies disclosed herein.
• agonist LT ⁇ R binding proteins having these functional properties i.e., binding proteins that bind residues of CRD4 of LT ⁇ R and do not block LIGHT binding to LT ⁇ R or LT ⁇ 1 ⁇ 2 binding to LT ⁇ R, are defined by their shared heavy chain variable region (V H ) CDR structure.
• These agonist LT ⁇ R binding proteins can further be defined by their shared light chain variable region (V L ) CDR structure.
• This shared CDR structure which is presented as consensus V H CDR and V L CDR sequences, was derived from the alignment of VH and VL domains of exemplary agonist LT ⁇ R binding proteins disclosed herein (Table 4), which (i) possess the above noted functional characteristics and (ii) comprise V H and V L amino acid sequences that have at least 90% sequence identity to the V H and V L amino acid sequences, respectively, of the LT ⁇ R binding proteins LIBC219081 and LIBC 218979.
• agonist LT ⁇ R binding proteins of the present disclosure that (i) bind residues of LT ⁇ R CRD4 and (ii) do not block LIGHT binding to LT ⁇ R and do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a shared V H CDR structure defined by the HCDR1 amino acid sequence of: X 1 YX 3 MX 5 (SEQ ID NO: 5), where X 1 is S or N; X 3 is G, D, or A; and X 5 is H or Y; the HCDR2 amino acid sequence of: X1IX3YDX6X7X8X9Y X11X12DSVKG (SEQ ID NO: 6), where X1 is A or V; X3 is W or R; X 6 is E or G; X 7 is S, R, or T; X 8 is N or K; X 9 is K, R, or Q; X 11 is H or Y; and X 12 is A or E
• the agonist LT ⁇ R binding protein is an antibody. In some embodiments, the agonist LT ⁇ R binding protein is a bispecific binding protein.
• Heavy chain CDR sequences of exemplary agonist LT ⁇ R binding proteins e.g., antibodies or bispecific binding proteins that bind CRD4 of LT ⁇ R, do not block LIGHT or LT ⁇ 1 ⁇ 2 binding or activity, and comprises a HCDR1 of SEQ ID NO: 5, a HCDR2 of SEQ ID NO: 6, and a HCDR3 of SEQ ID NO: 7 are provided in Table 2 below. TABLE 2: Heavy Chain CDR Sequences of LT ⁇ R CRD4 Binders comprising 90% Sequence Identity to the Fv Domains of LIBC219081 and LIBC218979
• agonist LT ⁇ R binding proteins that (i) bind residues of LT ⁇ R CRD4, and (ii) do not block LIGHT binding to LT ⁇ R and do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a shared V L CDR structure defined by the LCDR1 amino acid sequence of: SGDX 4 LPX 7 X 8 YX 10 Y (SEQ ID NO: 62), where X 4 is A or T; X 7 is E, K, Q, D or N; X 8 is Q or H; and X 10 is A or T; the LCDR2 amino acid sequence of: KDNERPS (SEQ ID NO: 63); and the LCDR3 amino acid sequence of: QSX 3 DX 5 SX 7 X 8 YX 10 X 11 (SEQ ID NO: 64), where X 3 is A or T; X 5 is S, G, or N; X 7 is G or A; X 8 is T, S, or A; X 10 is V or
• Light chain CDR sequences of exemplary agonist LT ⁇ R binding proteins that bind CRD4 of LT ⁇ R, do not block LIGHT or LT ⁇ 1 ⁇ 2 binding or activity, and comprises a LCDR1 of SEQ ID NO: 62, a LCDR2 of SEQ ID NO: 63, and a LCDR3 of SEQ ID NO: 64 are provided in Table 3 below.
• Table 3 Light Chain CDRs of Exemplary LT ⁇ R CRD4 Binders Comprising 90% Sequence Identity to the Fv Domains of LIBC219081 and LIBC218979
• agonist LT ⁇ R binding proteins that bind residues of human LT ⁇ R CRD4 and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R include those binding proteins possessing VH and VL amino acid sequences that have at least 80% sequence identity to the VH and VL amino acid sequences, respectively, of the LT ⁇ R binding protein LIBC219081 (SEQ ID NOs: 121 and 122, respectively).
• agonist LT ⁇ R binding proteins that bind human LT ⁇ R CRD4 and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R include those binding proteins possessing VH and VL amino acid sequences that have at least 80% sequence identity to the VH and VL amino acid sequences, respectively, of the LT ⁇ R binding protein LIBC218979 (SEQ ID NOs: 123 and 124, respectively).
• agonist LT ⁇ R binding proteins that bind human LT ⁇ R CRD4 and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R include those binding proteins possessing VH and VL amino acid sequences that have at least 80% sequence identity to the VH and VL amino acid sequences, respectively, of LT ⁇ R binding proteins LIBC219081 (SEQ ID NOs: 121 and 122, respectively) and LIBC218979 (SEQ ID NOs: 123 and 124, respectively).
• agonist LT ⁇ R binding proteins that bind human LT ⁇ R CRD4 and (a) do not block LIGHT binding to LT ⁇ R or (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R include those binding proteins possessing VH and VL amino acid sequences that have at least 85% sequence identity to the VH and VL amino acid sequences, respectively, of the LT ⁇ R binding protein LIBC219081 (SEQ ID NOs: 121 and 122, respectively).
• agonist LT ⁇ R binding proteins that bind human LT ⁇ R CRD4 and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R include those binding proteins possessing VH and VL amino acid sequences that have at least 85% sequence identity to the VH and VL amino acid sequences, respectively, of the LT ⁇ R binding protein LIBC218979 (SEQ ID NOs: 123 and 124, respectively).
• agonist LT ⁇ R binding proteins that bind human LT ⁇ R CRD4 and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R include those binding proteins possessing VH and VL amino acid sequences that have at least 85% sequence identity to the VH and VL amino acid sequences, respectively, of the LT ⁇ R binding proteins LIBC219081 (SEQ ID NOs: 121 and 122, respectively) and LIBC218979 (SEQ ID NOs: 123 and 124, respectively).
• agonist LT ⁇ R binding proteins that bind human LT ⁇ R CRD4 and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R include those binding proteins possessing VH and VL amino acid sequences that have at least 90% sequence identity to the VH and VL amino acid sequences, respectively, of the LT ⁇ R binding proteins LIBC219081 and LIBC218979.
• an agonist LT ⁇ R binding protein of the present disclosure comprises a VH amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 121.
• an agonist LT ⁇ R binding protein of the present disclosure comprises a VH amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 123. In one embodiment, an agonist LT ⁇ R binding protein of the present disclosure comprises a VH amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 121 and the amino acid sequence of SEQ ID NO: 123. Exemplary VH amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 121 and SEQ ID NO: 123 are provided in Table 4 below.
• agonist LT ⁇ R binding proteins that bind human LT ⁇ R CRD4 and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R include those binding proteins comprising a VH amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 121.
• an agonist LT ⁇ R binding protein of the present disclosure comprises a VH amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 123.
• an agonist LT ⁇ R binding protein of the present disclosure comprises a VH amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 121 and the amino acid sequence of SEQ ID NO: 123.
• VH amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 121 and SEQ ID NO: 123 are provided in Table 4 below.
• agonist LT ⁇ R binding proteins that bind human LT ⁇ R CRD4 and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R include those binding proteins comprising a VL amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 122.
• an agonist LT ⁇ R binding protein of the present disclosure comprises a VL amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 124.
• an agonist LT ⁇ R binding protein of the present disclosure comprises a VL amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 122 and the amino acid sequence of SEQ ID NO: 124.
• VL amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 122 and SEQ ID NO: 124 are provided in Table 4 below.
• agonist LT ⁇ R binding proteins that bind human LT ⁇ R CRD4 and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a VH amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 121 and a VL amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 122.
• an agonist LT ⁇ R binding protein of the present disclosure comprises a VH amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 121 and a VL amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 122. In one embodiment, an agonist LT ⁇ R binding protein of the present disclosure comprises a VH amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 121 and a VL amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 122.
• agonist LT ⁇ R binding proteins that bind human LT ⁇ R CRD4 and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a VH amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 123 and a VL amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 124.
• an agonist LT ⁇ R binding protein of the present disclosure comprises a VH amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 123 and a VL amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 124. In one embodiment, an agonist LT ⁇ R binding protein of the present disclosure comprises a VH amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 123 and a VL amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 124.
• agonist LT ⁇ R binding proteins that bind human LT ⁇ R CRD4 and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a VH amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 121 and the amino acid sequence of SEQ ID NO: 123 and a VL amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 122 and the amino acid sequence of SEQ ID NO: 124.
• an agonist LT ⁇ R binding protein of the present disclosure comprises a VH amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 121 and the amino acid sequence of SEQ ID NO: 123 and a VL amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 122 and the amino acid sequence of SEQ ID NO: 124.
• an agonist LT ⁇ R binding protein of the present disclosure comprises a VH amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 121 and the amino acid sequence of SEQ ID NO: 123 and a VL amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 122 and the amino acid sequence of SEQ ID NO: 124.
• agonist LT ⁇ R binding proteins that bind human LT ⁇ R CRD4 and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a VH amino acid sequence having at least 90% sequence identity to the VH amino acid sequence of SEQ ID NO: 121 or SEQ ID NO: 123 and comprise the HCDR1 amino acid sequence of: X 1 YX 3 MX 5 (SEQ ID NO: 5), where X 1 is S or N; X 3 is G, D, or A; and X 5 is H or Y; the HCDR2 amino acid sequence of: X 1 IX 3 YDX 6 X 7 X 8 X 9 Y X 11 X 12 DSVKG (SEQ ID NO: 6), where X 1 is A or V; X3 is W or R; X6 is E or G; X7 is S, R, or T
• VH amino acid sequences of agonist LT ⁇ R binding proteins comprising 90% sequence identity to the VH of amino acid sequence of SEQ ID NO: 121 or SEQ ID NO: 123 and comprising the HCDR sequences of SEQ ID NOs: 5, 6, and 7 are provided in Table 4 below.
• These agonist LT ⁇ R binding proteins may further comprise a VL amino acid sequence having at least 90% sequence identity to the VL amino acid sequence of SEQ ID NO: 122 or SEQ ID NO: 124 and comprise the LCDR1 amino acid sequence of: SGDX 4 LPX 7 X 8 YX 10 Y (SEQ ID NO: 62), where X 4 is A or T; X 7 is E, K, Q, D or N; X 8 is Q or H; and X 10 is A or T; the LCDR2 amino acid sequence of: KDNERPS (SEQ ID NO: 63); and the LCDR3 amino acid sequence of: QSX 3 DX 5 SX 7 X 8 YX 10 X 11 (SEQ ID NO: 64), where X 3 is A or T; X 5 is S, G, or N; X 7 is G or A; X 8 is T, S, or A; X 10 is V or M; and X 11 is I, or V.
• VL amino acid sequences of agonist LT ⁇ R binding proteins comprising 90% sequence identity to the VL of amino acid sequence of SEQ ID NO: 122 or SEQ ID NO: 124 and comprising the LCDR sequences of SEQ ID NOs: 62, 63, and 64 are provided in Table 4 below.
• an agonist LT ⁇ R binding protein that binds human LT ⁇ R CRD4 and (a) does not block LIGHT binding to LT ⁇ R and (b) does not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprises a VH amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASGFTFSX 31 YX 33 MX 35 WVRQAPGKGLEWVA X 50 IX 52 YDX 55 X 56 X 57 X 58 YX 60 X 61 DSVKGRFTISRDNSKNTLSLQMNSLRAEDTAVYYCARX 99 RX 101 X 1 02 X 103 X 104 X 105 X 106 X 107 YYGX 111 X 112 VWGQGTTVTVSS (SEQ ID NO: 119), where X 31 is S or N; X 33 is G or A; X 35 is H or Y;
• an agonist LT ⁇ R binding protein that binds human LT ⁇ R CRD4 and (a) does not block LIGHT binding to LT ⁇ R and (b) does not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprises a VL amino acid sequence of: SYELTQPPSVSVSPRQTARITCSGDX 26 LPX 29 X 30 YX 32 YWYQQKPGQAPVLVIYKDNERPSGIPERFS GSSSGTTVTLTISGVQAEDEADYYCQSX 90 DX 92 SX 94 X 95 YX 97 X 98 FGGGTKLTVLG (SEQ ID NO: 120), where X 26 is A or T; X 29 is E, K, Q, D or N; X 30 is Q or H; X 32 is A or T; X 90 is A or T; X 92 is S, G, or N; X 94 is G or A
• an agonist LT ⁇ R binding protein that binds human LT ⁇ R CRD4 and (a) does not block LIGHT binding to LT ⁇ R and (b) does not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprises a VH, where the VH comprises an amino acid sequence of SEQ ID NO: 119 and shares at least 90% sequence identity to the VH amino acid sequence of SEQ ID NO: 121 and/or the VH amino acid sequence of SEQ ID NO: 123.
• the agonist LT ⁇ R binding protein further comprises a VL, where the VL comprises an amino acid sequence of SEQ ID NO: 120 and shares at least 90% sequence identity to the VL amino acid sequence of SEQ ID NO: 122 and/or the VL amino acid sequence of SEQ ID NO: 124.
• an agonist LT ⁇ R binding protein that binds human LT ⁇ R CRD4 and (a) does not block LIGHT binding to LT ⁇ R and (b) does not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprises a VH, where the VH comprises an amino acid sequence of SEQ ID NO: 119 and shares at least 95% sequence identity to the VH amino acid sequence of SEQ ID NO: 121 and/or the VH amino acid sequence of SEQ ID NO: 123.
• the agonist LT ⁇ R binding protein further comprises a VL, where the VL comprises an amino acid sequence of SEQ ID NO: 120 and shares at least 95% sequence identity to the VL amino acid sequence of SEQ ID NO: 122 and/or the VL amino acid sequence of SEQ ID NO: 124.
• the agonist LT ⁇ R binding proteins of the present disclosure that bind human LT ⁇ R CRD4 and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a variable light (VL) domain, a variable heavy (VH) domain, or a combination of VL and VH domain.
• VH and VL amino acid sequences of exemplary agonist LT ⁇ R binding proteins that share 90% sequence identity with the VH and VL amino acid sequences of LIBC219081 and LIBC218979 are provided in Table 4.
• the VH domain of the agonist LT ⁇ R binding protein comprises any one of the VH amino acid sequences provided in Table 4, or an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of the VH amino acid sequences listed in Table 4.
• the VL domain of the agonist LT ⁇ R binding protein comprises any one of the VL amino acid sequences provided in Table 4 below, or an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical to any one of the VL amino acid sequences listed in Table 4.
• Table 4 VH and VL Sequences of LT ⁇ R CRD4 Binding Proteins Sharing 90% Sequence Identity
• the agonist LT ⁇ R binding protein (e.g., an agonist LT ⁇ R antibody or agonist LT ⁇ R bispecific binding protein) of the present disclosure binds to human LT ⁇ R CRD4, including one or more residues corresponding to residues 198-209 of SEQ ID NO: 1 (residues 168-179 of SEQ ID NO: 4), and (a) does not block LIGHT binding to LT ⁇ R and (b) does not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R.
• human LT ⁇ R CRD4 including one or more residues corresponding to residues 198-209 of SEQ ID NO: 1 (residues 168-179 of SEQ ID NO: 4)
• This agonist LT ⁇ R binding protein comprises a VH, where the VH comprises a HCDR1 sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 8, a HCDR2 sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 9, and a HCDR3 sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 10.
• the VH of this agonist LT ⁇ R binding protein comprises the HCDR1 sequence of SEQ ID NO: 5, the HCDR2 sequence of SEQ ID NO: 6, and the HCDR3 sequence of SEQ ID NO: 7. In one embodiment, the VH of this agonist LT ⁇ R binding protein comprises the HCDR1 sequence of SEQ ID NO: 8, the HCDR2 sequence of SEQ ID NO: 9, and the HCDR3 sequence of SEQ ID NO: 10.
• this agonist LT ⁇ R binding protein of the present disclosure further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 65, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 66, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 67.
• the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%
• the VL comprises the LCDR1 sequence of SEQ ID NO: 62, the LCDR2 of SEQ ID NO: 63, and the LCDR3 sequence of SEQ ID NO: 64. In one embodiment, the VL comprises the LCDR1 sequence of SEQ ID NO: 65, the LCDR2 of SEQ ID NO: 66, and the LCDR3 sequence of SEQ ID NO: 67.
• An exemplary LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.219081 (BR# 19325; 41B2).
• the agonist LT ⁇ R binding protein (e.g., an agonist LT ⁇ R antibody or agonist LT ⁇ R bispecific binding protein) of the present disclosure binds to human LT ⁇ R CRD4, including one or more residues corresponding to residues 197–209 of SEQ ID NO: 1) (i.e., residues 167- 179 of SEQ ID NO: 4), and (a) does not block LIGHT binding to LT ⁇ R and (b) does not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R.
• human LT ⁇ R CRD4 including one or more residues corresponding to residues 197–209 of SEQ ID NO: 1) (i.e., residues 167- 179 of SEQ ID NO: 4), and (a) does not block LIGHT binding to LT ⁇ R and (b) does not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R.
• This LT ⁇ R binding protein comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 11, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 12, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 13.
• the VH of this LT ⁇ R binding protein comprises the HCDR1 sequence of SEQ ID NO: 5, the HCDR2 sequence of SEQ ID NO: 6, and a HCDR3 sequence of SEQ ID NO: 7. In one embodiment, the VH of this LT ⁇ R binding protein comprises the HCDR1 sequence of SEQ ID NO: 11, the HCDR2 sequence of SEQ ID NO: 12, and the HCDR3 sequence of SEQ ID NO: 13.
• this agonist LT ⁇ R binding protein of the present disclosure further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 68, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 69, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 70.
• the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%
• the VL comprises the LCDR1 sequence of SEQ ID NO: 62, the LCDR2 of SEQ ID NO: 63, and the LCDR3 sequence of SEQ ID NO: 64. In one embodiment, the VL comprises the LCDR1 sequence of SEQ ID NO: 68, the LCDR2 of SEQ ID NO: 69, and the LCDR3 sequence of SEQ ID NO: 70.
• An exemplary LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No. 218979 (BR#19319; 23E9).
• the agonist LT ⁇ R binding protein (e.g., an agonist LT ⁇ R antibody or agonist LT ⁇ R bispecific binding protein) of the present disclosure that binds to human LT ⁇ R CRD4, and (a) does not block LIGHT binding to LT ⁇ R and (b) does not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprises a VH amino acid sequence and a VL amino acid sequence selected from: a VH amino acid sequence of SEQ ID NO: 119 and a VL amino acid sequence of SEQ ID NO: 120; a VH amino acid sequence of SEQ ID NO: 121 and a VL amino acid sequence of SEQ ID NO: 122; a VH amino acid sequence of SEQ ID NO: 123 and a VL amino acid sequence of SEQ ID NO: 124; a VH amino acid sequence of SEQ ID NO: 125 and a VL amino acid sequence of SEQ ID NO: 126; a VH amino acid sequence of SEQ ID NO: 127
• the agonist LT ⁇ R binding protein (e.g., an agonist LT ⁇ R antibody or agonist LT ⁇ R bispecific binding protein) of the present disclosure that binds to human LT ⁇ R CRD4, and (a) does not block LIGHT binding to LT ⁇ R and (b) does not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprises a VH region and/or VL region as described supra and further comprises one or more heavy chain constant regions coupled to the VH region and/or a light chain constant region coupled to the VL region.
• the LT ⁇ R binding protein is a Fab comprising a VH and first heavy chain constant domain (CH1) coupled to a VL and light chain constant region (CL).
• the LT ⁇ R binding protein is a F(ab’)2 comprising both LT ⁇ R binding regions of a full antibody coupled by the hinge region, where each binding region comprises a VH-CH1 and VL-CL.
• the LT ⁇ R binding protein is an antibody comprising full light chains (VL-CL) and full heavy chains (VH-CH1- CH2-CH3).
• the agonist LT ⁇ R binding protein e.g., an agonist LT ⁇ R antibody or agonist LT ⁇ R bispecific binding protein
• the agonist LT ⁇ R binding protein that binds to human LT ⁇ R CRD4, and (a) does not block LIGHT binding to LT ⁇ R and (b) does not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a human IgG1 heavy chain.
• the agonist LT ⁇ R binding protein (e.g., an agonist LT ⁇ R antibody or agonist LT ⁇ R bispecific binding protein) of the present disclosure that binds to human LT ⁇ R CRD4, and (a) does not block LIGHT binding to LT ⁇ R and (b) does not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a human IgG2 heavy chain.
• the human IgG heavy chain is modified to prevent or reduce interaction with Fc gamma receptors.
• the human IgG is a Stable Effector Functionless (SEFL) IgG (Liu et al., J Biol. Chem. 292(5):1876-1883), which is hereby incorporated by reference in its entirety).
• SEFL Stable Effector Functionless
• LT ⁇ R antibodies of the present disclosure that bind to human LT ⁇ R CRD4, and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R are provided in Table 9 herein.
• an agonist LT ⁇ R binding protein of the present disclosure e.g., agonist LT ⁇ R antibody or agonist LT ⁇ R bispecific binding protein
• agonist LT ⁇ R binding proteins having these functional properties i.e., compete with the LT ⁇ R binders LIBC219081 and LIBC218979 for binding to CRD4 of LT ⁇ R and do not block LIGHT or LT ⁇ 1 ⁇ 2 binding to LT ⁇ R, are defined by their structurally similar VH and VL domains.
• This shared variable region structure presented as consensus VH and VL CDR sequences as well as consensus VH and VL sequences, was derived from alignment of exemplary agonist LT ⁇ R binding proteins disclosed herein (see Tables 6, 7, and 8), which (i) possess these functional characteristics and (ii) comprise VH and VL amino acid sequences that share at least 90% sequence identity across their respective entire length, i.e. from N-terminus to C-terminus across the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
• the agonist LT ⁇ R binding proteins e.g., agonist LT ⁇ R antibodies or agonist LT ⁇ R bispecific binding proteins
• bind to human LT ⁇ R CRD4 and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a shared VH CDR structure defined by the HCDR1 amino acid sequence of: GX 2 YMH (SEQ ID NO: 159),where X 2 is Y or F; the HCDR2 amino acid sequence of: WINPNX 6 GGTNYAQKFQG (SEQ ID NO: 160), where X 6 is S, N, or R; and the HCDR3 amino acid sequence of: X 1 X 2 X 3 X 4 GX 6 X 7 X 8 YYGMDV (SEQ ID NO: 161), where X 1 is D or A; X 2 is R or D; X 3 is N, S,
• Heavy chain CDR sequences of exemplary agonist LT ⁇ R binding proteins that bind to human LT ⁇ R CRD4, do not block LIGHT binding to LT ⁇ R, do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R, and comprise a HCDR1 of SEQ ID NO: 159, a HCDR2 of SEQ ID NO: 160, and a HCDR3 of SEQ ID NO: 161 are provided in Table 6 below.
• the agonist LT ⁇ R binding proteins of the present disclosure comprising the HCDRs 1–3 of SEQ ID NOs: 159-161, respectively, further comprise a shared VL CDR structure defined by the LCDR1 amino acid sequence of TGTX 4 SDVGSYNLVS (SEQ ID NO: 278), where X 4 is T N, or S; the LCDR2 amino acid sequence of EVX 3 X 4 RPS (SEQ ID NO: 279), where X 3 is T or S and X 4 is K or V; and the LCDR3 amino acid sequence of CSYX 4 X 5 SX 7 TX 9 V (SEQ ID NO: 280), where X 4 is A or V; X 5 is D or E; X 7 is S or K; X 9 is L or W.
• Light chain CDR sequences of exemplary agonist LT ⁇ R binding proteins that bind to human LT ⁇ R CRD4, do not block LIGHT binding to LT ⁇ R, do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R, and comprise a LCDR1 of SEQ ID NO: 278, a LCDR2 of SEQ ID NO: 279, and a LCDR3 of SEQ ID NO: 280 are provided in Table 7 below.
• the agonist LT ⁇ R binding proteins of the present disclosure comprising the HCDRs 1–3 of SEQ ID NOs.
• SGDX 4 LPX 7 X 8 YX 10 Y SEQ ID NO: 308
• X 4 is A or T
• X 7 is Q, E, N, R, or K
• Light chain CDR sequences of exemplary agonist LT ⁇ R binding proteins that bind to human LT ⁇ R CRD4, do not block LIGHT binding to LT ⁇ R, do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R, and comprise a LCDR1 of SEQ ID NO: 308, a LCDR2 of SEQ ID NO: 309, and a LCDR3 of SEQ ID NO: 310 are provided in Table 7 below. TABLE 6. Heavy Chain CDR Sequences of agonist LT ⁇ R binding proteins that bind human LT ⁇ R CRD4 and do not block LIGHT or LT ⁇ 1 ⁇ 2 binding to LT ⁇ R
• the agonist LT ⁇ R binding proteins e.g., agonist LT ⁇ R antibodies or agonist LT ⁇ R bispecific binding proteins
• bind to human LT ⁇ R CRD4 and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a shared VH CDR structure defined by the HCDR1 amino acid sequence of: X 1 YGMH (SEQ ID NO: 195), where X 1 is A or S; the HCDR2 amino acid sequence of: X 1 IWYDGNNKYYX 12 DSVKG (SEQ ID NO: 196), where X 1 is L or V, and X 12 is E or A; and the HCDR3 amino acid sequence of: DRITX 5 VRGVTNYGMDV (SEQ ID NO: 197), where X 5 is M or R.
• Heavy chain CDR sequences of exemplary agonist LTßR binding proteins that bind to human LTßR CRD4, do not block LIGHT binding to LTßR, do not block LTa1ß2 binding to LTßR, and comprise a HCDR1 of SEQ ID NO: 195, a HCDR2 of SEQ ID NO: 196, and a HCDR3 of SEQ ID NO: 197 are provided in Table 6 above.
• the agonist LT ⁇ R binding proteins of the present disclosure comprising the HCDRs 1–3 of SEQ ID NOs: 195-197, respectively, further comprise a shared VL CDR structure defined by the LCDR1 amino acid sequence of SGDX 4 LPX 7 X 8 YX 10 Y (SEQ ID NO: 308), where X 4 is A or T; X 7 is Q, E, N, R, or K; X 8 is H or Q; and X 10 is V, T, or A; the LCDR2 amino acid sequence of KDX3X4RPS (SEQ ID NO: 309), where X3 is N or S, and X4 is D or E; and the LCDR3 amino acid sequence of QSADX 5 SGX 8 X 9 VV (SEQ ID NO: 310), where X 5 is I, N, or S; X 8 is S, A, I, or T; X 9 is F or Y.
• agonist LT ⁇ R binding proteins that bind to human LT ⁇ R CRD4, and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a shared VH CDR structure defined by the HCDR1 amino acid sequence of: SX 2 AMH (SEQ ID NO: 224),where X 2 is Y or F; the HCDR2 amino acid sequence of: VIWYX 5 X 6 X 7 NX 9 FYADSVKG (SEQ ID NO: 225), where X 5 is D, A, or N; X 6 is R or E; X 7 is N or S; X 9 is K or N; the HCDR3 amino acid sequence of: GDX 3 X 4 YX 6 YX 8 YGX 11 DX 13 (SEQ ID NO: 226), where X 3 is W or R
• Heavy chain CDR sequences of exemplary agonist LT ⁇ R binding proteins that bind to human LT ⁇ R CRD4, do not block LIGHT binding to LT ⁇ R, do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R, and comprise a HCDR1 of SEQ ID NO: 224, a HCDR2 of SEQ ID NO: 225, and a HCDR3 of SEQ ID NO: 226 are provided in Table 6 above.
• the agonist LT ⁇ R binding proteins of the present disclosure comprising the HCDRs 1–3 of SEQ ID NOs.
• 224-226, respectively, further comprise a shared VL CDR structure defined by the LCDR1 amino acid sequence of TGX 3 X 4 SDVGSYNLVS (SEQ ID NO: 356), where X 3 is S or T; X 4 is N, S, or I; the LCDR2 amino acid sequence of: EVX 3 KRPS (SEQ ID NO: 357), where X 3 is T, N or S; and the LCDR3 amino acid sequence of: CSYAX 5 X 6 X 7 TYV (SEQ ID NO: 358), where X 5 is D or G; X 6 is T or S; X 7 is R, S, or K.
• the agonist LT ⁇ R binding proteins of the present disclosure e.g., agonist LT ⁇ R antibodies or agonist LT ⁇ R bispecific binding proteins
• agonist LT ⁇ R antibodies or agonist LT ⁇ R bispecific binding proteins that bind to human LT ⁇ R CRD4, and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a variable light (VL) domain, a variable heavy (VH) domain, or a combination of VL and VH domain.
• the VH domain of the agonist LT ⁇ R binding protein comprises any one of the VH amino acid sequences provided in Table 8 below, or an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of the VH amino acid sequences listed in Table 8.
• the VL domain of the agonist LT ⁇ R binding protein comprises any one of the VL amino acid sequences provided in Table 8 below, or an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical to any one of the VL amino acid sequences listed in Table 8.
• Exemplary agonist LT ⁇ R binding proteins of the present disclosure that bind to human LT ⁇ R CRD4, and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences selected from: (i) SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 278, SEQ ID NO: 279, and SEQ ID NO: 280, respectively; (ii) SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 281, SEQ ID NO: 282, and SEQ ID NO: 283, respectively; (iii) SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ
• agonist LT ⁇ R binding proteins bind to human LT ⁇ R CRD4, and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R include those binding proteins that comprise HCDR1, HCDR2, and HCDR3 amino acid sequences of SEQ ID NOs: 159-161, respectively, and LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NOs: 278-280, respectively.
• These agonist LT ⁇ R binding proteins further comprise a VH and VL amino acid sequence selected from: a VH amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VH amino acid sequence of SEQ ID NO: 401 and a VL amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VL amino acid sequence of SEQ ID NO: 402; a VH amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VH amino acid sequence of SEQ ID NO: 403 and a VL amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VL amino acid sequence of SEQ ID NO: 404; a VH amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VH amino acid sequence of SEQ ID NO: 405 and a VL amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VL amino acid
• exemplary agonist LT ⁇ R binding proteins of the present disclosure that bind to human LT ⁇ R CRD4, and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences selected from: (i) SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 308, SEQ ID NO: 309, and SEQ ID NO: 310, respectively; (ii) SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 314, SEQ ID NO: 315, and SEQ ID NO: 316, respectively; (iii)
• agonist LT ⁇ R binding proteins of the present disclosure that bind to human LT ⁇ R CRD4, and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R include those binding proteins that comprise HCDR1, HCDR2, and HCDR3 amino acid sequences of SEQ ID NOs: 159-161, respectively, and LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NOs: 308-310, respectively.
• These agonist LT ⁇ R binding proteins further comprise a VH and VL amino acid sequence selected from: a VH amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VH amino acid sequence of SEQ ID NO: 417 and a VL amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VL amino acid sequence of SEQ ID NO: 418; a VH amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VH amino acid sequence of SEQ ID NO: 419 and a VL amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VL amino acid sequence of SEQ ID NO: 420; and a VH amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VH amino acid sequence of SEQ ID NO: 421 and a VL amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VL amino
• exemplary agonist LT ⁇ R binding proteins of the present disclosure that bind to human LT ⁇ R CRD4, and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences selected from: (i) SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 308, SEQ ID NO: 309, and SEQ ID NO: 310; (ii) SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 320, SEQ ID NO: 321, and SEQ ID NO: 322; (iii) SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 308, SEQ ID NO: 309, and SEQ ID NO: 310; (ii)
• agonist LT ⁇ R binding proteins of the present disclosure that bind to human LT ⁇ R CRD4, and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise HCDR1, HCDR2, and HCDR3 amino acid sequences of SEQ ID NOs: 195-197, respectively, and LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NOs: 308-310, respectively.
• These agonist LT ⁇ R binding proteins further comprise a VH and VL amino acid sequence selected from: a VH amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VH amino acid sequence of SEQ ID NO: 423 and a VL amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VL amino acid sequence of SEQ ID NO: 424; a VH amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VH amino acid sequence of SEQ ID NO: 425 and a VL amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VL amino acid sequence of SEQ ID NO: 426; a VH amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VH amino acid sequence of SEQ ID NO: 427 and a VL amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VL amino acid
• exemplary agonist LT ⁇ R binding proteins of the present disclosure that bind to human LT ⁇ R CRD4, and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences selected from: (i) SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 356, SEQ ID NO: 357, and SEQ ID NO: 358, respectively; (ii) SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 359, SEQ ID NO: 360, and SEQ ID NO: 361, respectively; (iii)
• agonist LT ⁇ R binding proteins of the present disclosure that bind to human LT ⁇ R CRD4, and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise HCDR1, HCDR2, and HCDR3 amino acid sequences of SEQ ID NOs: 224-226, respectively, and LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NOs: 356-358, respectively.
• These agonist LT ⁇ R binding proteins further comprise a VH and VL amino acid sequence selected from: a VH amino acid sequence having at least 90% sequence identity to the VH amino acid sequence of SEQ ID NO: 441 and a VL amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VL amino acid sequence of SEQ ID NO: 442; a VH amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VH amino acid sequence of SEQ ID NO: 443 and a VL amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VL amino acid sequence of SEQ ID NO: 444; a VH amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VH amino acid sequence of SEQ ID NO: 445 and a VL amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to the VL amino acid sequence of SEQ ID NO: 446;
• exemplary agonist LT ⁇ R binding proteins of the present disclosure that bind to human LT ⁇ R CRD4, and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprise a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences selected from (i) SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 389, SEQ ID NO: 390, and SEQ ID NO: 391, respectively (1319279); (ii) SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 347, SEQ ID NO: 348 and SEQ ID NO: 349, respectively (13182
• VH and VL amino acid sequences described herein are provided in Table 8 below.
• Table 8 VH and VL amino acid sequences of LT ⁇ R binding proteins that compete for binding to CRD4 of LT ⁇ R with the LT ⁇ R binders LIBC219081 and LIBC218979 and do not block LIGHT or LT ⁇ 1 ⁇ 2 binding
• the agonist LT ⁇ R binding protein e.g., an agonist LT ⁇ R antibody or agonist LT ⁇ R bispecific binding protein
• the present disclosure that binds to human LT ⁇ R CRD4, and (a) does not block LIGHT binding to LT ⁇ R and (b) does not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R comprises a VH region and/or VL region as described supra and further comprises one or more heavy chain constant regions coupled to the VH region and/or a light chain constant region coupled to the VL region.
• the LT ⁇ R binding protein is a Fab comprising a VH and first heavy chain constant domain (CH1) coupled to a VL and light chain constant region (CL).
• the LT ⁇ R binding protein is a F(ab’)2 comprising both LT ⁇ R binding regions of a full antibody coupled by the hinge region, where each binding region comprises a VH-CH1 and VL-CL.
• the LT ⁇ R binding protein is an antibody comprising full light chains (VL-CL) and full heavy chains (VH-CH1- CH2-CH3).
• Exemplary amino acid sequences of LT ⁇ R antibodies of the present disclosure that bind to human LT ⁇ R CRD4, and (a) do not block LIGHT binding to LT ⁇ R and (b) do not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R are provided in Table 9 below.
• Table 9 Antibody HC and LC Sequences of LT ⁇ R CRD4 Binding Antibodies
• this VH comprises a HCDR1 amino acid sequence of any one of SEQ ID NOs: 475-498,740-742, or a modified sequence thereof, wherein said modified sequence contains 1, 2, or 3 amino acid residue modifications as compared to any one of SEQ ID NOs: 475-498 and 740-742.
• the VH comprises a HCDR2 amino acid sequence of any one of SEQ ID NOs: 499-528, 743, or a modified sequence thereof, wherein said modified sequence contains 1, 2, or 3 amino acid residue modifications as compared to any one of SEQ ID NOs: 499-528 and 743.
• the VH comprises a HCDR3 amino acid sequence of any one of SEQ ID NOs: 529-558, 744-746, or a modified sequence thereof, wherein said modified sequence contains 1, 2, or 3 amino acid residue modifications as compared to any one of SEQ ID NOs: 529-558 and 744-746.
• the heavy chain CDR sequences of exemplary agonist LT ⁇ R binding proteins described herein are provided in Table 10 below. [0158] As described in the Examples herein, paratope mapping studies of several LT ⁇ R antibodies disclosed herein showed that, in some cases, binding of a particular LT ⁇ R antibody to LT ⁇ R did not involve or require all three heavy chain CDR regions of the antibody and/or did not involve all residues of one or more heavy chain CDRs.
• Heavy chain CDRs shown not to be involved in the LT ⁇ R antibody binding interaction are indicated with an asterisk in Table 10 below. Residues of heavy chain CDR regions shown not to be involved in or required for the LT ⁇ R antibody binding interaction as identified by paratope mapping or mutational analysis are identified as variable residue (X). The identity of X residues within the sequences of Table 10 is provided in Tables 18-25 herein. Table 10.
• the agonist LT ⁇ R binding protein of the present disclosure comprises a light chain variable region (VL), where the VL comprises one or more complementarity determining regions (i.e., LCDR1, LCDR2, and/or LCDR3), or portions thereof, that alone or together with the VH CDRs bind to LT ⁇ R.
• the VL comprises a LCDR1 amino acid sequence of any one of SEQ ID NOs: 559-589 or a modified sequence thereof, wherein said modified sequence contains 1, 2, or 3 amino acid residue modifications as compared to any one of SEQ ID NOs: 559-589.
• the VL comprises a LCDR2 amino acid sequence of any one of SEQ ID NOs: 590-618 or a modified sequence thereof, wherein said modified sequence contains 1, 2, or 3 amino acid residue modifications as compared to any one of SEQ ID NOs: 590-618.
• the VL comprises a LCDR3 amino acid sequence of any one of SEQ ID NOs: 619-643 or a modified sequence thereof, wherein said modified sequence contains 1, 2, or 3 amino acid residue modifications as compared to any one of SEQ ID NOs: 619-643.
• the light chain CDR sequences of exemplary agonist LT ⁇ R binding proteins described herein are provided in Table 11 below.
• the agonist LT ⁇ R binding protein of the present disclosure binds to CRD2 and CRD3 of human LT ⁇ R, including one or more residues corresponding to residues 56-64 and 81- 101 of SEQ ID NO: 4 (corresponding to residues 86-94 and 111-131 of SEQ ID NO: 1), and permits endogenous LT ⁇ R ligand binding activity as defined herein.
• This agonist LT ⁇ R binding protein comprises a variable heavy domain (VH) comprising a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 501, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 531.
• VH variable heavy domain
• this VH further comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 475.
• the VH of this LT ⁇ R binding protein comprises the HCDR2 sequence of SEQ ID NO: 499 and the HCDR3 sequence of SEQ ID NO: 529; or as further defined by the HCDR2 sequence of SEQ ID NO: 500 and the HCDR3 sequence of SEQ ID NO: 530.
• the VH of this LT ⁇ R binding protein comprises the HCDR2 sequence of SEQ ID NO: 501 and the HCDR3 sequence of SEQ ID NO: 531.
• the VH of this exemplary agonist LT ⁇ R binding protein further comprises the HCDR1 of SEQ ID NO: 475.
• this agonist LT ⁇ R binding protein optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 561, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 619.
• the VL further comprises an LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 590.
• the VL comprises the LCDR1 sequence of SEQ ID NO: 559 and the LCDR3 sequence of SEQ ID NO: 619; or as further defined by the LCDR1 sequence of SEQ ID NO: 560 and the LCDR3 sequence of SEQ ID NO: 619; or as further defined by the LCDR1 sequence of SEQ ID NO: 560 and the LCDR3 sequence of SEQ ID NO: 619.
• the VL further comprises an LCDR2 of SEQ ID NO: 590.
• an exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.218990 (19320; 30H1).
• the agonist LT ⁇ R binding protein of the present disclosure is a binding protein that binds to the CRD1 of LT ⁇ R, including one or more residues corresponding to residues 3-9, 20-29, and 38-47 of SEQ ID NO: 4 (corresponding to residues 33-39, 50-59, and 68-77 of SEQ ID NO: 1).
• this LT ⁇ R binding protein does not block or inhibit endogenous LIGHT binding to LT ⁇ R to occur and at least or about 80% of endogenous LT ⁇ 1 ⁇ 2 binding to LT ⁇ R to occur.
• This LT ⁇ R binding protein comprises a VH, where the VH comprises HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 478, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 504, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 534
• the VH of this LT ⁇ R binding protein comprises the HCDR1 sequence of SEQ ID NO: 476, the HCDR2 sequence of SEQ ID NO: 502, and the HCDR3 sequence of SEQ ID NO: 532; or as further defined by the HCDR1 sequence of SEQ ID NO: 477, the HCDR2 sequence of SEQ ID NO: 503, and the HCDR3 sequence of SEQ ID NO: 533; or as further defined by the HCDR1 sequence of SEQ ID NO: 478, the HCDR2 sequence of SEQ ID NO: 504, and the HCDR3 sequence of SEQ ID NO: 534.
• this agonist LT ⁇ R binding protein of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 564, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 593, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 622.
• the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at
• the VL comprises the LCDR1 sequence of SEQ ID NO: 562, the LCDR2 of SEQ ID NO: 591, and the LCDR3 sequence of SEQ ID NO: 620; or as further defined by the LCDR1 sequence of SEQ ID NO: 563, the LCDR2 of SEQ ID NO: 592, and the LCDR3 sequence of SEQ ID NO: 621; or as further defined by the LCDR1 sequence of SEQ ID NO: 564, the LCDR2 of SEQ ID NO: 593, and the LCDR3 sequence of SEQ ID NO: 622.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.218994 (19321; 31A3) [0165]
• the agonist LT ⁇ R binding protein of the present disclosure binds to CRD2 and CRD3 of human LT ⁇ R, including one or more residues corresponding to residues 56-64 and 81- 101 of SEQ ID NO: 4 (corresponding to residues 86-94 and 111-131 of SEQ ID NO: 1).
• this agonist LT ⁇ R binding protein does not block endogenous LIGHT or LT ⁇ 1 ⁇ 2 binding to LT ⁇ R at concentrations of ⁇ 8nM as measured by the cell based receptor-ligand assay described herein (see e.g., Example 8.2).
• This LT ⁇ R binding protein comprises a VH, where the VH comprises HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 479, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 507, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 537.
• the VH of this LT ⁇ R binding protein comprises the HCDR1 sequence of SEQ ID NO: 479, a HCDR2 sequence of SEQ ID NO: 505, and a HCDR3 sequence of SEQ ID NO: 535; or as further defined by the HCDR1 sequence of SEQ ID NO: 479, a HCDR2 sequence of SEQ ID NO: 506, and a HCDR3 sequence of SEQ ID NO: 536; or as further defined by the HCDR1 sequence of SEQ ID NO: 479, a HCDR2 sequence of SEQ ID NO: 507, and a HCDR3 sequence of SEQ ID NO: 537.
• this agonist LT ⁇ R binding protein of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 596.
• the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 565, and optionally comprises an LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 623.
• the VL comprises the LCDR2 of SEQ ID NO: 594, or as further defined by the LCDR2 of SEQ ID NO: 595, or as further defined by the LCDR2 of SEQ ID NO: 596.
• the VL comprises the LCDR1 sequence of SEQ ID NO: 565, the LCDR2 of SEQ ID NO: 596, and the LCDR3 sequence of SEQ ID NO: 623.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.219058 (19324; 36G2).
• the agonist LT ⁇ R binding protein of the present disclosure is a binding protein that binds to LT ⁇ R and does not block or inhibit LIGHT binding to LT ⁇ R to occur.
• This agonist LT ⁇ R binding protein comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 480, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 508, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 538.
• the VH comprises the HCDR1 sequence of SEQ ID NO: 408, the HCDR2 of SEQ ID NO: 508, and the HCDR3 sequence of SEQ ID NO: 538.
• this agonist LT ⁇ R binding protein of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 567, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 600, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%,
• the VL comprises the LCDR1 sequence of SEQ ID NO: 567, the LCDR2 of SEQ ID NO: 600, and the LCDR3 sequence of SEQ ID NO: 625.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.219037 (19322; 34D1).
• the agonist LT ⁇ R binding protein of the present disclosure is a binding protein that binds to LT ⁇ R and does not block or inhibit LIGHT binding to LT ⁇ R as defined herein. In one embodiment, this agonist LT ⁇ R binding protein and does not block or inhibit LIGHT binding to LT ⁇ R.
• This LT ⁇ R binding protein comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 4812, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 509, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 539.
• the VH comprises the HCDR1 sequence of SEQ ID NO: 481, the HCDR2 of SEQ ID NO: 509, and the HCDR3 sequence of SEQ ID NO: 539 or as further defined by the HCDR1 sequence of SEQ ID NO: 481, the HCDR2 of SEQ ID NO: 509, and the HCDR3 sequence of SEQ ID NO: 540; or as further defined by the HCDR1 sequence of SEQ ID NO: 481, the HCDR2 of SEQ ID NO: 509, and the HCDR3 sequence of SEQ ID NO: 541.
• this agonist LT ⁇ R binding protein of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 570, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 601, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 626.
• the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
• the VL comprises the LCDR1 sequence of SEQ ID NO: 568, the LCDR2 of SEQ ID NO: 601, and the LCDR3 sequence of SEQ ID NO: 626; or as further defined by the LCDR1 sequence of SEQ ID NO: 569, the LCDR2 of SEQ ID NO: 601, and the LCDR3 sequence of SEQ ID NO: 626; or as further defined by the LCDR1 sequence of SEQ ID NO: 570, the LCDR2 of SEQ ID NO: 601, and the LCDR3 sequence of SEQ ID NO: 626; or as further defined by the LCDR1 sequence of SEQ ID NO: 571, the LCDR2 of SEQ ID NO: 602, and the LCDR3 sequence of SEQ ID NO: 627.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.219098 (19327; 44B1).
• the agonist LT ⁇ R binding protein of the present disclosure binds to human LT ⁇ R CRD1 including one or more residues corresponding to residues 1-19 of SEQ ID NO: 4 (corresponding to residues 31-49 of SEQ ID NO: 1).
• This agonist LT ⁇ R binding protein does not block LIGHT binding to LT ⁇ R and does not block LT ⁇ 1 ⁇ 2 binding to LT ⁇ R.
• This agonist LT ⁇ R binding protein comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 483, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 513, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 543.
• the VH comprises the HCDR1 sequence of SEQ ID NO: 483, the HCDR2 of SEQ ID NO: 511, and the HCDR3 sequence of SEQ ID NO: 543; or as further defined the HCDR1 sequence of SEQ ID NO: 483, the HCDR2 of SEQ ID NO: 512, and the HCDR3 sequence of SEQ ID NO: 543; or as further defined the HCDR1 sequence of SEQ ID NO: 483, the HCDR2 of SEQ ID NO: 513, and the HCDR3 sequence of SEQ ID NO: 543.
• the agonist LT ⁇ R binding protein of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 574, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 603, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 628.
• the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
• the VL comprises the LCDR1 sequence of SEQ ID NO: 57217, the LCDR2 of SEQ ID NO: 603, and the LCDR3 sequence of SEQ ID NO: 628; or as further defined by the LCDR1 sequence of SEQ ID NO: 573, the LCDR2 of SEQ ID NO: 603, and the LCDR3 sequence of SEQ ID NO: 628; or as further defined by the LCDR1 sequence of SEQ ID NO: 574, the LCDR2 of SEQ ID NO: 603, and the LCDR3 sequence of SEQ ID NO: 628.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.219051 (19323; 35F5).
• the LT ⁇ R binding protein of the present disclosure is an antibody- based molecule that binds to LT ⁇ R CRD1 and CRD2, including one or more residues corresponding to residues 38-42 and 56-70 of SEQ ID NO: 4.
• This LT ⁇ R antibody-based molecule comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 742, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 746.
• the VH of this LT ⁇ R binding protein further comprises a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 743.
• the VH of this LT ⁇ R binding protein comprises the HCDR1 sequence of SEQ ID NO: 740 and the HCDR3 sequence of SEQ ID NO: 744; or as further defined by the HCDR1 sequence of SEQ ID NO: 741 and the HCDR3 sequence of SEQ ID NO: 745; or as further defined by the HCDR1 sequence of SEQ ID NO: 742 and the HCDR3 sequence of SEQ ID NO: 746.
• the VH of this exemplary LT ⁇ R binding protein further comprises the HCDR2 of SEQ ID NO: 743.
• this LT ⁇ R antibody-based molecule of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 566, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 599.
• the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 599.
• the VL further comprises a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 624.
• the VL comprises the LCDR1 sequence of SEQ ID NO: 566 and the LCDR2 of SEQ ID NO: 597; or as further defined by the LCDR1 sequence of SEQ ID NO: 566 and the LCDR2 of SEQ ID NO: 598; or as further defined by the LCDR1 sequence of SEQ ID NO: 566 and the LCDR2 of SEQ ID NO: 599.
• the VL further comprises a LCDR3 of SEQ ID NO: 624.
• an exemplary LT ⁇ R antibody-based molecule comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.219097 (19326; 43D9).
• the agonist LT ⁇ R binding protein comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 484, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 514, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence
• the VH comprises the HCDR1 sequence of SEQ ID NO: 484, the HCDR2 of SEQ ID NO: 514, and the HCDR3 sequence of SEQ ID NO: 544.
• This agonist LT ⁇ R binding protein further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 575, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 604, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 629.
• the VL comprises the LCDR1 sequence of SEQ ID NO: 575, the LCDR2 of SEQ ID NO: 604, and the LCDR3 sequence of SEQ ID NO: 629.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.218989.
• the agonist LT ⁇ R binding protein comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 485, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 515, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 545.
• the VH comprises the HCDR1 sequence of SEQ ID NO: 485, the HCDR2 of SEQ ID NO: 515, and the HCDR3 sequence of SEQ ID NO: 545.
• This agonist LT ⁇ R binding protein of the present disclosure further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 576, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 605, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO:
• the VL comprises the LCDR1 sequence of SEQ ID NO: 576, the LCDR2 of SEQ ID NO: 605, and the LCDR3 sequence of SEQ ID NO: 630.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.218967.
• the agonist LT ⁇ R binding protein of the present disclosure comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 486, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 516, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 546.
• the VH comprises the HCDR1 sequence of SEQ ID NO: 486, the HCDR2 of SEQ ID NO: 516, and the HCDR3 sequence of SEQ ID NO: 546.
• This agonist LT ⁇ R binding protein of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 577, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 606, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID
• the VL comprises the LCDR1 sequence of SEQ ID NO: 577, the LCDR2 of SEQ ID NO: 6068, and the LCDR3 sequence of SEQ ID NO: 631.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.218981.
• the agonist LT ⁇ R binding protein of the present disclosure comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 487, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 517, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 547.
• the VH comprises the HCDR1 sequence of SEQ ID NO: 487, the HCDR2 of SEQ ID NO: 517 and the HCDR3 sequence of SEQ ID NO: 547.
• This agonist LT ⁇ R binding protein of the present disclosure further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 578, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 607, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO
• the VL comprises the LCDR1 sequence of SEQ ID NO: 578, the LCDR2 of SEQ ID NO: 607, and the LCDR3 sequence of SEQ ID NO: 632.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.218995.
• the agonist LT ⁇ R binding protein of the present disclosure comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 488, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 518, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 548.
• the VH comprises the HCDR1 sequence of SEQ ID NO: 488, the HCDR2 of SEQ ID NO: 518, and the HCDR3 sequence of SEQ ID NO: 548.
• This agonist LT ⁇ R binding protein of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 579, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 608, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ
• the VL comprises the LCDR1 sequence of SEQ ID NO: 579, the LCDR2 of SEQ ID NO: 608, and the LCDR3 sequence of SEQ ID NO: 633.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.218996.
• the VH comprises the HCDR1 sequence of SEQ ID NO: 489, the HCDR2 of SEQ ID NO: 519, and the HCDR3 sequence of SEQ ID NO: 549.
• This agonist LT ⁇ R binding protein of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 580, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 609, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of
• the VL comprises the LCDR1 sequence of SEQ ID NO: 580, the LCDR2 of SEQ ID NO: 609, and the LCDR3 sequence of SEQ ID NO: 634.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.218998.
• the agonist LT ⁇ R binding protein of the present disclosure comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 490, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 520, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 550.
• the VH comprises the HCDR1 sequence of SEQ ID NO: 490, the HCDR2 of SEQ ID NO: 520, and the HCDR3 sequence of SEQ ID NO: 550.
• This agonist LT ⁇ R binding protein of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 581, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 610, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of
• the VL comprises the LCDR1 sequence of SEQ ID NO: 581, the LCDR2 of SEQ ID NO: 610, and the LCDR3 sequence of SEQ ID NO: 635.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.219083.
• the agonist LT ⁇ R binding protein of the present disclosure comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 491, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 521, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 551.
• the VH comprises the HCDR1 sequence of SEQ ID NO: 491, the HCDR2 of SEQ ID NO: 521, and the HCDR3 sequence of SEQ ID NO: 551.
• This agonist LT ⁇ R binding protein of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 582, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 611, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID
• the VL comprises the LCDR1 sequence of SEQ ID NO: 582, the LCDR2 of SEQ ID NO: 611, and the LCDR3 sequence of SEQ ID NO: 636.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.219092.
• the agonist LT ⁇ R binding protein of the present disclosure is a binding protein that binds to LT ⁇ R and permits endogenous LT ⁇ R ligand binding activity as defined herein.
• This LT ⁇ R binding protein comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 492, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 522, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 552.
• the VH comprises the HCDR1 sequence of SEQ ID NO: 492, the HCDR2 of SEQ ID NO: 522, and the HCDR3 sequence of SEQ ID NO: 552.
• the HCDR2 of SEQ ID NO: 522 is modified to substitute the cysteine residues at positions 4 and 8 in SEQ ID NO: 522.
• the HCDR2 of this LT ⁇ R binding protein comprises an HCDR2 of SEQ ID NO: 523.
• This agonist LT ⁇ R binding protein of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 583, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 612, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 637.
• the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at
• the VL comprises the LCDR1 sequence of SEQ ID NO: 583, the LCDR2 of SEQ ID NO: 612, and the LCDR3 sequence of SEQ ID NO: 637.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.219023.
• the agonist LT ⁇ R binding protein of the present disclosure comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 494, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 524, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 554.
• the VH comprises the HCDR1 sequence of SEQ ID NO: 494, the HCDR2 of SEQ ID NO: 524, and the HCDR3 sequence of SEQ ID NO: 554.
• This agonist LT ⁇ R binding protein of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 585, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 614, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ
• the VL comprises the LCDR1 sequence of SEQ ID NO: 585, the LCDR2 of SEQ ID NO: 614, and the LCDR3 sequence of SEQ ID NO: 639.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.219095.
• the agonist LT ⁇ R binding protein of the present disclosure comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 495, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 525, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 555.
• the VH comprises the HCDR1 sequence of SEQ ID NO: 495, the HCDR2 of SEQ ID NO: 525, and the HCDR3 sequence of SEQ ID NO: 555.
• This agonist LT ⁇ R binding protein of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 586, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 615, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of
• the VL comprises the LCDR1 sequence of SEQ ID NO: 586, the LCDR2 of SEQ ID NO: 615, and the LCDR3 sequence of SEQ ID NO: 640.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.219102.
• the agonist LT ⁇ R binding protein of the present disclosure comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 496, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 526, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 556.
• the VH comprises the HCDR1 sequence of SEQ ID NO: 496, the HCDR2 of SEQ ID NO: 526, and the HCDR3 sequence of SEQ ID NO: 556.
• This agonist LT ⁇ R binding protein of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 587, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 616, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of
• the VL comprises the LCDR1 sequence of SEQ ID NO: 587, the LCDR2 of SEQ ID NO: 616, and the LCDR3 sequence of SEQ ID NO: 641.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.218973.
• the agonist LT ⁇ R binding protein of the present disclosure comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 497, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 527, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 557.
• the VH comprises the HCDR1 sequence of SEQ ID NO: 497, the HCDR2 of SEQ ID NO: 527, and the HCDR3 sequence of SEQ ID NO: 557.
• This agonist LT ⁇ R binding protein of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 588, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 617, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of
• the VL comprises the LCDR1 sequence of SEQ ID NO: 588, the LCDR2 of SEQ ID NO: 617, and the LCDR3 sequence of SEQ ID NO: 642.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.219044.
• the agonist LT ⁇ R binding protein of the present disclosure comprises a VH, where the VH comprises a HCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 498, a HCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 528, and a HCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence SEQ ID NO: 558.
• the VH comprises the HCDR1 sequence of SEQ ID NO: 498, the HCDR2 of SEQ ID NO: 528, and the HCDR3 sequence of SEQ ID NO: 558.
• This agonist LT ⁇ R binding protein of the present disclosure optionally further comprises a VL, where the VL comprises a LCDR1 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 589, a LCDR2 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 618, and a LCDR3 sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of
• the VL comprises the LCDR1 sequence of SEQ ID NO: 589, the LCDR2 of SEQ ID NO: 618, and the LCDR3 sequence of SEQ ID NO: 643.
• An exemplary agonist LT ⁇ R binding protein comprising a VH and VL of this embodiment includes, without limitation, the LT ⁇ R antibody identified herein as LIBC No.218997.
• Suitable amino acid modifications to the heavy chain CDR sequences and/or the light chain CDR sequences of the LT ⁇ R binding protein disclosed herein include, for example, conservative substitutions or functionally equivalent amino acid residue substitutions that result in variant CDR sequences having similar or enhanced binding characteristics to those of the CDR sequences described above.
• CDRs of Tables 2, 3, 6, 7, 9 and 10 containing 1, 2, 3, 4, 5, or more amino acid substitutions (depending on the length of the CDR) that maintain or enhance LT ⁇ R binding of the antibody.
• the resulting modified CDRs are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% similar in sequence to the CDRs as provided in Tables 2, 3, 6, 7, 9 and 10.
• Suitable amino acid modifications to the heavy chain and light chain CDR sequences disclosed herein include, for example, conservative substitutions or functionally equivalent amino acid residue substitutions that result in variant CDR sequences having similar or enhanced binding characteristics to those of the CDR sequences disclosed herein.
• Conservative substitutions are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids can be divided into four families: (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
• the amino acid repertoire can be grouped as (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), with serine and threonine optionally grouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur-containing (cysteine and methionine) (Stryer (ed.), Biochemistry, 2nd ed, WH Freeman and Co., 1981, which is hereby incorporated by reference in its entirety).
• Non- conservative substitutions can also be made to the heavy chain CDR sequences and the light chain CDR sequences disclosed herein.
• Non-conservative substitutions involve substituting one or more amino acid residues of the CDR with one or more amino acid residues from a different class of amino acids to improve or enhance the binding properties of CDR.
• the amino acid sequences of the heavy chain variable region CDRs and/or the light chain variable region CDRs disclosed herein may further comprise one or more internal neutral amino acid insertions or deletions that maintain or enhance LT ⁇ R binding.
• the LT ⁇ R binding proteins that bind LT ⁇ R as described herein may comprise a variable light (VL) chain, a variable heavy (VH) chain, or a combination of VL and VH chains.
• the VH chain of the LT ⁇ R binding protein comprises any one of the VH amino acid sequences provided in Table 12 below, or an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of the VH amino acid sequences listed in Table 12.
• the VL chain of the LT ⁇ R binding protein comprises any one of the VL amino acid sequences provided in Table 12 below, or an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical to any one of the VL amino acid sequences listed in Table 12.
• an agonist LT ⁇ R binding protein of the present disclosure binds to and agonizes human LT ⁇ R (SEQ ID NO: 1) while allowing endogenous LT ⁇ R ligand binding and activity (i.e., LIGHT or LT ⁇ 1 ⁇ 2 binding) as described herein, and comprises a heavy chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to any one of the VH domain amino acid sequences set forth in Table 12.
• an agonist LT ⁇ R binding protein of the present disclosure binds to and agonizes human LT ⁇ R (SEQ ID NO: 1) while allowing endogenous LT ⁇ R ligand binding and activity (i.e., LIGHT or LT ⁇ 1 ⁇ 2 binding) as described herein, and comprises a light chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to any one of the VL domain amino acid sequences set forth in Table 12.
• the agonist LT ⁇ R binding protein of the present disclosure binds to and agonizes human LT ⁇ R (SEQ ID NO: 1) while allowing endogenous LT ⁇ R ligand binding and activity (i.e., LIGHT or LT ⁇ 1 ⁇ 2 binding) as described herein, and comprises a heavy chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to any one of the VH domain amino acid sequences set forth in Table 12, and a light chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to the corresponding VL domain amino acid sequence as set forth in Table 12. TABLE 12.
• an agonist LT ⁇ R binding protein of the present disclosure binds to and agonizes human and cyno LT ⁇ R.
• This agonist LT ⁇ R binding protein binds CRD2 and CRD3 of human LT ⁇ R at an epitope comprising or consisting of one or more residues at positions 56-64 and 81-101 of SEQ ID NO: 4 (corresponding to residues 86-94 and 111-131 of SEQ ID NO: 1).
• This agonist LT ⁇ R binding protein comprises a heavy chain variable domain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the VH domain amino acid sequence of SEQ ID NO: 644 and a light chain variable domain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the VL domain amino acid sequence of SEQ ID NO: 645.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 644 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 645.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 644 and a VL amino acid sequence of SEQ ID NO: 645 (30H1/19320).
• an agonist LT ⁇ R binding protein of the present disclosure binds to and agonizes human and cyno LT ⁇ R. This agonist LT ⁇ R binding protein binds CRD1 of human LT ⁇ R at an epitope comprising or consisting of one or more residues at positions 3-9, 12-29, and 38-47 of SEQ ID NO: 4 (corresponding to residues 33-39, 50-59, and 68-77 of SEQ ID NO: 1).
• This agonist LT ⁇ R binding protein allows 100% of LIGHT and/or at least or about 80% of LT ⁇ 1 ⁇ 2 binding to LT ⁇ R to occur and comprises a heavy chain variable domain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the VH domain amino acid sequence of SEQ ID NO: 646.
• This LT ⁇ R binding protein further comprises a light chain variable domain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the VL domain amino acid sequence of SEQ ID NO: 647.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 646 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 647. In one embodiment, the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 646 and a VL amino acid sequence of SEQ ID NO: 647 (19321). [0195] In one embodiment, an agonist LT ⁇ R binding protein of the present disclosure binds to and agonizes human and cyno LT ⁇ R.
• This agonist LT ⁇ R binding protein binds to CRD2 and CRD3 of human LT ⁇ R at an epitope comprising or consisting of one or more residues at positions 56-64 and 81-101 of SEQ ID NO: 4 (corresponding to residues 86-94 and 111-131 of SEQ ID NO: 1).
• This agonist LT ⁇ R binding protein does not inhibit LIGHT and LT ⁇ 1 ⁇ 2 binding to LT ⁇ R at concentrations of ⁇ 8 nM in the cell based receptor-ligand assay as described herein.
• This LT ⁇ R binding protein and comprises a heavy chain variable domain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the VH domain amino acid sequence of SEQ ID NO: 648 and a light chain variable domain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the VL domain amino acid sequence of SEQ ID NO: 649.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 648 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 649.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 648 and a VL amino acid sequence of SEQ ID NO: 649 (19324).
• an agonist LT ⁇ R binding protein of the present disclosure binds to and agonizes human and cyno LT ⁇ R. This LT ⁇ R binding protein binds to CRD1 and CRD2 of human LT ⁇ R at an epitope comprising or consisting of one or more residues at positions 38-42, 56-65, and 65-70 of SEQ ID NO: 4.
• This agonist LT ⁇ R binding protein comprises a heavy chain variable domain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the VH domain amino acid sequence of SEQ ID NO: 650 and a light chain variable domain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the VL domain amino acid sequence of SEQ ID NO: 651.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 650 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 651.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 650 and a VL amino acid sequence of SEQ ID NO: 651.
• an agonist LT ⁇ R binding protein of the present disclosure binds to and agonizes human and cyno LT ⁇ R, and does not inhibit endogenous LIGHT binding to LT ⁇ R.
• This agonist LTßR binding protein comprises a heavy chain variable domain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the VH domain amino acid sequence of SEQ ID NO: 652 and a light chain variable domain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the VL domain amino acid sequence of SEQ ID NO: 653.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 652 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 653.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 652 and a VL amino acid sequence of SEQ ID NO: 653 (19322).
• an agonist LT ⁇ R binding protein of the present disclosure binds to and agonizes human and cyno LT ⁇ R, and does not inhibit endogenous LIGHT binding to LT ⁇ R.
• This agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 654 and a VL amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 655 or SEQ ID NO: 657.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 654 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 657. In one embodiment, the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 654 and a VL amino acid sequence of SEQ ID NO: 657 (19327). [0199] In one embodiment, an LT ⁇ R binding protein of the present disclosure binds to and agonizes human and cyno LT ⁇ R, and does not inhibit LT ⁇ 1 ⁇ 2 binding to LT ⁇ R and does not inhibit LIGHT binding to LT ⁇ R to occur.
• This agonist LT ⁇ R binding protein comprises a heavy chain variable domain having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the VH domain amino acid sequence of SEQ ID NO: 658 and a light chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to the VL domain amino acid sequence of SEQ ID NO: 659.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 658 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 659.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 658 and a VL amino acid sequence of SEQ ID NO: 659. (35F5/19323) [0200]
• the agonist LT ⁇ R binding protein of the present disclosure binds to and agonizes human and cyno LT ⁇ R and comprises a VH amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 660 and a VL amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 661.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 660 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 661. In one embodiment, the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 660 and a VL amino acid sequence of SEQ ID NO: 661.
• the agonist LT ⁇ R binding protein of the present disclosure binds to and agonizes human and cyno LT ⁇ R and comprises a VH amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 662 and a VL amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 663.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 662 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 663.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 662 and a VL amino acid sequence of SEQ ID NO: 663.
• the agonist LT ⁇ R binding protein of the present disclosure is an agonist binding protein that binds to LT ⁇ R and comprises a VH amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 664 and a VL amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 665.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 664 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 665. In one embodiment, the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 664 and a VL amino acid sequence of SEQ ID NO: 665.
• the agonist LT ⁇ R binding protein of the present disclosure is an agonist binding protein that binds to LT ⁇ R and comprises a VH amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 666 and a VL amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 667.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 666 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 667.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 666 and a VL amino acid sequence of SEQ ID NO: 667.
• the agonist LT ⁇ R binding protein of the present disclosure is an agonist binding protein that binds to LT ⁇ R and comprises a VH amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 668 and a VL amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 669.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 668 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 669. In one embodiment, the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 668 and a VL amino acid sequence of SEQ ID NO: 669.
• the agonist LT ⁇ R binding protein of the present disclosure is an agonist binding protein that binds to LT ⁇ R and comprises a VH amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 670 and a VL amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 671.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 670 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 671.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 670 and a VL amino acid sequence of SEQ ID NO: 671.
• the agonist LT ⁇ R binding protein of the present disclosure is an agonist binding protein that binds to LT ⁇ R and comprises a VH amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 672 and a VL amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 673.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 672 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 673. In one embodiment, the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 672 and a VL amino acid sequence of SEQ ID NO: 673.
• the agonist LT ⁇ R binding protein of the present disclosure is an agonist binding protein that binds to LT ⁇ R and comprises a VH amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 674 and a VL amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 675.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 674 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 675.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 674 and a VL amino acid sequence of SEQ ID NO: 675.
• the agonist LT ⁇ R binding protein of the present disclosure is an agonist binding protein that binds to LT ⁇ R and comprises a VH amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 676 and a VL amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 677.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 676 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 677. In one embodiment, the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 676 and a VL amino acid sequence of SEQ ID NO: 677.
• the agonist LT ⁇ R binding protein of the present disclosure is an agonist binding protein that binds to LT ⁇ R and comprises a VH amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 678 and a VL amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 679.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 678 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 679.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 678 and a VL amino acid sequence of SEQ ID NO: 679.
• the agonist LT ⁇ R binding protein of the present disclosure is an agonist binding protein that binds to LT ⁇ R and comprises a VH amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 680 and a VL amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 681.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 680 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 681. In one embodiment, the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 680 and a VL amino acid sequence of SEQ ID NO: 681.
• the agonist LT ⁇ R binding protein of the present disclosure is an agonist binding protein that binds to LT ⁇ R and comprises a VH amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 682 and a VL amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 683 or SEQ ID NO: 685.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 682 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 683 or SEQ ID NO: 685.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 682 and a VL amino acid sequence of SEQ ID NO: 683 or SEQ ID NO: 685.
• the agonist LT ⁇ R binding protein of the present disclosure is an agonist binding protein that binds to LT ⁇ R and comprises a VH amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 686 and a VL amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 687.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 686 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 687. In one embodiment, the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 686 and a VL amino acid sequence of SEQ ID NO: 687.
• the agonist LT ⁇ R binding protein of the present disclosure is an agonist binding protein that binds to LT ⁇ R and comprises a VH amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 688 and a VL amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 689.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 688 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 689.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 688 and a VL amino acid sequence of SEQ ID NO: 689.
• the agonist LT ⁇ R binding protein of the present disclosure is an agonist binding protein that binds to LT ⁇ R and comprises a VH amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 690 and a VL amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 691.
• the agonist LT ⁇ R binding protein comprises a VH amino acid sequence that is at least 95% identical to SEQ ID NO: 690 and a VL amino acid sequence that is at least 95% identical to SEQ ID NO: 691. In one embodiment, the agonist LT ⁇ R binding protein comprises a VH amino acid sequence of SEQ ID NO: 690 and a VL amino acid sequence of SEQ ID NO: 691. [0215] In one embodiment, the agonist LT ⁇ R binding protein of the present disclosure comprises a VH region and/or VL region as described supra and further comprises one or more heavy chain constant regions coupled to the VH region and/or a light chain constant region coupled to the VL region.
• the agonist LT ⁇ R binding protein is a Fab comprising a VH and first heavy chain constant domain (CH1) coupled to a VL and light chain constant region (CL).
• the agonist LT ⁇ R binding protein is a F(ab’)2 comprising both LT ⁇ R binding regions of a full antibody coupled by the hinge region, where each binding region comprises a VH-CH1 and VL-CL.
• the agonist LT ⁇ R binding protein is a multi- specific agonist LT ⁇ R binding protein.
• the multi-specific agonist LT ⁇ R binding protein comprises a first binding domain that binds to human LT ⁇ R (SEQ ID NO: 1), and a second binding domain that binds a non-LT ⁇ R protein target (i.e., a protein other than LT ⁇ R), where the multi-specific binding protein agonizes LT ⁇ R activity and (a) does not inhibit LIGHT binding to LTßR and (b) does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R.
• the second binding domain of an exemplary LT ⁇ R multi-specific binding protein as described herein, binds to a non-LT ⁇ R protein target.
• the second binding domain of the LT ⁇ R multi-specific binding protein binds to a protein or antigen preferentially expressed by a tumor cell or other cells of the tumor microenvironment (TME).
• TEE tumor microenvironment
• the multi-specific agonist LT ⁇ R binding protein is monovalent for each of the protein targets bound by the first and second binding domains multi-specific binding protein.
• the multi-specific agonist LT ⁇ R binding protein is monovalent for the LT ⁇ R ligand binding domain and bivalent for the non-LT ⁇ R binding domain.
• the multi-specific agonist LT ⁇ R binding protein of the present disclosure further comprises one or more additional binding domains.
• the multi- specific agonist LT ⁇ R binding protein described herein comprises a third binding domain, where the third binding domain binds the same protein target as the first or second binding domain to form a bi-specific binding protein that is bivalent for one of the protein targets bound by the bi-specific binding protein.
• the third binding domain binds a different protein target than the first and second binding domains, thereby forming a monovalent trispecific binding protein.
• the multi- specific agonist LT ⁇ R binding protein can further comprise a fourth binding domain, where the fourth binding domain binds to the same protein target as the first, second, or third binding domain.
• the multi-specific agonist LT ⁇ R binding protein can assume any multi-specific binding protein format known in the art.
• the multi-specific agonist LT ⁇ R binding protein is an agonist LT ⁇ R bispecific binding protein or a trispecific agonist LT ⁇ R binding protein.
• the first, second, and/or third binding domain of the multi-specific agonist LT ⁇ R binding domain is a Fab.
• the first, second, and/or third binding domain of a multi-specific agonist LT ⁇ R binding domain is a scFv.
• the first binding domain is a Fab
• the second binding domain is a scFv
• an optionally present third binding domain is a Fab or scFv.
• the second binding domain is a Fab
• the first binding domain is a scFv
• an optionally present third binding domain is a Fab or scFv.
• both the first and second binding domains are Fabs
• the optionally present third binding domain is a Fab or scFv.
• both the first and second binding domains are scFvs
• the optionally present third binding domain is a Fab or scFv.
• the multi-specific agonist LT ⁇ R binding protein is a bispecific agonist LT ⁇ R binding protein.
• a “bispecific binding protein” as referred to herein is a binding protein that binds to two different antigens at the same time.
• Bispecific agonist LT ⁇ R binding proteins of the present disclosure encompass any bispecific molecular format known in the art (see e.g., Spiess et al., Mol. Immunol. 67(2): 95-106 (2015), which is hereby incorporated by reference in its entirety).
• the first and/or second binding domain of the bispecific agonist LT ⁇ R binding protein is a Fab.
• the first and/or second binding domain of the bispecific agonist LT ⁇ R binding protein is a scFv.
• the first binding domain is a Fab and the second binding domain is a scFv.
• the second binding domain is a Fab and the first binding domain is a scFv.
• both the first and second binding domains are Fabs. In one embodiment, both the first and second binding domains are scFvs.
• the bispecific agonist LT ⁇ R binding protein of the present disclosure is a full-length bispecific IgG or comprises an IgG-like structure. In one embodiment, the bispecific agonist LT ⁇ R binding protein is a hetero-IgG that is monovalent for each target protein bound by the bispecific binding protein. Exemplary hetero-IgG bispecific formats comprising Fc domains engineered to enhance heterodimer formation are well known in the art and are suitable formats for the bispecific agonist LT ⁇ R binding protein of the present disclosure.
• hetero-IgG formats include, without limitation, knob-into- holes format (see e.g., Ridgway et al., “‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization,” Protein Eng. 9:617–621 (1996) and Atwell et al., “Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library,” J. Mol. Biol. 270:26–35 (1997), which are hereby incorporated by reference in their entirety), DuoBody format (see e.g., Labrijn et al., “Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange,” Proc.
• knob-into- holes format see e.g., Ridgway et al., “‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization,” Protein Eng. 9:617–62
• the bispecific agonist LT ⁇ R binding protein of the present disclosure is a construct comprising one or more binding protein fragments.
• Suitable LT ⁇ R bispecific binding protein fragments constructs of the present disclosure include, without limitation, nanobodies comprising two single variable (VHH) domains connected via a peptide linker (Els Conrath et al., “Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs,” J. Biol.
• Suitable diabody LT ⁇ R bispecific constructs include, without limitation, single chain diabodies (see e.g., Alt et al., “Novel tetravalent and bispecific IgG-like antibody molecules combining single-chain diabodies with the immunoglobulin gamma1 Fc or CH3 region,” FEBS Lett.454: 90-94 (1999), which is hereby incorporated by reference in its entirety), dual-affinity re-targeting diabodies (DART) (see e.g., Johnson et al., “Effector cell recruitment with novel Fv-based dual-affinity re-targeting protein leads to potent tumor cytolysis and in vivo B-cell depletion,” J. Mol.
• DART dual-affinity re-targeting diabodies
• tandem diabodies and tetravalent tandem diabodies (TandAb) comprising two pairs of VL and VH domains connected in a single polypeptide chain (see e.g., Arndt et al., “A bispecific diabody that mediates natural killer cell cytotoxicity against xenotransplanted human Hodgkin's tumors,” Blood 94: 2562-2568 (1999), which is hereby incorporated by reference in its entirety).
• Suitable LT ⁇ R bispecific constructs also include scFv fragments or diabodies coupled to an Fc portion (e.g., minibodies, Diabody-CH3, scDiabody-CH3, scFv-CH3, minibodies) or fused to one or more other moieties to extend half-life (e.g., fusion to serum albumin or albumin binding proteins).
• Fc portion e.g., minibodies, Diabody-CH3, scDiabody-CH3, scFv-CH3, minibodies
• one or more other moieties to extend half-life e.g., fusion to serum albumin or albumin binding proteins.
• the bispecific agonist LT ⁇ R binding protein of the present disclosure is a monospecific IgG antibody (e.g., containing the first or second binding domain of the bispecific antibody) engineered for bispecificity with the coupling of an additional binding domain (e.g., the second or first binding domain, respectively) comprising, e.g., a VHH, scFv, 2scFv, Fv, Fab, or antibody mimetic binding domain to either the amino or carboxy termini of either the light or heavy chain(s) of the antibody.
• an additional binding domain e.g., the second or first binding domain, respectively
• Exemplary engineered bispecific binding proteins include, without limitation dual variable domain (DVD)- IgG, IgG(H)-scFv, ScFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG((L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)- V, V(L)-IgG, IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig (see e.g., Spiess et al., “Alternative molecular formats and therapeutic applications for bispecific antibodies,” Mol.
• DVD dual variable domain
• the multi-specific agonist LT ⁇ R binding protein of the present disclosure is a bispecific agonist LT ⁇ R binding protein.
• This bispecific agonist LT ⁇ R binding protein comprises a (i) LT ⁇ R binding domain, where the LT ⁇ R binding domain binds one or more amino acid residues of human LT ⁇ R CRD4 comprising amino acid residues 169-211 of SEQ ID NO: 1, and (ii) a tumor associated antigen binding domain.
• This bispecific LT ⁇ R binding protein agonizes LT ⁇ R activity and (a) does not inhibit LIGHT to LT ⁇ R and (b) does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R.
• Exemplary LT ⁇ R binding domains i.e., HCDRs and LCDRs, and VH and VL domains, that bind CRD4 of human LT ⁇ R are described supra, and include, without limitation the binding domains of LIBC219081 and LIBC218979 as well as binding domains of the LT ⁇ R binding proteins provided in Tables 2–4 and 6–8.
• suitable first binding domains of the bispecific agonist LT ⁇ R binding protein as described herein may comprise a heavy chain variable domain (VH) or fragment thereof comprising one or more of the HCDR1, HCDR2, and/or HCDR3 or portions thereof as set forth in a single row of Table 2 or Table 6.
• VH heavy chain variable domain
• the first binding domain of the bispecific agonist LT ⁇ R binding protein comprises all three of the HCDR1, HCDR2, and HCDR3 or portions thereof as set forth in a single row of Table 2 or Table 6.
• the first binding domain of the bispecific agonist LT ⁇ R binding protein comprises a light chain variable domain (VL) or fragment thereof comprising one or more of the LCDR1, LCDR2, and/or LCDR3 or portions thereof as set forth in a single row in Table 3 or Table 7.
• VL light chain variable domain
• the first binding domain of the bispecific agonist LT ⁇ R binding protein comprises all three of the LCDR1, LCDR2, and LCDR3 or portions thereof as set forth in a single row of Table 3 or Table 7.
• the first binding domain of the bispecific agonist LT ⁇ R binding protein as described herein comprises a VH or fragment thereof comprising one or more of the HCDR1, HCDR2, and/or HCDR3 or portions thereof as set forth in a single row of Table 2 or Table 6 and a VL or fragment thereof comprising one or more of the LCDR1, LCDR2, and/or LCDR3 or portions thereof of a corresponding binding molecule as set forth in a single row of Table 3 or Table 7.
• the first binding domain of the bispecific agonist LT ⁇ R binding protein as described herein comprises a VH or fragment thereof comprising all three of the HCDR1, HCDR2, and HCDR3 or portions thereof as set forth in a single row of Table 2 or Table 6 and a VL or fragment thereof comprising all three of the LCDR1, LCDR2, and LCDR3 or portions of a corresponding binding molecule as set forth in a single row of Table 3 or Table 7.
• This bispecific agonist LT ⁇ R binding protein (a) does not inhibit LIGHT binding to LT ⁇ R and (b) does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R.
• the first binding domain of a bispecific agonist LT ⁇ R binding protein that binds CRD4 of human LT ⁇ R comprises a heavy chain variable domain (VH), a light chain variable domain (VL), or a combination of VH and VL domains.
• VH domain of the first binding domain of the bispecific agonist LT ⁇ R binding protein comprises any one of the VH amino acid sequences provided in Table 4 or Table 8, or an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical to any one of the VH amino acid sequences listed in Table 4 or Table 8.
• the VL domain of the first binding domain of the bispecific agonist LT ⁇ R binding protein comprises any one of the VL amino acid sequences provided in Table 4 or Table 8, or an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical to any one of the VL amino acid sequences listed in Table 4 or Table 8.
• This bispecific agonist LT ⁇ R binding protein (a) does not inhibit LIGHT binding to LT ⁇ R and (b) does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R.
• the first binding domain of a bispecific agonist LT ⁇ R binding protein that binds CRD4 of human LT ⁇ R comprises a heavy chain variable domain having at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of the VH domain amino acid sequences set forth in Table 4 or Table 8, and a light chain variable domain having at least 85%, at least 90%, at least 95%, or 100% sequence identity to the corresponding VL domain amino acid sequence as set forth in Table 4 or Table 8.
• This bispecific agonist LT ⁇ R binding protein (a) does not inhibit LIGHT binding to LT ⁇ R and (b) does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R.
• the LT ⁇ R binding domain of the bispecific agonist LT ⁇ R binding protein that binds to CRD4 of human LT ⁇ R comprises a VH comprising the HCDR1 amino acid sequence of: X 1 YX 3 MX 5 (SEQ ID NO: 5), where X 1 is S or N; X 3 is G, D, or A; and X 5 is H or Y; the HCDR2 amino acid sequence of: X 1 IX 3 YDX 6 X 7 X 8 X 9 Y X 11 X 12 DSVKG (SEQ ID NO: 6), where X 1 is A or V; X 3 is W or R; X 6 is E or G; X 7 is S, R, or T; X 8 is N or K; X 9 is K, R, or Q; X 11 is H or Y; and X 12 is A or E; and the HCDR3 amino acid sequence of: X1RX3X4X5X6 X7X
• the LT ⁇ R binding domain of the bispecific agonist LT ⁇ R binding protein that binds to CRD4 of human LT ⁇ R further comprises a VL comprising the LCDR1 amino acid sequence of: SGDX 4 LPX 7 X 8 YX 10 Y (SEQ ID NO: 62), where X 4 is A or T; X 7 is E, K, Q, D or N; X 8 is Q or H; and X 10 is A or T; the LCDR2 amino acid sequence of: KDNERPS (SEQ ID NO: 63); and the LCDR3 amino acid sequence of: QSX 3 DX 5 SX 7 X 8 YX 10 X 11 (SEQ ID NO: 64), where X 3 is A or T; X 5 is S, G, or N; X 7 is G or
• the LT ⁇ R binding domain of the bispecific agonist LT ⁇ R binding protein that binds to CRD4 of human LT ⁇ R comprises a VH amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 121.
• an LT ⁇ R binding domain comprises a VH amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 123.
• an LT ⁇ R binding domain comprises a VH amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 121 and the amino acid sequence of SEQ ID NO: 123.
• Exemplary VH amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 121 and SEQ ID NO: 123 are provided in Table 4 supra.
• This bispecific agonist LT ⁇ R binding protein (a) does not inhibit LIGHT binding to LT ⁇ R and (b) does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R.
• the LT ⁇ R binding domain of the bispecific agonist LT ⁇ R binding protein that binds to CRD4 of human LT ⁇ R comprises a VL amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 122. In one embodiment, the LT ⁇ R binding domain comprises a VL amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 124. In one embodiment, the LT ⁇ R binding domain comprises a VL amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 122 and the amino acid sequence of SEQ ID NO: 124.
• VL amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 122 and SEQ ID NO: 124 are provided in Table 4 supra.
• This bispecific agonist LT ⁇ R binding protein (a) does not inhibit LIGHT binding to LT ⁇ R and (b) does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R.
• the LT ⁇ R binding domain of the bispecific agonist LT ⁇ R binding protein that binds to CRD4 of human LT ⁇ R comprises a VH amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 121 and a VL amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 122.
• the LT ⁇ R binding domain comprises a VH amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 121 and a VL amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 122.
• an LT ⁇ R binding domain comprises a VH amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 121 and a VL amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 122.
• This bispecific agonist LT ⁇ R binding protein (a) does not inhibit LIGHT binding to LT ⁇ R and (b) does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R.
• the LT ⁇ R binding domain of the bispecific agonist LT ⁇ R binding protein comprises a VH amino acid sequence having at least 90% sequence identity to SEQ ID NO: 123 and a HCDR1 amino acid sequence of SEQ ID NO: 5 (X 1 YX 3 MX 5 ), a HCDR2 amino acid sequence of SEQ ID NO: 6 (X 1 IX 3 YDX 6 X 7 X 8 X 9 YX 11 X 12 DSVKG), and a HCDR3 amino acid sequence of SEQ ID NO: 7 (X1RX3X4X5X6X7X8X9YYGX13X14V).
• this LT ⁇ R binding domain further comprises a VL amino acid sequence having at least 90% sequence identity to SEQ ID NO: 124 and a LCDR1 amino acid sequence of SEQ ID NO: 62 (SGDX4LPX7X8YX10Y), a LCDR2 amino acid sequence of SEQ ID NO: 63 (KDX 3 ERPS), and a LCDR3 amino acid sequence of SEQ ID NO: 64 (QSX3DX5SX7X8YX10X11).
• This bispecific agonist LT ⁇ R binding protein (a) does not inhibit LIGHT binding to LT ⁇ R and (b) does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R.
• the LT ⁇ R binding domain of the bispecific agonist LT ⁇ R binding protein comprises a VH, which comprises the HCDR1, HCDR2, and HCDR3 of amino acid sequences of SEQ ID NO: 11-13, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NO: 68-70, respectively.
• the LT ⁇ R binding domain comprises a VH comprising the HCDR1, HCDR2, and HCDR3 of amino acid sequences of SEQ ID NO: 8-10, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NO: 65-67, respectively.
• This bispecific agonist LT ⁇ R binding protein (a) does not inhibit LIGHT binding to LT ⁇ R and (b) does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R. [0236]
• the LT ⁇ R binding domain of the bispecific agonist LT ⁇ R binding protein competes for binding to LT ⁇ R with the exemplary LT ⁇ R binders of LIBC219081 and LIBC218979.
• this first binding domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences selected from (i) SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 278, SEQ ID NO: 279, and SEQ ID NO: 280; (ii) SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 299, SEQ ID NO: 300, and SEQ ID NO: 301; (iii) SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 308, SEQ ID NO: 309, and SEQ ID NO: 310; (iv) SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 308, SEQ ID NO: 309, and SEQ ID NO: 310; (v) SEQ ID NO: 224, SEQ ID NO:
• Bispecific agonist LT ⁇ R binding proteins comprising a first binding domain with the aforementioned heavy chain and light chain CDRs does not inhibit LIGHT binding to LT ⁇ R and does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R.
• the LT ⁇ R binding domain of the bispecific agonist LT ⁇ R binding protein binds human LT ⁇ R CRD1 and comprises a heavy chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to the VH domain amino acid sequence of SEQ ID NO: 646 and a light chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to the VL domain amino acid sequence of SEQ ID NO: 647.
• the LT ⁇ R binding domain of the bispecific agonist LT ⁇ R binding protein binds to human LT ⁇ R, and comprises a heavy chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to the VH domain amino acid sequence of SEQ ID NO: 652 and a light chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to the VL domain amino acid sequence of SEQ ID NO: 653.
• This bispecific LT ⁇ R binding protein does not inhibit endogenous LIGHT binding to LT ⁇ R.
• the LT ⁇ R binding domain of the bispecific agonist LT ⁇ R binding protein binds to human LT ⁇ R, and comprises a heavy chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to the VH domain amino acid sequence of SEQ ID NO: 654 and a light chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to the VL domain amino acid sequence of SEQ ID NO: 655.
• the LT ⁇ R binding domain of the bispecific agonist LT ⁇ R binding protein binds to human LT ⁇ R CRD1, and comprises a heavy chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to the VH domain amino acid sequence of SEQ ID NO: 658 and a light chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to the VL domain amino acid sequence of SEQ ID NO: 659.
• the LT ⁇ R binding domain of the bispecific agonist LT ⁇ R binding protein binds to human LT ⁇ R CRD2 and CRD3 and comprises a heavy chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to the VH domain amino acid sequence of SEQ ID NO: 648 and a light chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to the VL domain amino acid sequence of SEQ ID NO: 649.
• the LT ⁇ R binding domain of the bispecific agonist LT ⁇ R binding protein binds to human LT ⁇ R CRD2 and CRD3 and comprises a heavy chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to the VH domain amino acid sequence of SEQ ID NO: 644 and a light chain variable domain having at least 85%, at least 90%, or at least 95% sequence identity to the VL domain amino acid sequence of SEQ ID NO: 645.
• the tumor-associated binding domain of the bispecific agonist LT ⁇ R binding protein binds to a tumor-associated antigen (TAA).
• TAA tumor-associated antigen
• the TAA is solid tumor TAA.
• Suitable TAAs are known in the art and include, without limitation, alpha-fetoprotein (AFP), CD44v6, carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD133, hepatocyte growth factor receptor (c-MET), claudin 18.2 (CLDN18.2), claudin 6 (CLDN6), leucine-rich repeat containing protein 15 (LRRC15), epidermal growth factor receptor (EGFR), type III variant epidermal growth factor receptor (EGFRvIII), erythropoietin producing hepatocellular carcinoma A2 (EphA2), epithelial cell adhesion molecule (EpCAM), fetal acetylcholine receptor, folate receptor alpha (FR ⁇ ), ganglio
• TAA binding domain may comprise the CLDN6 binding domain, the CLDN18.2 binding domain, the LRRC15 binding domain, or the MUC17 binding domain described in Example 7 herein.
• suitable TAA binding domains include, without limitation, an anti- alpha-fetoprotein (AFP) antibody or binding domain thereof (see e.g., U.S.
• Patent No.8,268,312 to Hansen et al. which is hereby incorporated by reference in its entirety
• an anti-CD44v6 antibody or binding domain thereof see e.g., U.S. Patent No.6,972,324 to Adolf et al., which is hereby incorporated by reference in its entirety
• an anti-carbonic anhydrase IX (CAIX) antibody or binding domain thereof see e.g., WO2011139375 to Renner and U.S. Patent No.10,487,153 to Lenferink, which are hereby incorporate by reference in their entirety
• an anti-carcinoembryonic antigen (CEA) antibody or binding domain thereof see e.g., U.S. Patent Appl. Publ.
• Patent No.9,068,011 to Neijssen et al. which are hereby incorporate by reference in their entirety
• an anti-claudin 18.2 antibody or binding domain thereof see e.g., U.S. Patent App. Publ. No.20200055932 to Dahlhoff et al., and U.S. Patent No. 10,421,817 to Hu et al., which are hereby incorporate by reference in their entirety
• an anti- claudin 6 antibody or binding domain thereof see e.g., U.S. Patent No.
• an anti-erythropoietin producing hepatocellular carcinoma A2 (EphA2) antibody or binding domain thereof see e.g., U.S. Patent No.9,676,864 to Bouchard et al., and U.S. Patent No. 10,406,225 to Zhou and Marks, which are hereby incorporate by reference in their entirety
• an anti-epithelial cell adhesion molecule (EpCAM) antibody or binding domain thereof see e.g., U.S. Patent No.8,637,017 to Gunnarsson et al., and U.S.
• Patent No.9,790,274 to Harvey et al. which are hereby incorporate by reference in their entirety
• an anti-fetal acetylcholine receptor antibody or binding domain thereof see e.g., WO2013011030 to Martinez-Martinez which is hereby incorporated by reference in its entirety
• anti-folate receptor alpha (FR ⁇ ) binding domain see e.g., U.S. Patent No. 8,475,795 to O’Shannessy, which is hereby incorporated by reference in its entirety
• an anti-ganglioside GD2 (GD2) antibody or binding domain thereof see e.g., U.S. Patent App. Publ. No.20210189000 to Scholz et al., and U.S. Patent App.
• Patent Appl. Publ. No. 20180100022 to Bossenmaier et al. which is hereby incorporated by reference in its entirety
• an anti- human epidermal growth factor receptor 2 (ERBB2/HER2) antibody or binding domain thereof see e.g., U.S. Patent No. 11,046,771 to Goeij et al. and U.S. Patent No. 8,722,362 to Alper, which are hereby incorporate by reference in their entirety
• an anti-intercellular adhesion molecule 1 (ICAM-1) antibody or binding domain thereof see e.g., U.S. Patent Appl. Publ. No.20160280788 Hansson et al., and U.S. Patent No.
• an anti- interleukin 13 receptor ⁇ 2 (IL13R ⁇ 2) antibody or binding domain thereof see e.g., U.S. Patent Appl. Publ. No.20200181227 to Balyasnikova et al., which is hereby incorporated by reference in its entirety
• an anti- interleukin 11 receptor ⁇ (IL11R ⁇ ) antibody or binding domain thereof see e.g., U.S.
• Patent No.9,340,618 to Edwards et al. which is hereby incorporated by reference in its entirety
• an anti-Kirsten rat sarcoma viral oncogene homolog (KRAS) antibody or binding domain thereof see e.g., U.S. Patent No.11,174,314 to Zhou et al., which is hereby incorporated by reference in its entirety
• an anti-L1-cell adhesion molecule (L1CAM) antibody or binding domain thereof see e.g., U.S. Patent Appl. Publ. No.20220033494 to Hong et al., which is hereby incorporated by reference in its entirety
• an anti-MAGE antibody or binding domain thereof see e.g., U.S.
• an anti-natural killer group 2 member D (NKG2D) antibody or binding domain thereof see e.g., U.S. Patent No. 10,526,409 to Urso et al., which is hereby incorporated by reference in its entirety
• an anti-cancer/testis antigen 1 (CT6.1) antibody or binding domain thereof see e.g., U.S. Patent Appl. Publ. No.20190382504, which is hereby incorporated by reference in its entirety
• an anti-prostate stem cell antigen (PSCA) antibody or binding domain thereof see e.g., U.S.
• Patent No.8,013,128 to Gudas et al. which is hereby incorporated by reference in its entirety
• WT-1 anti-Wilms tumor 1
• PDT-1 anti-Wilms tumor 1
• PD-1 anti-program cell death 1 ligand 1
• Patent No.9,988,452 to Freeman et al. which is hereby incorporated by reference in its entirety
• an anti-trophoblast glycoprotein (5T4 oncofetal antigen) antibody or binding domain thereof see e.g., U.S. Patent No.8,044,178 to Boghaert et al., which is hereby incorporated by reference in its entirety
• an anti-folate receptor alpha (FOLR1) antibody or binding domain thereof see e.g., U.S.
• the tumor-associated antigen binding domain of the bispecific LT ⁇ R binding protein binds to a stromal associated antigen (SAA) of the tumor microenvironment (TME).
• Suitable SAAs include, without limitation, fibronectin (FN1), matrix metalloproteinase-2 (MMP2), platelet derived growth factor receptor- ⁇ (PDGFR ⁇ ), dickkopf-related protein 3 (DKK3), platelet-derived growth factor subunit B (PDGFB), NUAK family SNF1-like kinase 1 (NUAK1), fibroblast growth factor (FGF1), PDZ and LIM domain protein 4 (PDLIM4), gremlin 1(Grem1), and periostin (POSTN).
• FN1 fibronectin
• MMP2 matrix metalloproteinase-2
• PDGFR ⁇ platelet derived growth factor receptor- ⁇
• DKK3 platelet-derived growth factor subunit B
• NUAK1 NUAK family SNF1-like kinase 1
• FGF1 fibroblast growth factor
• PDLIM4 LIM domain protein 4
• POSTN periostin
• the second binding domain may comprise an anti-fibronectin antibody or binding domain thereof (see e.g., U.S. Patent Appl. Publ. No.20100248262 to Kato et al., which is hereby incorporated by reference in its entirety), an anti-matrix metalloproteinase-2 antibody or binding domain thereof (see e.g., U.S. Patent No. 8,013,125 to Devy which is hereby incorporated by reference in its entirety), an anti-platelet derived growth factor receptor- ⁇ (PDGFR ⁇ ) antibody or binding domain thereof (see e.g., U.S. Patent No.
• PDGFR ⁇ anti-platelet derived growth factor receptor- ⁇
• NUAK1 anti-NUAK family SNF1-like kinase 1
• NUAK1 anti-fibroblast growth factor antibody or binding domain thereof
• PDLIM4 anti-PDZ and LIM domain protein 4
• the multi-specific LT ⁇ R binding protein of the present disclosure is a bispecific LT ⁇ R binding protein that is monovalent for LT ⁇ R binding.
• bispecific molecules comprising these LT ⁇ R binding domains in monovalent form (but not bivalent form) exhibit cross-linking dependent LT ⁇ R agonism.
• bispecific molecules comprising monovalent LT ⁇ R binding domains require binding of both the first and second binding domains to induce LT ⁇ R receptor clustering and achieve LT ⁇ R agonism.
• This bispecific construct, having monovalency for LT ⁇ R is particularly beneficial for targeting LT ⁇ R agonism to a particular tissue, such as the tumor microenvironment, because activity is dependent on binding of the first and second binding domains.
• the LT ⁇ R binders disclosed herein were specifically screened and selected based on their binding to LT ⁇ R at a region, e.g., CRD4, that that does not inhibit endogenous LT ⁇ R ligand binding and signaling.
• CRD4 a region that does not inhibit endogenous LT ⁇ R ligand binding and signaling.
• the LT ⁇ R binding proteins disclosed herein ensure that if a LT ⁇ R bispecific binding protein binds to LT ⁇ R outside of the targeted tumor microenvironment, (i) binding will not induce off-target LT ⁇ R signaling in the absence of the second binding domain binding to its target, and (ii) such binding will not block endogenous LT ⁇ R ligand (i.e., LIGHT and LT ⁇ 1 ⁇ 2) signaling in the off-target tissue.
• endogenous LT ⁇ R ligand i.e., LIGHT and LT ⁇ 1 ⁇ 2
• the LT ⁇ R binding proteins described herein comprise one or more amino acid modifications in the heavy chain constant regions that improve half-life/stability or render the antibody more suitable for expression/manufacturability.
• the LT ⁇ R binding protein is designed to prevent or reduce interaction with Fc receptors.
• the binding protein is a Stable Effector Functionless (SEFL) binding protein comprising a constant region that lacks the ability to interact with Fc ⁇ receptors.
• SEFL Stable Effector Functionless
• an LT ⁇ R binding protein described herein is modified to comprise one or more of the following mutations, numbered according to the EU system: L242C, A287C, R292C, N297G, V302C, L306C, and/or K334C.
• an LT ⁇ R binding protein described herein comprises an N297G substitution to form a SEFL binding protein.
• an LT ⁇ R binding protein described herein comprises A287C, N297G, and L306C substitutions to form a SEFL binding protein.
• the LT ⁇ R binding protein comprises R292C, N297G, and V302C substitutions to form a SEFL2-2 antibody based molecule.
• the agonist LT ⁇ R binding proteins as described herein may comprise other half-life extension (HLE) modifications.
• the HLE modification occurs in the heavy chain constant region and comprises one or more of the following amino acid substitutions, numbered according to the EU system: M252Y, S254T, and T256E.
• the LT ⁇ R binding proteins comprise one or two of M252Y, S254T, and T256E substitutions.
• the LT ⁇ R binding proteins of the present disclosure comprise all three of M252Y, S254T, and T256E.
• the HLE modification occurs in the heavy chain constant region and comprises one or more of the following substitutions, numbered according to the EU system: L309D, Q311H, and N434S.
• the LT ⁇ R binding proteins of the present disclosure comprise one, two or all three of the L309D, Q311H, and N434S substitutions. In exemplary instances, the LT ⁇ R binding proteins comprise all three of L309D, Q311H, and N434S substitutions. In one embodiment, the LT ⁇ R binding proteins disclosed herein comprise SEFL or SEFL2-2 modifications in combination with any of the herein described HLE modifications. [0250] In certain embodiments, variants of the agonist LT ⁇ R binding protein include glycosylation variants wherein the number and/or type of glycosylation site has been altered compared to the amino acid sequences of a parent polypeptide.
• variants comprise a greater or a lesser number of N-linked glycosylation sites than the native protein.
• substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain.
• rearrangement of N- linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created.
• Additional binding protein variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the parent amino acid sequence. Cysteine variants may be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies.
• Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.
• the agonist LT ⁇ R binding proteins disclosed herein include post translationally modified variants.
• the agonist LT ⁇ R binding proteins comprising a heavy chain have the C- terminal lysine residue deleted.
• Other desired amino acid substitutions (whether conservative or non-conservative) and deletions can be determined by those skilled in the art at the time such substitution or deletion is desired.
• amino acid substitutions can be used, for example, to identify important residues of a binding protein associated with function, to increase or decrease the affinity of the antibodies to the target of interest described herein, reduce susceptibility to proteolysis, reduce susceptibility to oxidation, alter binding affinity for forming protein complexes, and/or confer or modify other physiochemical or functional properties on such polypeptides.
• single or multiple amino acid substitutions may be made in the naturally-occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts).
• a conservative amino acid substitution typically may not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence or disrupt other types of secondary structure that characterizes the parent sequence).
• a replacement amino acid should not tend to break a helix that occurs in the parent sequence or disrupt other types of secondary structure that characterizes the parent sequence.
• nucleic acid molecules comprising a nucleotide sequence encoding an agonist LT ⁇ R binding protein of the present disclosure.
• a “nucleic acid molecule” as used herein encompasses polynucleotides and oligonucleotides and generally refers to a polymer of DNA or RNA, or modified forms thereof, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered inter-nucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
• the nucleic acid molecule can comprise any nucleotide sequence which encodes any of the agonist LT ⁇ R binding proteins of the present disclosure.
• the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions.
• the nucleic acid comprises one or more insertions, deletions, inversions, and/or substitutions.
• the nucleic acid molecules of the present disclosure are recombinant.
• the term “recombinant” refers to molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments using laboratory methods to form nucleic acid molecules that are not otherwise found in nature.
• nucleic acid molecules of the present disclosure in some aspects are constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.
• a nucleic acid molecule can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides).
• modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5- fluorouracil, 5-bromouracil, 5-chIorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridme, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N 6 - isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N -substituted adenine, 7-methylguanine, 5- methylammomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannos
• the nucleic acid molecule comprises one or more polynucleotides that encode all or part of an agonist LT ⁇ R binding protein, for example, one or both chains of a binding protein as disclosed herein.
• the polynucleotide can be any length as appropriate for the desired use or function, and can be operably coupled to one or more additional sequences, for example, regulatory sequences, and/or be part of a larger nucleic acid molecule, for example, a vector.
• a polynucleotide is “operably coupled” or “operably linked” to a regulatory sequence if the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the polynucleotide sequence.
• polynucleotide molecule can be single-stranded or double-stranded and can comprise RNA and/or DNA nucleotides, and artificial variants thereof (e.g., peptide nucleic acids).
• a polynucleotide of the present disclosure encodes an agonist LT ⁇ R binding protein disclosed supra and comprises a sequence encoding any one, any two, any three, any four, any five, or any six of the CDRs described supra, including the heavy chain CDRs provided in Tables 2, 6, and 10, and the light chain CDRs provided in Tables 3, 7 and 11.
• polynucleotide molecules of the present disclosure comprise a nucleotide sequence encoding a VH domain and a VL domain of an LT ⁇ R binding protein that binds to the CRD4 of LT ⁇ R.
• the polynucleotide molecule comprises a nucleotide sequence encoding a VH and VL domain selected from: a VH amino acid sequence of SEQ ID NO: 119 and a VL amino acid sequence of SEQ ID NO: 120; a VH amino acid sequence of SEQ ID NO: 121 and a VL amino acid sequence of SEQ ID NO: 122; a VH amino acid sequence of SEQ ID NO: 123 and a VL amino acid sequence of SEQ ID NO: 124; a VH amino acid sequence of SEQ ID NO: 125 and a VL amino acid sequence of SEQ ID NO: 126; a VH amino acid sequence of SEQ ID NO: 127 and a VL amino acid sequence of SEQ ID NO: 128; a VH amino acid sequence of SEQ ID NO: 129 and a VL amino acid sequence of SEQ ID NO: 130; a VH amino acid sequence of SEQ ID NO: 131 and a VL amino acid sequence of SEQ ID NO: 120;
• the polynucleotide molecule of the present disclosure comprises a nucleotide sequence encoding a VH domain and a VL domain of an agonist LT ⁇ R binding protein that competes for binding to CRD4 of LT ⁇ R with the exemplary LT ⁇ R binders of LIBC219081 and LIBC218979.
• the polynucleotide molecule comprises a nucleotide sequence encoding a VH and VL domain selected from: a VH amino acid sequence of SEQ ID NO: 401 and a VL amino acid sequence of SEQ ID NO: 402; a VH amino acid sequence of SEQ ID NO: 403 and a VL amino acid sequence of SEQ ID NO: 404; a VH amino acid sequence of SEQ ID NO: 405 and a VL amino acid sequence of SEQ ID NO: 406; a VH amino acid sequence of SEQ ID NO: 407 and a VL amino acid sequence of SEQ ID NO: 408; a VH amino acid sequence of SEQ ID NO: 409 and a VL amino acid sequence of SEQ ID NO: 410; a VH amino acid sequence of SEQ ID NO: 411 and a VL amino acid sequence of SEQ ID NO: 412; a VH amino acid sequence of SEQ ID NO: 413 and a VL amino acid sequence
• the polynucleotide molecule of the present disclosure comprises a nucleotide sequence encoding a VH domain and a VL domain of an agonist LT ⁇ R binding protein that binds human LT ⁇ R.
• the polynucleotide comprises a nucleotide sequence encoding a VH and VL domain selected from: a VH amino acid sequence of SEQ ID NO: 644 and a VL amino acid sequence of SEQ ID NO: 645; a VH amino acid sequence of SEQ ID NO: 646 and a VL amino acid sequence of SEQ ID NO: 647; a VH amino acid sequence of SEQ ID NO: 648 and a VL amino acid sequence of SEQ ID NO: 649; a VH amino acid sequence of SEQ ID NO: 650 and a VL amino acid sequence of SEQ ID NO: 651; a VH amino acid sequence of SEQ ID NO: 652 and a VL amino acid sequence of SEQ ID NO: 653; a VH amino acid sequence of SEQ ID NO: 654 and a VL amino acid sequence of SEQ ID NO: 655; a VH amino acid sequence of SEQ ID NO: 656 and a VL amino acid sequence of SEQ ID NO: 645
• Changes can be introduced into the nucleic acid molecules disclosed herein by mutation, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., the LT ⁇ R binding protein) that it encodes. Mutations can be introduced using any technique known in the art. In one embodiment, one or more particular amino acid residues are changed using, for example, a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues is changed using, for example, a random mutagenesis protocol. Irrespective of how it is made, a mutant polypeptide can be expressed and screened for a desired property.
• Mutations can be introduced into a nucleic acid molecule disclosed herein without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues.
• a nucleotide sequence provided herein for one of the agonist LT ⁇ R binding proteins of the present disclosure is mutated such that it encodes an amino acid sequence comprising one or more deletions or substitutions of amino acid residues that are shown herein for the light chains and/or the heavy chains of the LT ⁇ R binding proteins of the present disclosure.
• the mutagenesis inserts an amino acid adjacent to one or more amino acid residues shown herein for the light and/or heavy chains of the agonist LT ⁇ R binding proteins of the present disclosure.
• one or more mutations can be introduced into the nucleic acid molecules as disclosed herein that selectively changes the biological activity of the agonist LT ⁇ R binding protein that it encodes.
• nucleotide sequences of the agonist LT ⁇ R binding proteins of the present disclosure can be altered, for example, by random mutagenesis or by site-directed mutagenesis to create an altered polynucleotide comprising one or more particular nucleotide substitutions, deletions, or insertions as compared to the non- mutated polynucleotide. Examples of techniques for making such alterations are described in Walder et al., Gene 42:133 (1986); Bauer et al. Gene 37:73 (1985); Smith et al., 1981, Genetic Engineering: Principles and Methods, Plenum Press; and U.S.
• the present disclosure provides vectors comprising a polynucleotide encoding an agonist LT ⁇ R binding protein as disclosed herein.
• a vector is a nucleic acid molecule used to introduce another nucleic acid into a cell.
• vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors and expression vectors, for example, recombinant expression vectors.
• vectors comprising a polynucleotide of the present disclosure can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual.3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (2001); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY (1994), which are hereby incorporated by reference in their entirety.
• Constructs of expression vectors which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell.
• Replication systems can be derived, e.g., from CoIEl, 2 ⁇ plasmid, ⁇ , SV40, bovine papilloma virus, and the like.
• the expression vector is a circular plasmid (see, e.g., Muthumani et al., “Optimized and Enhanced DNA Plasmid Vector Based In vivo Construction of a Neutralizing anti-HIV-1 Envelope Glycoprotein Fab,” Hum. Vaccin. Immunother. 9: 2253-2262 (2013), which is hereby incorporated by reference in its entirety).
• Plasmids can transform a target cell by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).
• Exemplary plasmid vectors include, without limitation, pCEP4, pREP4, pVAX, pcDNA3.0, provax, or any other expression vector capable of expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.
• the expression vector is a linear expression cassette (“LEC”).
• LECs are capable of being efficiently delivered to a subject via electroporation to express the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.
• the LEC may be any linear DNA devoid of a phosphate backbone. In one embodiment, the LEC does not contain any antibiotic resistance genes and/or a phosphate backbone. In another embodiment, the LEC does not contain other nucleic acid sequences unrelated to the desired gene expression.
• the LEC may be derived from any plasmid capable of being linearized. The plasmid may be capable of expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.
• Exemplary plasmids include, without limitation, pNP (Puerto Rico/34), pM2 (New Caledonia/99), WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.
• the expression vector is a viral vector.
• Suitable viral vectors that are capable of expressing full length antibodies or binding portions thereof include, for example, an adeno- associated virus (AAV) vector (see, e.g., Lewis et al., “Generation of Neutralizing Activity against Human Immunodeficiency Virus Type I in Serum by Antibody Gene Transfer,” J. Virol.76:8769-775 (2002); Fang et al., “An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In vivo,” Mol. Ther. 15(6): 1153-9 (2007); Buning et al, “Recent Developments in Adeno- associated Virus Vector Technology,” J.
• AAV adeno- associated virus
• AAVs adeno-associated viruses
• the expression vector construct encoding the agonist LT ⁇ R binding protein as described herein can include the polynucleotide encoding a heavy chain polypeptide, a heavy chain variable region, or a fragment thereof.
• the heavy chain polypeptide can include a variable heavy chain (VH) region and/or at least one constant heavy chain (CH) region.
• the at least one constant heavy chain region can include a constant heavy chain region 1 (CH1), a constant heavy chain region 2 (CH2), and a constant heavy chain region 3 (CH3), and/or a hinge region.
• the heavy chain polypeptide can include a VH region and a CH1 region.
• the heavy chain polypeptide can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.
• the expression construct can also include a polynucleotide sequence encoding a light chain polypeptide, a light chain variable region, or a fragment thereof.
• the light chain polypeptide can include a variable light chain (VL) region and/or a constant light chain (CL) region.
• the vector comprises one or more regulatory sequences.
• a “regulatory sequence” is a nucleic acid that affects the expression (e.g., the level, timing, or location of expression) of a nucleic acid to which it is operably linked.
• Exemplary regulatory sequences include transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA-based.
• Suitable regulatory sequences are selected on the basis of the host cells to be used for expression and are operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells (e.g., SV40 early gene enhancer, Rous sarcoma virus promoter and cytomegalovirus promoter), those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue- specific regulatory sequences), and those that direct inducible expression of a nucleotide sequence in response to particular treatment or condition (e.g., the metallothionein promoter in mammalian cells and the tet-responsive and/or streptomycin responsive promoter in both prokaryotic and eukaryotic systems (see e.g., Voss et al., Trends Biochem.
• SV40 early gene enhancer e.g., Rous sarcoma virus promoter and cytomegalo
• the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
• the expression vectors of the disclosure can be introduced into host cells to thereby produce proteins or peptides encoded by nucleic acids as described herein.
• the expression construct can further encode a protease cleavage site.
• the protease cleavage site can be recognized by a protease or peptidase.
• the protease can be an endopeptidase or endoprotease, for example, but not limited to, furin, elastase, HtrA, calpain, trypsin, chymotrypsin, trypsin, and pepsin.
• the protease can be a serine protease, a threonine protease, cysteine protease, aspartate protease, metalloprotease, glutamic acid protease, or any protease that cleaves an internal peptide bond (i.e., does not cleave the N-terminal or C-terminal peptide bond).
• the protease cleavage site can include one or more amino acid sequences that promote or increase the efficiency of cleavage.
• the expression construct can further encode a linker sequence.
• the linker sequence can encode an amino acid sequence that spatially separates and/or links the one or more components of the expression construct (heavy chain and light chain components of the encoded antibody).
• a first expression vector construct encodes a heavy chain polypeptide that includes a VH and CH1
• a second expression vector construct encodes a light chain polypeptide that includes a VL and CL.
• An alternative arrangement includes a first vector encoding a heavy chain polypeptide that includes VH, CH1, hinge region, CH2, and CH3, and a second vector encoding the light chain polypeptide that includes VL and CL.
• the expression vector construct encodes a heavy chain polypeptide that includes VH and CH1, and a light chain polypeptide that includes VL and CL, and a linker sequence is positioned between the nucleic acid sequence encoding the heavy chain polypeptide and the nucleic acid sequence encoding the light chain polypeptide.
• the expression vector construct encodes a heavy chain polypeptide that includes VH and CH1, and a light chain polypeptide that includes VL and CL, and a nucleic acid sequence encoding a protease cleavage site is positioned between the nucleic acid sequence encoding the heavy chain polypeptide and the nucleic acid sequence encoding the light chain polypeptide.
• the expression vector construct encodes a heavy chain polypeptide that includes VH, CH1, hinge region, CH2, and CH3, and a light chain polypeptide that includes VL and CL, and a linker sequence is positioned between the nucleic acid sequence encoding the heavy chain polypeptide and the nucleic acid sequence encoding the light chain polypeptide.
• the expression vector construct encodes a heavy chain polypeptide that includes VH, CH1, hinge region, CH2, and CH3, and a light chain polypeptide that includes VL and CL, and a heterologous nucleic acid sequence encoding the protease cleavage site is positioned between the nucleic acid sequence encoding the heavy chain polypeptide and the nucleic acid sequence encoding the light chain polypeptide.
• the vector can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like.
• Suitable marker genes for the presently disclosed expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.
• the vector can comprise a native or normative promoter operably linked to the nucleotide sequence encoding the agonist LT ⁇ R binding protein.
• the selection of promoters e.g., strong, weak, inducible, tissue-specific, and developmental-specific, is within the ordinary skill of the artisan.
• the combining of a nucleotide sequence with a promoter is also within the skill of the artisan.
• the promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus.
• CMV cytomegalovirus
• SV40 SV40 promoter
• RSV RSV promoter
• a promoter found in the long-terminal repeat of the murine stem cell virus e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus.
• CMV cytomegalovirus
• RSV RSV promoter
• the present disclosure provides host cells into which a recombinant expression vector of the disclosure has been introduced.
• a host cell is a cell that is used to express a nucleic acid, e.g., a polyn
• a host cell refers not only to the particular cell containing a nucleic acid molecule of interest, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to, e.g., mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of “host cell” as used herein.
• Prokaryotic host cells include gram negative or gram positive organisms, for example E. coli or bacilli.
• the cell is a eukaryotic cell, including, but not limited to, a yeast cell, filamentous fungi cell, protozoa cell, algae cell, insect cell, or mammalian cell.
• the eukaryotic cells are mammalian cells.
• the mammalian cells are non-human mammalian cells.
• the cells are Chinese Hamster Ovary (CHO) cells and derivatives thereof (e.g., CHO-K1, CHO pro-3, CS9), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2/0), cells engineered to be deficient in dihydrofolatereductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or derivatives thereof (e.g., HEK293T, HEK293-EBNA), green African monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human bone osteosarcoma epithelial cells U2-OS, adenocarcinomic human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, mouse embryo fibroblast cells NIH 3
• the host cell is CS9 (a CHO cell line).
• Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Additional selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
• LT ⁇ R binding proteins as disclosed herein can be produced by any method known in the art for the synthesis of binding proteins and/or proteins, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
• Recombinant expression techniques involve the construction of an expression vector containing a polynucleotide that encodes an agonist LT ⁇ R binding protein as described herein. Once a polynucleotide encoding the agonist LT ⁇ R binding protein has been obtained, the vector for the production of the agonist LT ⁇ R binding protein may be produced by recombinant DNA technology.
• An expression vector is constructed containing the agonist LT ⁇ R binding protein coding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. [0287] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an agonist LT ⁇ R binding protein of the disclosure. In one embodiment of the disclosure, vectors encoding both the heavy and light chains of an antibody may be co-expressed in the host cell for expression of the entire immunoglobulin molecule. [0288] A variety of host-expression vector systems may be utilized to express the agonist LT ⁇ R binding proteins of the disclosure.
• Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express binding protein of the disclosure in situ.
• Bacterial cells such as E. coli, and eukaryotic cells are commonly used for the expression of a recombinant antibody molecule, especially for the expression of whole recombinant antibody molecule.
• mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Nat. Biotech. 8:2 (1990)).
• a host cell strain which modulates the expression of the inserted sequences or modifies and processes the gene product in a desired fashion may also be selected. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the binding protein.
• Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
• eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
• mammalian host cells include, but are not limited to, CHO cells, COS cells, 293 cells, 3T3 cells, NS0 cells, VERO cells, BHK cells, or myeloma cells.
• cell lines which stably express the LT ⁇ R antibody molecule may be engineered.
• host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
• expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
• engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
• the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
• This method may advantageously be used to engineer cell lines which express the LT ⁇ R binding protein. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
• a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (see e.g., Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (see e.g., Szybalska & Szybalski, Proc. Natl. Acad. Sci.
• adenine phosphoribosyltransferase genes can be employed in tk, hgprt or aprt-cells, respectively.
• antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (see e.g., Wigler et al., Proc. Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci.
• the host cell may be co-transfected with two expression vectors of the disclosure, for example, the first vector encoding an agonist LT ⁇ R binding protein heavy chain or variable domain thereof as described herein and the second vector encoding a LT ⁇ R binding protein light chain or variable domain thereof as described herein.
• the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
• a single vector may be used which encodes, and is capable of expressing, for example, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (see e.g., Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad.
• LT ⁇ R single chain binding proteins may be formed by linking heavy and light chain variable domain (Fv region) fragments via an amino acid bridge (short peptide linker), resulting in a single polypeptide chain.
• Fv region heavy and light chain variable domain
• amino acid bridge short peptide linker
• the resulting polypeptides can fold back on themselves to form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers), depending on the length of a flexible linker between the two variable domains (see e.g., Kortt et al., Prot. Eng.10:423 (1997); Kortt et al., Biomol. Eng.18:95-108 (2001)).
• V L and V H -comprising polypeptides By combining different V L and V H -comprising polypeptides, one can form multimeric scFvs that bind to different epitopes (Kriangkum et al., Biomol. Eng. 18:31-40 (2001)).
• a bispecific LT ⁇ R binding protein capable of binding LT ⁇ R and a TAA or SAA as described herein can be produced using methods known in the art, see e.g., U.S. Patent Appl. Publ.
• a bispecific agonist LT ⁇ R binding protein as described herein is produced using the methods described in U.S. Patent Appl. Publ. No.20100233173 to Wu et al.; U.S. Patent Appl. Publ. No.20100105873 to Allan et al.; or U.S. Patent Appl. Publ. No.20090155275 to Wu et al.
• a bispecific agonist LT ⁇ R binding protein capable of binding LT ⁇ R and a TAA or SAA as described herein is produced using the methods described in U.S. Patent Appl.
• the bispecific agonist LT ⁇ R binding protein described herein can alternatively be produced by the direct recovery of Fab′ fragments recombinantly expressed, e.g., in E. coli, and can be chemically coupled to form bispecific antibodies (see e.g., Carter et al., Applications for Escherichia coli-Derived Humanized Fab’ Fragments: Efficient Construction of Bispecific Antibodies. In: Rosenberg, M., Moore, G.P. (eds) The Pharmacology of Monoclonal Antibodies.
• an agonist LT ⁇ R binding protein is purified using standard immunoglobulin purification methods. Suitable purification methods include, without limitation, chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and size-exclusion chromatography), centrifugation, or differential solubility.
• chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and size-exclusion chromatography
• centrifugation e.g., centrifugation, or differential solubility.
• the binding proteins of the present disclosure can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
• compositions comprising an agonist LT ⁇ R binding protein (e.g., an agonist LT ⁇ R antibody or bispecific agonist LT ⁇ R binding protein), in the form of a polypeptide, a nucleic acid, a vector, a host cell, or a combination thereof, are provided herein.
• a pharmaceutical composition of the present disclosure comprises the agonist LT ⁇ R binding protein, or a polynucleotide encoding the same, in an isolated and/or purified form.
• the composition comprises a combination of two or more different binding proteins (e.g., different structures) and/or polynucleotides of the present disclosure.
• compositions of the present disclosure comprise agents which enhance the chemico-physico features of the agonist LT ⁇ R binding protein or polynucleotide encoding the same, e.g., via stabilizing at certain temperatures (e.g., room temperature), increasing shelf life, reducing degradation, e.g., oxidation protease mediated degradation, increasing half-life of the agonist LT ⁇ R binding protein, etc.
• the composition additionally comprises a pharmaceutically acceptable carrier, diluents, or excipient.
• the LT ⁇ R binding protein or polynucleotide encoding the same as presently disclosed are formulated into a pharmaceutical composition comprising the active agent, along with a pharmaceutically acceptable carrier, diluent, or excipient.
• active agents are formulated into a pharmaceutical composition comprising the active agent, along with a pharmaceutically acceptable carrier, diluent, or excipient.
• the present disclosure further provides pharmaceutical compositions comprising an active agent which pharmaceutical composition is intended for administration to a subject, e.g., a mammal.
• the active agent is present in the pharmaceutical composition at a purity level suitable for administration to a patient.
• the active agent has a purity level of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%, and a pharmaceutically acceptable diluent, carrier or excipient.
• the compositions contain an active agent at a concentration of about 0.001 to about 30.0 mg/ml.
• the pharmaceutical compositions comprise a pharmaceutically acceptable carrier.
• the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.
• the term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.
• the pharmaceutical composition can comprise any pharmaceutically acceptable ingredients, including, for example, acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, bases, binders, buffering agents, chelating agents, coating agents, coloring agents, desiccants, detergents, diluents, disinfectants, disintegrants, dispersing agents, dissolution enhancing agents, dyes, emollients, emulsifying agents, emulsion stabilizers, fillers, film forming agents, flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesive, ointment bases, ointments, oleaginous vehicles, organic bases, pastille bases, pigments, plasticizers, polishing agents, preservatives, sequestering agents,
• the pharmaceutical composition comprises formulation materials that are nontoxic to recipients at the dosages and concentrations employed.
• pharmaceutical compositions comprising an active agent and one or more pharmaceutically acceptable salts, polyols, surfactants, osmotic balancing agents, tonicity agents, anti-oxidants, antibiotics, antimycotics, bulking agents; lyoprotectants; anti-foaming agents; chelating agents; preservatives; colorants; analgesics; or additional pharmaceutical agents.
• the pharmaceutical composition comprises one or more polyols and/or one or more surfactants, optionally, in addition to one or more of the above noted excipients.
• the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
• suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents;
• amino acids
• the pharmaceutical compositions can be formulated to achieve a physiologically compatible pH.
• the pH of the pharmaceutical composition can be for example between about 4 or about 5 and about 8.0 or about 4.5 and about 7.5 or about 5.0 to about 7.5.
• Another aspect of the present disclosure is directed methods of treating a subject in need thereof, where the method comprises administering an effective amount of an agonist LT ⁇ R binding protein as described herein, e.g., an agonist LT ⁇ R antibody or a bispecific agonist LT ⁇ R binding protein, or pharmaceutical composition comprising the same to the subject in need thereof.
• the agonist LT ⁇ R binding protein is one that binds to CRD4 of human LT ⁇ R and (a) does not inhibit LIGHT binding to LT ⁇ R and (b) does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R.
• this agonist LT ⁇ R binding protein is an agonist LT ⁇ R antibody.
• the agonist LT ⁇ R binding protein is a bispecific LT ⁇ R binding protein comprising (i) a LT ⁇ R binding domain, wherein the LT ⁇ R binding domain binds one or more amino acid residues of human LT ⁇ R CRD4 comprising amino acid residues 169-211 of SEQ ID NO: 1; and (ii) a tumor-associated antigen binding domain.
• This bispecific LT ⁇ R binding protein agonizes LT ⁇ R activity and (a) does not inhibit LIGHT binding to LT ⁇ R and (b) does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R.
• this bispecific agonist LT ⁇ R binding protein that is monovalent for LT ⁇ R binding.
• a subject in need thereof is a subject having cancer.
• the subject has a solid tumor.
• agonizing LT ⁇ R activity in the tumor microenvironment enhances T cell infiltration to the tumor microenvironment and induces the formation of tertiary lymphoid structures (TLS).
• TLS tertiary lymphoid structures
• This activity of LT ⁇ R enhances and/or provides anti-tumor T cell activity in the tumor microenvironment.
• administration of an agonist LT ⁇ R binding protein or pharmaceutical composition comprising the same is suitable for inducing and/or enhancing an anti-tumor immune response in a subject having a tumor.
• the term “subject” refers to a mammal, including humans, and can be used interchangeably with the term “patient”.
• the methods comprise administering to the subject the pharmaceutical composition of the present disclosure in an effective amount.
• the T cell activity or immune response is directed against a cancer cell or cancer tissue or a tumor cell or tumor.
• the immune response is a humoral immune response.
• the immune response is an innate immune response.
• the immune response which is enhanced is a T-cell mediated immune response.
• the term “enhance” and words stemming therefrom do not require or infer a 100% or complete enhancement or increase. Rather, there are varying degrees of enhancement of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect.
• the pharmaceutical compositions of the present disclosure may enhance, e.g., T cell activity, or enhance an immune response, to any amount or level beyond the amount or level of T cell activity or immune response present in the absence of treatment with an LT ⁇ R binding protein as described herein.
• the enhancement of anti-tumor immunity provided by the methods of the present disclosure is at least or about a 10% enhancement, at least or about a 20% enhancement, at least or about a 30% enhancement, at least or about a 40% enhancement, at least or about a 50% enhancement, at least or about a 60% enhancement, at least or about a 70% enhancement, at least or about a 80% enhancement, at least or about a 90% enhancement, at least or about a 95% enhancement, at least or about a 98% enhancement in anti-tumor immunity.
• Methods of measuring T cell activity and immune responses are known in the art.
• T cell activity can be measured by, for example, a cytotoxicity assay, such as those described in Fu et al., PLoS ONE 5(7): e11867 (2010).
• Other T cell activity assays that are suitable for measuring a response are described in Bercovici et al., Clin Diagn Lab Immunol. 7(6): 859–864 (2000).
• Suitable methods of measuring immune responses include those described in e.g., Macatangay et al., Clin Vaccine Immunol 17(9): 1452-1459 (2010) and Clay et al., Clin Cancer Res.7(5):1127-35 (2001).
• a subject in need of treatment with the agonist LT ⁇ R binding protein as described herein include, without limitation, subjects having cancer, and particularly subjects having a solid tumor.
• the method comprises administering to the subject an agonist LT ⁇ R binding protein or a pharmaceutical composition comprising an agonist LT ⁇ R binding protein in an amount effective to treat the cancer or the solid tumor in the subject.
• the cancer treatable by the methods and agonist LT ⁇ R binding proteins disclosed herein can be any cancer, e.g., any malignant growth or tumor caused by abnormal and uncontrolled cell division that may spread to other parts of the body through the lymphatic system or the blood stream.
• the cancer or solid tumor is a “cold” tumor, i.e., a tumor surrounded by cells that suppress an anti-tumor immune response.
• the subject to be treated with an agonist LT ⁇ R binding protein as described herein has a tumor selected from mesothelioma, a pancreatic tumor, an ovarian tumor, a lung tumor, an esophageal tumor, a gastric tumor, a hepatic tumor, a colorectal tumor, a cervical tumor, an endometrial tumor, a breast tumor, a renal tumor, a bladder tumor, a testicular tumor, a prostate tumor, a brain tumor, a bone tumor, and a head and neck tumor.
• the cancer is selected from alveolar rhabdomyosarcoma, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), malignant mesothelioma, melanoma, nasopharynx cancer, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue
• the subject has a solid tumor and the agonist LT ⁇ R binding protein (e.g., agonist LT ⁇ R antibody or bispecific agonist LT ⁇ R binding protein) is administered to the subject in an amount effective to treat the tumor in the subject.
• treatment encompasses alleviation of at least one symptom or other embodiment of a disorder, or reduction of disease severity, and the like.
• An agonist LT ⁇ R binding protein in particular an agonist LT ⁇ R antibody or bispecific binding protein that binds CRD4 of human LT ⁇ R and does not block endogenous LT ⁇ R ligand binding according to the present disclosure, need not effect a complete cure, or eradicate every symptom or manifestation of a disease, to constitute a viable therapeutic agent.
• therapeutic agents may reduce the severity of a given disease state, but need not abolish every manifestation of the disease to be regarded as useful therapeutic agents. Simply reducing the impact of a disease (for example, by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood that the disease will occur or worsen in a subject, is sufficient.
• One embodiment of the disclosure is directed to a method comprising administering to a patient an agonist LT ⁇ R binding protein in an amount and for a time sufficient to induce a sustained improvement over baseline of an indicator that reflects the severity of the particular disorder.
• the method comprising administering to a patient an agonist LT ⁇ R binding protein as disclosed herein in an amount and for a time sufficient to convert a cold tumor into a hot tumor by increasing the immune cell infiltration and/or immune cell anti-tumor activity in the tumor microenvironment.
• the method further involves administering the agonist LT ⁇ R binding protein in combination with one or more immunomodulators, e.g., PD(L)1-axis inhibitors, to further stimulate the immune cells infiltrating into the tumor microenvironment.
• the term “prevention” encompasses prevention of at least one symptom or other embodiment of a disorder, and the like.
• a prophylactically administered treatment incorporating an agonist LT ⁇ R binding protein e.g., an agonist LT ⁇ R antibody or bispecific agonist LT ⁇ R binding protein
• an agonist LT ⁇ R binding protein e.g., an agonist LT ⁇ R antibody or bispecific agonist LT ⁇ R binding protein
• prophylactic administration of the agonist LT ⁇ R binding protein as described herein is effective at preventing a tumor from turning into a cold tumor by enhancing immune cell infiltration into the tumor microenvironment.
• prophylactic administration of the agonist LT ⁇ R binding protein as described herein is effective at preventing a tumor growth by enhancing immune cell infiltration and enhancing the anti-tumor immune response in the tumor microenvironment.
• a pharmaceutical composition comprising an agonist LT ⁇ R binding protein as disclosed herein is administered to a subject in a manner appropriate to the indication and the composition.
• compositions comprising an agonist LT ⁇ R binding protein that binds to CRD4 of LT ⁇ R and does not inhibit endogenous LT ⁇ R ligand binding (e.g., an agonist LT ⁇ R antibody or bispecific agonist LT ⁇ R binding protein) is administered by any suitable technique, including but not limited to parenterally, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes, by bolus injection, or continuous infusion. Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, inhalation of the agonist LT ⁇ R binding protein in aerosol form, and the like. Other alternatives include oral preparations including pills, syrups, or lozenges.
• the agonist LT ⁇ R binding proteins can be administered in the form of a composition comprising one or more additional components such as a physiologically acceptable carrier, excipient or diluent.
• the composition additionally comprises one or more physiologically active agents.
• the composition comprises one, two, three, four, five, or six physiologically active agents in addition to one or more agonist LT ⁇ R binding proteins.
• the kit includes a sterile preparation of one or more agonist LT ⁇ R binding proteins, which may be in the form of a composition as disclosed herein, and may be in one or more vials.
• dosesages and the frequency of administration may vary according to such factors as the route of administration, the particular agonist LT ⁇ R binding proteins employed, the nature and severity of the disease to be treated, whether the condition is acute or chronic, and the size and general condition of the subject. Appropriate dosages can be determined by procedures known in the pertinent art, e.g., in clinical trials that may involve dose escalation studies.
• An agonist LT ⁇ R binding protein that binds to CRD4 of LT ⁇ R and does not inhibit endogenous LT ⁇ R ligand binding activity as disclosed herein may be administered, for example, once or more than once, e.g., at regular intervals over a period of time.
• a LT ⁇ R binding protein is administered over a period of at least once a month or more, e.g., for one, two, or three months or even indefinitely.
• long-term treatment is generally most effective.
• administration for shorter periods e.g., from one to six weeks, may be sufficient.
• the agonist LT ⁇ R binding protein is administered until the patient manifests a medically relevant degree of improvement over baseline for the chosen indicator or indicators.
• An agonist LT ⁇ R binding protein that binds CRD4 of LT ⁇ R and/or does not inhibit endogenous LT ⁇ R ligand binding activity as disclosed herein, may preferably be administered in conjunction with the administration of an immunomodulatory therapeutic.
• the immunomodulatory therapeutic is a checkpoint inhibitor. Suitable checkpoint inhibitors are known in the art and include, for example and without limitation, a PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, TIM-3 inhibitor, LAG-3 inhibitor, NKG2A inhibitor, CD73 inhibitor, and TIGIT inhibitor, and any combination thereof.
• Combination Therapies involve the use of at least one agonist LT ⁇ R binding protein and one or more other therapeutics useful for treating or preventing cancer.
• agonist LT ⁇ R binding protein is administered alone or in combination with other agents useful for treating a subject having a solid tumor. Examples of such agents include both proteinaceous and non-proteinaceous drugs.
• dosages may be adjusted accordingly, as is recognized in the pertinent art.
• Co-administration and combination therapy are not limited to simultaneous administration, but also include treatment regimens in which an agonist LT ⁇ R binding protein is administered at least once during a course of treatment that involves administering at least one other therapeutic agent to the patient.
• an agonist LT ⁇ R binding protein is administered prior to the administration of at least one other therapeutic agent.
• agonist LT ⁇ R binding protein is administered concurrent with the administration of at least one other therapeutic agent.
• an agonist LT ⁇ R binding protein is administered subsequent to the administration of at least one other therapeutic agent.
• the at least one other therapeutic agent is an immunomodulatory therapeutic, in particular a checkpoint inhibitor or a T cell engaging therapeutic.
• Suitable checkpoint inhibitors are known in the art and include, for example and without limitation, a PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, TIM-3 inhibitor, LAG-3 inhibitor, NKG2A inhibitor, CD73 inhibitor, and TIGIT inhibitor, and any combination thereof.
• the agonist LT ⁇ R binding protein that binds CRD4 of LT ⁇ R and does not inhibit endogenous LT ⁇ R ligand binding activity as disclosed herein is administered in combination with a PD-1 inhibitor, e.g., an anti-PD-1 antibody.
• Suitable anti-PD-1 antibodies include, without limitation, nivolumab (Opdivo ® /KEGG D10316), pembrolizumab (Keytruda ® /KEGG D10574), Cemiplimab (Libtayo ® /KEGG D11108), Dostarlimab (TSR-042/KEGG D11366), Spartalizumab (KEGG D11605), and pidilizumab (CT- 011; KEGG D10390).
• nivolumab Opdivo ® /KEGG D10316
• pembrolizumab Keytruda ® /KEGG D10574
• Cemiplimab Libtayo ® /KEGG D11108
• Dostarlimab TSR-042/KEGG D11366
• Spartalizumab KEGG D11605
• pidilizumab CT- 011; KEGG D10390.
• the agonist LT ⁇ R binding protein that binds CRD4 of LT ⁇ R and does not inhibit endogenous LT ⁇ R ligand binding activity as disclosed herein is administered in combination with a PD-L1 inhibitor, e.g., an anti-PD-L1 antibody.
• a PD-L1 inhibitor e.g., an anti-PD-L1 antibody.
• Suitable anti-PD-L1 antibodies include, without limitation, Atezolizumab (Tecentriq ® ; KEGG D10773), Avelumab (Bavencio ® ; KEGG D10817), and Durvalumab (Imfinzi ® ; KEGG D10808) [0325]
• the agonist LT ⁇ R binding protein that binds CRD4 of LT ⁇ R and does not inhibit endogenous LT ⁇ R ligand binding activity as disclosed herein is administered in combination with a CTLA-4 inhibitor.
• a suitable CTLA-4 inhibitor is the monoclonal antibody Ipilimumab (Yervoy; KEGG D04603).
• the agonist LT ⁇ R binding protein that binds CRD4 of LT ⁇ R and does not inhibit endogenous LT ⁇ R ligand binding activity as disclosed herein is administered in combination with a TIM-3 inhibitor, e.g., an anti-TIM-3 antibody.
• a TIM-3 inhibitor e.g., an anti-TIM-3 antibody.
• suitable anti-TIM-3 antibodies include, without limitation, sabatolimab (MBG453; Novartis); LY3321367 (Eli Lilly), and TSR-022 (Tesaro).
• the agonist LT ⁇ R binding protein that binds CRD4 of LT ⁇ R and/or does not inhibit endogenous LT ⁇ R ligand binding activity as disclosed herein is administered in combination with a LAG-3 inhibitor, e.g., an anti-LAG-3 antibody.
• a LAG-3 inhibitor e.g., an anti-LAG-3 antibody.
• An exemplary anti-LAG-3 antibody suitable for use in combination with the LT ⁇ R binding protein described herein includes, without limitation, Relatlimab (KEGG D11350).
• the agonist LT ⁇ R binding protein that binds CRD4 of LT ⁇ R and does not inhibit endogenous LT ⁇ R ligand binding activity as disclosed herein is administered in combination with an inhibitor of NKG2-A/NKG2-B type II integral membrane protein (NKG2A), e.g., an anti- NKG2A antibody.
• NKG2-A/NKG2-B type II integral membrane protein e.g., an anti- NKG2A antibody.
• An exemplary anti- NKG2A antibody suitable for use in combination with the LT ⁇ R binding protein described herein includes, without limitation, BMS-986315 (Bristol Myers Squibb ® ).
• the agonist LT ⁇ R binding protein that binds CRD4 of LT ⁇ R and does not inhibit endogenous LT ⁇ R ligand binding activity as disclosed herein is administered in combination with a CD73 inhibitor.
• Suitable CD73 inhibitors for use in combination with the LT ⁇ R binding protein described herein include, without limitation, the small molecule CD73 inhibitors OP-5244 (Du et al., J. Med. Chem. 63:10433-10459 (2020), which is hereby incorporated by reference in its entirety) and LY- 3475070 (Eli Lilly).
• the agonist LT ⁇ R binding protein that binds CRD4 of LT ⁇ R and does not inhibit endogenous LT ⁇ R ligand binding activity as disclosed herein is administered in combination with a TIGIT inhibitor, e.g., an anti-TIGIT antibody.
• a TIGIT inhibitor e.g., an anti-TIGIT antibody.
• suitable anti-TIGIT antibodies include, without limitation, BMS-986207 (Bristol-Myers Squibb); Tiragolumab (Roche)), and MK-7684 (Merck), and COM902 (Compugen).
• Embodiment 1 of the disclosure is directed to an agonist Lymphotoxin ⁇ Receptor (LT ⁇ R) binding protein, wherein said binding protein binds an epitope comprising one or more residues of human LT ⁇ R cysteine-rich domain 4 (CRD4), wherein human LT ⁇ R CRD4 comprises amino acid residues 169- 211 of SEQ ID NO: 1, and wherein said binding protein (a) does not inhibit LIGHT binding to LT ⁇ R or (b) does not inhibit LT ⁇ 1 ⁇ 2 binding to LT ⁇ R.
• LT ⁇ R Lymphotoxin ⁇ Receptor
• Embodiment 2 is the agonist LT ⁇ R binding protein of embodiment 1, wherein said binding protein (a) does not inhibit LIGHT binding to LT ⁇ R and (b) does not inhibit LT ⁇ 1 ⁇ 2 binding to LT ⁇ R.
• Embodiment 3 is the agonist LT ⁇ R binding protein of embodiment 1 or 2, wherein said binding protein binds to one or more residues of LT ⁇ R CRD4 comprising residues 197-209 of SEQ ID NO: 1.
• Embodiment 4 is the agonist LT ⁇ R binding protein of embodiments 1-3, wherein inhibition of LIGHT and/or LT ⁇ 1 ⁇ 2 binding to LT ⁇ R in the presence of the agonist LT ⁇ R binding protein is measured in a cell-based receptor-ligand binding assay, said assay comprising the steps of: incubating LT ⁇ R expressing cells with media containing the agonist LT ⁇ R binding protein for 1 hour; contacting the LT ⁇ R expressing cells, after said incubating, with a detectable LIGHT ligand, a detectable LT ⁇ 1 ⁇ 2 ligand, or a combination thereof to allow the detectable ligands to bind to LT ⁇ R; detecting, after said contacting, the detectable LIGHT and/or LT ⁇ 1 ⁇ 2 ligands bound to LT ⁇ R expressing cells in the presence of the LT ⁇ R binding protein; and identifying the agonist LT ⁇ R binding protein as not inhibiting LIGHT or LT ⁇ 1 ⁇ 2 binding to LT ⁇ R
• Embodiment 5 is the agonist LT ⁇ R binding protein of any one of embodiments 1–4, wherein said binding protein comprises: a heavy chain variable domain (VH) amino acid sequence having at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 123 and a light chain variable domain (VL) amino acid sequence having at least 90% sequence identity to SEQ ID NO: 122 or SEQ ID NO: 124.
• VH heavy chain variable domain
• VL light chain variable domain
• Embodiment 6 is the agonist LT ⁇ R binding protein of any one of embodiments 1–4, wherein said binding protein comprises: a VH amino acid sequence having at least 90% sequence identity to SEQ ID NO: 121 and SEQ ID NO: 123, and a VL amino acid sequence having at least 90% sequence identity to SEQ ID NO: 122 and SEQ ID NO:124.
• Embodiment 7 is the agonist LT ⁇ R binding protein of any one of embodiments 1–4, wherein said binding protein comprises: a VH amino acid sequence having at least 95% sequence identity to SEQ ID NO: 121 and SEQ ID NO: 123, and a VL amino acid sequence having at least 95% sequence identity to SEQ ID NO: 122 and SEQ ID NO:124.
• Embodiment 8 is the agonist LT ⁇ R binding protein of any one of embodiments 1–7, wherein said binding protein comprises: a VH comprising the HCDR1 amino acid sequence of X 1 YX 3 MX 5 (SEQ ID NO: 5), wherein X 1 is S or N; X 3 is G, D, or A; and X 5 is H or Y; the HCDR2 amino acid sequence of X 1 IX 3 YDX 6 X 7 X 8 X 9 Y X 11 X 12 DSVKG (SEQ ID NO: 6), wherein X 1 is A or V; X 3 is W or R; X 6 is E or G; X 7 is S, R, or T; X 8 is N or K; X 9 is K, R, or Q; X 11 is H or Y; and X 12 is A or E; and the HCDR3 amino acid sequence of X 1 RX 3 X 4 X 5 X 6 X 7 X 8 X
• Embodiment 9 is the agonist LT ⁇ R binding protein of any one of embodiments 1–8, wherein said binding protein comprises: a VH amino acid sequence having at least 90% sequence identity to SEQ ID NO: 121 and a HCDR1 amino acid sequence of SEQ ID NO: 5, a HCDR2 amino acid sequence of SEQ ID NO: 6, and a HCDR3 amino acid sequence of SEQ ID NO: 7; and a VL amino acid sequence having at least 90% sequence identity to SEQ ID NO: 122 and a LCDR1 amino acid sequence of SEQ ID NO: 62, a LCDR2 amino acid sequence of SEQ ID NO: 63, and a LCDR3 amino acid sequence of SEQ ID NO: 64.
• Embodiment 10 is the agonist LT ⁇ R binding protein of any one of embodiments 1–9, wherein said binding protein comprises: a VH comprising the HCDR1, HCDR2, and HCDR3 of amino acid sequences of SEQ ID NO: 11-13, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NO: 68-70, respectively; or a VH comprising the HCDR1, HCDR2, and HCDR3 of amino acid sequences of SEQ ID NO: 8-10, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NO: 65-67, respectively.
• Embodiment 11 is the agonist LT ⁇ R binding protein of any one of embodiments 1–10, wherein the binding protein is an antibody.
• Embodiment 12 is the agonist LT ⁇ R binding protein of any one of embodiments 1–10, wherein the binding protein is a bispecific binding protein.
• Embodiment 13 is directed to a bispecific Lymphotoxin ⁇ Receptor (LT ⁇ R) binding protein, said binding protein comprising: an LT ⁇ R binding domain, wherein the LT ⁇ R binding domain binds an epitope comprising one or more residues of human LT ⁇ R cysteine-rich domain 4 (CRD4), wherein said LT ⁇ R CRD4 comprises amino acid residues 169-211 of SEQ ID NO: 1; and a tumor-associated antigen binding domain, wherein the bispecific binding protein agonizes LT ⁇ R activity, and (a) does not inhibit LIGHT binding to LT ⁇ R or (b) does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R.
• LT ⁇ R bispecific Lymphotoxin ⁇ Receptor
• Embodiment 14 is the bispecific agonist LT ⁇ R binding protein of embodiment 13, wherein said binding protein (a) does not inhibit LIGHT binding to LT ⁇ R and (b) does not inhibit LT ⁇ 1 ⁇ 2 binding to LT ⁇ R.
• Embodiment 15 is the bispecific agonist LT ⁇ R binding protein of embodiment 13, wherein the epitope comprises one or more residues of LT ⁇ R CRD4 at positions 197-209 of SEQ ID NO: 1.
• Embodiment 16 is the bispecific agonist LT ⁇ R binding protein of any one of embodiments 13–15, wherein inhibition of LIGHT and/or LT ⁇ 1 ⁇ 2 binding to LT ⁇ R in the presence of the bispecific agonist LT ⁇ R binding protein is measured in a cell-based receptor-ligand binding assay, said assay comprising the steps of: incubating LT ⁇ R expressing cells with media containing the bispecific agonist LT ⁇ R binding protein for 1 hour; contacting the LT ⁇ R expressing cells, after said incubating, with a detectable LIGHT ligand, a detectable LT ⁇ 1 ⁇ 2 ligand, or a combination thereof to allow the detectable ligands to bind to LT ⁇ R; detecting, after said contacting, the detectable LIGHT and/or LT ⁇ 1 ⁇ 2 ligands bound to LT ⁇ R expressing cells in the presence of the bispecific agonist LT ⁇ R binding protein; and identifying the bispecific agonist LT ⁇ R binding protein as not inhibiting
• Embodiment 17 is he bispecific agonist LT ⁇ R binding protein of any one of embodiments 13-15, wherein the LT ⁇ R binding domain comprises: a VH amino acid sequence having at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 123, and a VL amino acid sequence having at least 90% sequence identity to SEQ ID NO: 122 or SEQ ID NO: 124.
• Embodiment 18 is the bispecific agonist LT ⁇ R binding protein of embodiment 13, wherein the LT ⁇ R binding domain comprises: a VH amino acid sequence having at least 90% sequence identity to SEQ ID NO: 121 and SEQ ID NO: 123 and a VL amino acid sequence having at least 90% sequence identity to SEQ ID NO: 122 and SEQ ID NO: 124.
• Embodiment 19 is the bispecific agonist LT ⁇ R binding protein of embodiment 13, wherein the LT ⁇ R binding domain comprises: a VH amino acid sequence having at least 95% sequence identity to SEQ ID NO: 121 and SEQ ID NO: 123 and a VL amino acid sequence having at least 95% sequence identity to SEQ ID NO: 122 and SEQ ID NO: 124.
• Embodiment 20 is the bispecific agonist LT ⁇ R binding protein of any one of embodiments 13–19, wherein the LT ⁇ R binding domain comprises: a VH comprising the HCDR1 amino acid sequence of X 1 YX 3 MX 5 (SEQ ID NO: 5), wherein X 1 is S or N; X 3 is G, D, or A; and X 5 is H or Y; the HCDR2 amino acid sequence of X 1 IX 3 YDX 6 X 7 X 8 X 9 Y X 11 X 12 DSVKG (SEQ ID NO: 6), wherein X 1 is A or V; X 3 is W or R; X 6 is E or G; X 7 is S, R, or T; X 8 is N or K; X 9 is K, R, or Q; X 11 is H or Y; and X 12 is A or E; and the HCDR3 amino acid sequence of X 1 RX 3 X 4 X 5 X 6 X
• Embodiment 21 is he bispecific agonist LT ⁇ R binding protein of any one of embodiments 13–20, wherein said binding protein comprises: a VH amino acid sequence having at least 90% sequence identity to SEQ ID NO: 121 and a HCDR1 amino acid sequence of SEQ ID NO: 5, a HCDR2 amino acid sequence of SEQ ID NO: 6, and a HCDR3 amino acid sequence of SEQ ID NO: 7; and a VL amino acid sequence having at least 90% sequence identity to SEQ ID NO: 122 and a LCDR1 amino acid sequence of SEQ ID NO: 62, a LCDR2 amino acid sequence of SEQ ID NO: 63, and a LCDR3 amino acid sequence of SEQ ID NO: 64.
• Embodiment 22 is the bispecific agonist LT ⁇ R binding protein of embodiment 13, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 of amino acid sequences of SEQ ID NO: 11-13, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NO: 68-70, respectively; or the VH comprises the HCDR1, HCDR2, and HCDR3 of amino acid sequences of SEQ ID NO: 8-10, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NO: 65-67, respectively.
• Embodiment 23 is the bispecific agonist LT ⁇ R binding protein of any one of embodiments 13–22, wherein the bispecific binding protein comprises only one LT ⁇ R binding domain.
• Embodiment 24 is the bispecific agonist LT ⁇ R binding protein of any one of embodiments 13–22, wherein the LT ⁇ R binding domain is a Fab.
• Embodiment 25 is the bispecific agonist LT ⁇ R binding protein of any one of embodiments 13–24, wherein binding protein comprises one tumor-associated antigen binding domain.
• Embodiment 26 is the bispecific agonist LT ⁇ R binding protein of embodiment 25, wherein the tumor-associated antigen binding domain is a Fab.
• Embodiment 27 is the bispecific agonist LT ⁇ R binding protein of any one of embodiments 13-24, wherein binding protein comprises two tumor-associated antigen binding domains.
• Embodiment 28 is the bispecific agonist LT ⁇ R binding protein of any one of embodiments 13–22, wherein the LT ⁇ R binding domain is a Fab and the tumor-associated antigen binding domain is a Fab.
• Embodiment 29 is the bispecific agonist LT ⁇ R binding protein of any one of embodiments 13–28, wherein the LT ⁇ R binding domain and the tumor-associated antigen binding domain are each coupled to an Fc portion.
• Embodiment 30 is the bispecific agonist LT ⁇ R binding protein of embodiment 29, wherein the Fc portion does not bind to an Fc-gamma receptor.
• Embodiment 31 is directed to a polynucleotide encoding the agonist LT ⁇ R binding protein of any one of embodiments 1–12.
• Embodiment 32 is a vector comprising the polynucleotide of embodiment 31.
• Embodiment 33 is a host cell comprising the polynucleotide of embodiment 31 or the vector of embodiment 32.
• Embodiment 34 is one or more polynucleotides encoding the bispecific agonist LT ⁇ R binding protein of any one of embodiments 13–30.
• Embodiment 35 is a vector comprising the one or more polynucleotides of embodiment 34.
• Embodiment 36 is a host cell comprising the one or more polynucleotides of embodiment 34 or the vector of embodiment 35.
• Embodiment 37 is a pharmaceutical composition comprising: the agonist LT ⁇ R binding protein of any one of embodiments 1–12, the bispecific agonist LT ⁇ R binding protein of any one of embodiments 13–30, the one or more polynucleotides of embodiments 31 or 34, or the vector of embodiments 32 or 35, and a pharmaceutically acceptable carrier.
• Embodiment 38 is the agonist LT ⁇ R binding protein of any one of embodiments 1–12 for use as a medicament.
• Embodiment 39 is the agonist LT ⁇ R binding protein of any one of embodiments 1–12 for use in the treatment of cancer.
• Embodiment 40 is the agonist LT ⁇ R binding protein of any one of embodiments 1–12 for use in the manufacture of a medicament for the treatment of cancer
• Embodiment 41 is the bispecific agonist LT ⁇ R binding protein of any one of embodiments 13–30 for use as a medicament.
• Embodiment 42 is the bispecific agonist LT ⁇ R binding protein of any one of embodiments 13–30 for use in the treatment of cancer.
• Embodiment 43 is the bispecific agonist LT ⁇ R binding protein of any one of embodiments 13–30 for use in the manufacture of a medicament for the treatment of cancer.
• Embodiment 44 is directed to a method of treating cancer in a subject, said method comprising: administering, to the subject having cancer, an agonist Lymphotoxin ⁇ Receptor (LT ⁇ R) binding protein, wherein said binding protein binds an epitope comprising one or more residues of human LT ⁇ R cysteine-rich domain 4 (CRD4), wherein human LT ⁇ R CRD4 comprises amino acid residues 169- 211 of SEQ ID NO: 1, and wherein said binding protein (a) does not inhibit LIGHT binding to LT ⁇ R or (b) does not inhibit LT ⁇ 1 ⁇ 2 binding to LT ⁇ R.
• LT ⁇ R Lymphotoxin ⁇ Receptor
• Embodiment 45 is a method of treating cancer in a subject, said method comprising: administering, to the subject having cancer, a bispecific Lymphotoxin ⁇ Receptor (LT ⁇ R) binding protein, said binding protein comprising: an LT ⁇ R binding domain, wherein the LT ⁇ R binding domain binds an epitope comprising one or more amino acid residues of human LT ⁇ R cysteine-rich domain 4 (CRD4), wherein human LT ⁇ R CRD4 comprises amino acid residues 169-211 of SEQ ID NO: 1; and a tumor- associated antigen binding domain, wherein the bispecific binding protein agonizes LT ⁇ R activity, and (a) does not inhibit LIGHT binding to LT ⁇ R or (b) does not inhibit LT1 ⁇ 2 ⁇ binding to LT ⁇ R.
• LT ⁇ R bispecific Lymphotoxin ⁇ Receptor
• Embodiment 46 is the method of embodiment 44 or embodiment 45 further comprising: administering an immunomodulatory therapeutic in conjunction with said agonist LT ⁇ R binding protein or bispecific LT ⁇ R binding protein.
• Embodiment 47 is the method of embodiments 41 or 42, wherein the subject has a solid tumor selected from the group consisting of mesothelioma, a pancreatic tumor, an ovarian tumor, a lung tumor, an esophageal tumor, a gastric tumor, a hepatic tumor, a colorectal tumor, a cervical tumor, an endometrial tumor, a breast tumor, a renal tumor, a bladder tumor, a testicular tumor, a prostate tumor, a brain tumor, a bone tumor, and a head and neck tumor.
• Example 1 Tumor Associated Antigen (TAA)-targeted LT ⁇ R Agonism Exhibits Therapeutic Efficacy in In Vivo Tumor Models
• TAA Tumor Associated Antigen
• 1.1 EpCAM Targeted LT ⁇ R Agonism Induces HEV Formation and Increases Lymphocyte Infiltration in Murine B16F10 Melanoma Model.
• HEVs high endothelial venules
• FIG.2 huEpCAM-LT ⁇ R treatment induced the formation of high endothelial venules (HEVs) in the tumor environment (bottom image). The presence of HEVs was not observed in the isotype control treated tumor tissue (FIG.2, top image). The presence of HEVs was assessed by immunohistochemical staining using anti-PNAd (Meca79) mAb.
• 1.2 LRRC15 Targeted LT ⁇ R Agonism Induces Lymphocyte Infiltration and Inhibits Tumor Growth in KPC M5 Tumor Model.
• BsAbs bispecific antibodies
• Tumors were collected with skin attached on the periphery. The attached skin was removed and the tumor was dissociated for flow cytometry analysis. As seen in FIG.3, an increase in T cell infiltration (CD3 + cells) into the tumor following LRRC15 x LT ⁇ R bispecific antibody treatment compared to isotype control was observed.
• mice were dosed intraperitoneally, twice a week for 9 doses (or until they exited) with 3mg/kg of control isotype mIgG1 (11445-3), twice a week for 9 doses with 3 mg/kg of LRRC15 x LT ⁇ R BsAb, or once every three days for 3 doses with 100 ⁇ g/mouse of anti-PD1 antibody (7358-1).
• Tumors were measured and body weights were weighted twice a week until tumors reached greater or equal to 2000mm 3 or if animals reached twice the median survival limit.
• PD-1 antibody treatment did not significantly inhibit tumor growth compared to isotype ( ⁇ ), producing a tumor growth inhibition (TGI) of only 4.64%.
• TGI Tumors treated with LRRC15 x LT ⁇ R BsAb monotherapy ( ⁇ ) did produce a statistically significant TGI of 20.76% compared to isotype. The combination of LRRC15 x LT ⁇ R BsAb and PD-1 antibody did lead to a greater TGI of 32.37%, but there was no statistical significance between the two treatment groups.
• Transgenic XenoMouse ® that produce human immunoglobulin G (IgG) 2 or IgG 4 antibodies were immunized intradermally 12-16 times over 6-8 weeks with GeneGun (BioRad) protocol that delivered gold microparticles coated with plasmid DNA encoding human LT ⁇ R and molecular adjuvants.
• animals were subcutaneously immunized with CHO- S cells that were transiently transfected with human or cyno LT ⁇ R expression vectors.
• mice were subcutaneously injected with human LT ⁇ R recombinant protein at 5 ⁇ g protein per boost twice weekly for 6-8 weeks.
• Cyno LT ⁇ R antigen was introduced to mice at week 4 and was injected on an alternating schedule with human LT ⁇ R antigen.
• sera from immunized mice were tested for binding to the human and cyno LT ⁇ R expressed on transiently transfected HEK 293 cells and analyzed by fluorescence-activated cell sorting (FACS).
• FACS fluorescence-activated cell sorting
• the fold GeoMean shift over background was applied to select the responding mice with the highest native titer to human and cyno LT ⁇ R.
• the selected mice received a final boost 4 days before tissue harvest followed by B cell enrichment.
• the harvest and immunogen details are listed in Table 15. Table 15.
• Example 4 First Round of Screening Hybridoma Pools for LT ⁇ R Binder Identification
• XenoMouse ® hybridoma cultures from harvest 1 to 6 were thawed and grown in DMEM Selection Medium for 3-4 days. Culture medium was changed to BDQY Hybridoma Medium a day before FACS enrichment sorting. Cells were washed in 10 mL sterile FACS buffer and then incubated with biotin-labelled recombinant human LT ⁇ R (R&D Systems, Cat: 7538LR) at 2 to 5 ⁇ g/mL concentration in 1 mL reaction volume for 30 minutes at 4°C.
• biotin-labelled recombinant human LT ⁇ R R&D Systems, Cat: 7538LR
• the cells were washed in 10 mL FACS buffer, resuspended in 2 mL of BDQY Hybridoma Medium containing 5 ⁇ L of 7-AAD (BD Pharmingen, Cat: 559925) then put through a 40-micron cell strainer to remove any clumps.
• Cells were bulk sorted on BD FACSAria® by gating on live cell population dual positive for Alexa Fluor 488 and Alexa Fluor 647 fluorescence signals.
• LT ⁇ R Human LT ⁇ R (pTT5.2:VK1O2O12::huLT ⁇ R(31-435) C-173003 (DNA-38098)), was expressed on host Human Embryonic Kidney 293 cells by transfection using expression vectors, Opti-MEM® media (GibcoTM, Cat.
• Protein G (Sigma, Cat# P4689) was prepared in assay media (growth media contain 2% FBS) at final concentration of 0.5 ⁇ g/mL.
• Anti-LT ⁇ R mAb (R&D systems, Clone 71315 and Clone 31G4D8) was prepared at final concentration of 1 ⁇ g/mL and 5 ⁇ g/mL respectively, titrated 1:3 down for total 8 points.
• exhausted supernatant (ESN) containing anti- huLT ⁇ R antibody was added at final concentration of 1 ⁇ g/mL.
• ESN was first normalized to 10 ⁇ g/mL, then further diluted in assay medium to the final concentration of 1 ⁇ g/mL, titrated 1:3 down for total 8 points.
• Hybridoma supernatants were screened for the presence of monoclonal antibodies binding to cyno Major LT ⁇ R, cyno minor LT ⁇ R, Mu LT ⁇ R, TNRFSF1 and TNRFSF2 by incubating antibodies on each of the transfected cells for 1 hour, followed by two wash steps to remove the primary antibody. The cells were then incubated with a goat anti-human Fc antibody conjugated to Alexa647 (Jackson Immunochemicals 109-605-098) for 15 minutes and washed two times to remove unbound detection reagent.
• Biotinylated recombinant soluble human LT ⁇ R protein Fc Chimera (R&D Systems 7538-LR-100) was coupled at 2 ⁇ g/mL concentration to streptavidin coated, uniquely barcoded LumAvidin Beads® (LumAvidin Microspheres, Luminex Corp., Austin,Texas, U.S.A.) for 30 minutes in the dark at room temperature and washed twice.
• the reference antibody hybridoma supernatant samples at 5 ⁇ g/mL concentration were incubated with the antigen-coated beads for 1 hour in the dark at room temperature and washed three times.
• Beads were resuspended in FACS buffer containing an immunoassay stabilizer solution (StabilguardTM, SurModics).
• StabilguardTM an immunoassay stabilizer solution
• the antigen-coated, reference antibody-bound beads were pooled and then divided into individual sample wells containing a normalized (2 ⁇ g/ml) test antibody (hybridoma supernatant) sample (or negative control), incubated for 1 hour in the dark at room temperature and washed twice.
• the samples were then incubated with Mo anti-HuG2, G3, G4 (PL-46339, made from the sequence of Calbiochem HP6030 antibody which was discontinued) for 1 hour in the dark at room temperature and washed twice.
• Samples were incubated with a goat anti-mouse Fc antibody conjugated to Alexa488 (Jackson Immunochemicals 115-545-071) for 15 minutes in the dark at room temp and washed two times to remove unbound detection reagent and resuspended in FACS buffer. Samples were analyzed using an Intellicyt iQueTM Screener Platform. To determine the antibody competition/binding profiles of the individual test antibodies, the reference-only antibody binding signal was subtracted from the reference plus test antibody signal for each competition/binding reaction (i.e., across the entire reference antibody set). An individual antibody binding profile was defined as the collection of net binding values for each competition/binding reaction.
• the degree of similarity between individual profiles was then assessed by calculating the coefficient of determination between each of the test antibody profiles.
• Test antibodies showing high degrees of similarity (R 2 > 0.8) to each other were then grouped into common binning profiles.
• the LT ⁇ R binding antibodies were sub-divided into 6 unique binning profiles (A, B, C, D, E and F).
• the results for relative epitope binning of the LT ⁇ R antibodies are shown in Table A of FIG.5.
• Hybridoma supernatants containing human LT ⁇ R-binding antibodies were assayed for their ability to block human LIGHT and human LT ⁇ 1 ⁇ 2 binding to human LT ⁇ R-His via a FACS-based receptor-ligand assay. If a LT ⁇ R antibody prevented human LIGHT-Alexa 647 or human LT ⁇ 1 ⁇ 2-Alexa 647 from binding to human LT ⁇ R-His, the flow cytometry geometric mean (GeoMean) signal decreases relative to the GeoMean signal achieved in the absence of LT ⁇ R antibody thus resulting in a high percent inhibition value of ligand binding due to competition. Table A of FIG.
• the beads were then washed twice in FACS buffer by centrifugation for 2 minutes at 3500 RPM.
• the coated beads were resuspended in immunoassay stabilizing solution (StabilGuard TM SurModics, Cat. No. SG01).
• Human LT ⁇ R-specific monoclonal antibodies (15 ⁇ L at 10 ⁇ g/mL) were combined with 15 ⁇ L of coated beads such that the final concentration of LT ⁇ R antibody was at 5 ⁇ g/mL and incubated for 1 hour at room temperature using 96 well V-bottom polystyrene FACS plates (Corning, Cat. No. 3897).
• the maximum GeoMean signal for human LIGHT-Alexa 647 or human LT ⁇ 1 ⁇ 2-Alexa 647 was determined by averaging the GeoMean signals of the ligand-Alexa 647 signal over 12 replicates that were incubated with irrelevant exhaust supernatant (ESN) binding to biotinylated human LT ⁇ R-His.
• the minimum GeoMean signal for human LIGHT-Alexa 647 or human LT ⁇ 1 ⁇ 2-Alexa 647 was determined from GeoMean signal of the ligand-Alexa 647 signal incubated with an irrelevant human IgG2 control binding to irrelevant biotinylated human TREM1-His.
• Affinity Gap Analysis was measured on a BIAcore ® 3000 from GE Healthcare. Experiments were run at 25°C. The running buffer was HBS-P (10 mM HEPES, pH7.4, 150 mM NaCl, 0.05% Surfactant P-20; Cytiva cat# BR100827) supplemented with 0.5% BSA, and kinetics were at a high flow rate (100 ⁇ l/min). 10 mM Glycine, pH 1.7 was used for regeneration.
• a mouse anti- human antibody was diluted to 0.065mg/mL in 10nM Sodium Acetate, pH 5 and covalently coupled to the sample and reference flowcell (Fc1) of a CM5 sensor chip (Cytiva cat# 29104988) using amine coupling reagent (Cytiva cat# BR100050).
• Unpurified anti-LT ⁇ R mAbs were diluted in HBS-P to 1 ⁇ g/ml and captured on flowcell 2, 3, or 4 to obtain a capture level of 100 RU – 200 RU.
• Fc1 was used as a reference.
• the target, human or cyno LT ⁇ R was injected as analyte at 150 nM, 50 nM, 16.7 nM, 5.6 nM, and 1.85 nM with the 50 nM concentration run twice to gage reproducibility.
• the dissociation time was 10 minutes to allow for screening of the large antibody panel.
• the data was double background referenced in that both a reference Fc and a 0 nM analyte concentration were subtracted from the data.
• a 1:1 Langmuir binding model with mass transfer was used to analyze the data in the BIAcore ® evaluation software.
• the results for human and cyno major are shown in Table B of FIG.5.
• RNA total or mRNA
• RNA total or mRNA
• 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).
• RT-PCR polymerase chain reaction
• 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.
• 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 and lambda light chains were 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 light chain using multiplex PCR.
• the 5’ light chain-specific primer annealed to the signal sequence of the antibody light chain while the 3’ primer annealed to a constant region of the light chain.
• 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.
• Example 5 LT ⁇ R Antibody Engineering [0399] Select anti-LT ⁇ R antibodies were converted to a standard antibody format of the IgG1 subtype by fusing the VH domains to the CH1-CH2-CH3 sequence and VL domains to CL or CK sequence.
• the CH2 domain of this antibody isotype has been engineered for reduced effector function by incorporating an N297G mutation and for improved thermostability through an engineered disulfide bond (R292C, V302C); this antibody isotype is designated SEFL2.2.
• the anti-LT ⁇ R antibodies were additionally engineered to remove “hotspots,” or residues that were computationally predicted or empirically determined to impact the molecule’s expression, purification, homogeneity, thermal stability, colloidal stability, long-term storage stability, in vivo pharmacokinetics, and/or immunogenicity.
• a variety of amino acid mutations at these hotspots in the variable heavy (VH) and variable light (VL) domains were designed based on conservation, co-variation, chemical similarity, predictions from structural modeling, and prior knowledge from other antibody engineering campaigns.
• Engineered antibodies were designed that included both single mutations and combinations of mutations.
• Table 17 (below) provides a summary of the single and combination mutations introduced into a subset of LT ⁇ R antibodies and the binding affinity (KD) of the engineered antibodies. The position of the identified substitution is relative to a reference sequence derived from Honegger and Pluckthun, “Yet Another Numbering Scheme for Immunoglobulin Variable Domains: An Automatic Modeling and Analysis Tool,” J. Mol. Biol.309: 657-670 (2001), which is hereby incorporated by reference in its entirety. Table 17.
• HDX MS measures the deuterium exchange rate of protein back bone amide hydrogens.
• HDX MS experiments were performed with a Twin HTS PAL HDX system (LEAP Technologies, Carrboro. NC), configured to perform online digestion on a pepsin column, and interfaced with an Orbitrap mass spectrometer (Elite, ThermoFisher Scientific, San Jose, CA), as previously described (see e.g., Chalmers et al., Anal. Chem.
• LT ⁇ R/antibody complexes can be readily formed by preincubating LT ⁇ R and antibodies at the final concentrations of ⁇ 20 ⁇ M and ⁇ 10 ⁇ M, respectively.
• the H/D exchange reaction was initiated by 5-fold dilution protein samples with 10 mM acetate in D2O (pD 5.2) as indicated for a predetermined time (10 s, 1, 10 min, 1, 4, and 12 h) at 25°C.
• the exchange reaction was quenched by mixing 1:1 with ice-cold 200 mM sodium phosphate, 4 M guanidine HCl, 0.5 M Tris(2- carboxyethyl)phosphine (TCEP), pH 2.4.
• the quenched protein mixture was passed over a custom-packed 2 mm ⁇ 2 cm pepsin (Fisher Scientific, Pittsburgh, PA) column (Agilent Technologies, Santa Clara, CA) at a flow rate of 200 ⁇ L/min.
• Digested peptides were captured on a 2 mm ⁇ 1 cm C18 trap column (Waters Corporation, Milford, MA) and desalted for 3 minutes at a flow rate of 0.2 mL/min.
• Product-ion spectra were acquired in a data-dependent mode, and the 10 most abundant ions were selected for product-ion analysis. All data were processed with the software MassAnalyzer (Zhang et al., Anal. Chem.84: 4942-4949 (2012), which is hereby incorporated by reference in its entirety) for the peptide identification and the deuterium level calculation. Approximately 500 peptides were analyzed with sequence coverage of at least 97% for all mAb2 polypeptide chains. All HDX-MS data were normalized to 100% deuterium concentration and the percent deuterium incorporation was plotted against labeling time in log scale with Prism v 6.02 (GraphPad Software, La Jolla, CA).
• the labeling was quenched by diluting 4-fold into a quench/denaturation buffer (pH 2.7) containing 0.625 M tris(2-carboxyethyl)phosphine (TCEP) and ⁇ 5.8M urea at 1°C.
• TCEP tris(2-carboxyethyl)phosphine
• Pepsin was then added to the quenched solution, followed by injecting into the sample loop at 1°C, and a 6 min digestion delay before the loop was switched inline for gradient elution.
• LT ⁇ R/antibody complex was formed by incubating the LT ⁇ R ECD antigen with excess amount (1.5 fold) of each antibody Fab, and analyzed with the free antigen in parallel.
• H/D exchange labeling and digestion was performed the same way as described above, except that a 1:1 pepsin/protease type XIII mixture was used for digestion, and the digestion was performed at ⁇ 7°C for 6 min prior to injecting into the sample loop, due to the difficulty of digesting the LT ⁇ R ECD by pepsin using the standard condition.
• All MS data were collected on a Thermo Scientific Orbitrap Eclipse high-resolution mass spectrometer, with electrospray ionization interface. MS data were collected at 120k resolution setting. For each paratope or epitope experiment, data-dependent MS/MS were also collected for three injections from each of the two unlabeled samples (free and complex) for peptide identification.
• the N- terminal residue of a peptide does not have an amide hydrogen for measurement and is excluded in reporting the epitope regions.
• the LT ⁇ R antibodies LIBC218990-1 (19320) and LIBC219058 (19324) bound to residues within CRD2 of LT ⁇ R as shown by a reduction in deuterium present in complex peptides consisting of amino acid residues 56-64 and 81-101 of the LT ⁇ R ECD (SEQ ID NO: 4).
• the epitope for LIBC218990-1 and the epitope for LIBC219058 comprise one or more residues within the amino acid sequence of SYNEHWNY (residues 57–64 of SEQ ID NO: 4) and one or more residues within the amino acid sequence of EIAPCTSKRKTQCRCQPGMF (residues 82-101 of SEQ ID NO: 4). No other regions of the LT ⁇ R: LT ⁇ R-antibody complexes were protected from deuterium exchange (data not shown).
• LT ⁇ R antibody LIBC218994 (19321) bound residues within CRD1, particularly one or more residues within residues 4–9, 21–29, and 39–47 of the LT ⁇ R ECD (SEQ ID NO: 4) (see FIG.7).
• the epitope for LIBC218944 comprises one or more residues within the amino acid sequences of PPYASE (residues 4–9 of SEQ ID NO: 4), YEPQHRICC (residues 21–29 of SEQ ID NO: 4), and SAKCSRIRD (residues 39–47 of SEQ ID NO: 4).
• LT ⁇ R antibody LIBC219081 (19325) bound residues within CRD4, particularly residues within the stretch of residues at positions 168-179 of the LT ⁇ R ECD (SEQ ID NO: 4) (see FIG.9).
• the epitope for LIBC219081 comprises one or more residues within the amino acid sequence of EAAPGTAQSDTT (residues 168–179 of SEQ ID NO: 4).
• No other regions of the LT ⁇ R: LT ⁇ R-antibody complexes were protected from deuterium exchange (data not shown).
• the LT ⁇ R antibody LIBC219097 (19326) bound to a region of LT ⁇ R spanning CRD1 and CRD2 comprising residues in the region of 39–42 and 57–70 of the LT ⁇ R ECD (SEQ ID NO: 4) (see FIG. 10).
• a comparison of the deuterium uptake curves for peptides comprising residues 38-42 and residues 37-47 shows that the shorter peptide of residues 38-42 has relatively large protection from deuterium exchange, while the longer peptide 37-47 has relatively small protection. This indicates that the protection mainly occurred in the overlapping shorter peptide, and thus narrows the epitope region to residues 39-42 of the LT ⁇ R ECD.
• the epitope for LIBC219097 comprises one or more residues with the amino acid sequence of SAKC (residues 39–42 of SEQ ID NO: 4) and SYNEHWNYLTICQL (resides 57–70 of SEQ ID NO: 4). No other regions of the LT ⁇ R: LT ⁇ R-antibody complexes were protected from deuterium exchange (data not shown). [0412] The LT ⁇ R antibody LIBC218979 (19319) bound to residues of LT ⁇ R within CRD4 of the LT ⁇ R ECD (SEQ ID NO: 4).
• the epitope comprises one or more residues within the amino acid sequence of EAAPGTAQSDT (residues 168-179 of SEQ ID NO: 4).
• the epitope comprises one or more residues within the amino acid sequence of QAVPPYASENQTCRDQEKE (residues 1-19 of SEQ ID NO: 4). Note that Q1 has been converted to pyro-E, which contains a measurable amide hydrogen. Data also shows that the binding is independent of the glycosylation status on residue N10.
• FIG.13 shows a series of deuterium uptake graphs for peptides derived from unbound and bound LT ⁇ R antibody LIBC218990 (19320). Peptides of the bound antibody (i.e., antibody-LT ⁇ R complex) protected from deuterium exchange are identified within their corresponding heavy chain and light chain variable sequences at the bottom of the figure (see boxed residues). The location of the CDRs within each variable region sequence are underlined.
• the deuterium uptake graph of the heavy chain peptide consisting of residues 27-34 of SEQ ID NO: 644 shows no protection from deuterium uptake.
• This heavy chain peptide spans HCDR1 residues 31–35, indicating HCDR1 of LIBC218990 is not involved in the binding interaction with LT ⁇ R.
• deuterium uptake in the complexed light chain peptide of residues 50–70 of SEQ ID NO: 645 also shows no protection from deuterium uptake.
• This light chain peptide spans LCDR2 residues 51–57 indicating that LCDR2 of LIBC218990 is also not involved in the binding interaction of this antibody with LT ⁇ R.
• HCDR2 of LIBC218990 comprises residues 50–66 of SEQ ID NO: 644. However, only residues within the first half of this HCDR2 region are involved in the binding interaction (i.e., residues 51- 59) as shown by the protection from deuterium exchange observed for the complexed heavy chain peptide of residues 50–59 and lack of protection observed in the complexed heavy chain peptide of residues 60–66 of SEQ ID NO: 644 (FIG.13).
• LCDR1 of LIBC218990 spans VL residues 24–35 (SEQ ID NO: 645), based on deuterium uptake of peptides overlapping this region, only residues 29–33 of SEQ ID NO: 645 appear to be involved in LCDR1 interaction with LT ⁇ R (FIG.13).
• the peptides examined in the region of HCDR3 and LCDR3 of LIBC218990 include framework regions on one or both ends of the respective CDR region. While these framework residues have been included in the boxed region, their involvement in binding interaction cannot be resolved using this method. [0417] Similar to LIBC218990, paratope mapping of LIBC219058 (19324) (FIG.
• FIGs. 14 and 16 show deuterium uptake graphs for peptides derived from unbound and bound LT ⁇ R antibody LIBC218994 (19321) (FIGs.14A–14B) and LT ⁇ R antibody LIBC219081 (19325) (FIGs.16A–16B).
• Peptides of the bound antibody (i.e., antibody-LT ⁇ R complex) protected from deuterium exchange are identified within their corresponding heavy chain and light chain variable chain sequences at the bottom of each figure (see boxed residues). The location of the CDRs within each variable region sequence are underlined.
• FIGs 18A-18B show deuterium uptake graphs for peptides derived from unbound and bound LT ⁇ R antibody LIBC218979 (19319). Peptides of the bound antibody (i.e., antibody-LT ⁇ R complex) protected from deuterium exchange are identified within their corresponding heavy chain (FIG.18A) and light chain (FIG. 18B) variable chain sequences at the bottom of each figure (see boxed residues).
• FIGs 19A-19B show deuterium uptake graphs for peptides derived from unbound and bound LT ⁇ R antibody LIBC219051 (19323). Peptides of the bound antibody (i.e., antibody-LT ⁇ R complex) protected from deuterium exchange are identified within their corresponding heavy chain (FIG.19A) and light chain (FIG. 19B) variable chain sequences at the bottom of each figure (see boxed residues). The location of the CDRs within each variable region sequence are underlined.
• CDR residues of the antibodies not identified as being involved in or critical to the LT ⁇ R binding interaction can be substituted with any amino acid residue, with a conservative amino acid substitution, or with a hotspot remediated amino acid residue as identified in each of Tables 18-25 below.
• TAA Tumor antigen associated
• SAA stromal antigen associated
• LT ⁇ R binding proteins capable of binding (i) human LT ⁇ R (huLT ⁇ R) and human Claudin 18.2 (CLDN18.2), (ii) huLT ⁇ R and human Claudin 6 (CLDN6), (iii) huLT ⁇ R and human MUC17, or (iv) huLT ⁇ R and LRRC15 were generated in an AmAb format (i.e., Fab x scFv) or heteroIgG format.
• AmAb format i.e., Fab x scFv
• variable region binding domains of LT ⁇ R antibodies i.e., LT ⁇ R antibodies 36G2 (19324), 31A3 (19321), 23E9 (19319) and 41B2 (19325)
• variable regions for the respective SAA or TAA as provided in Table 26 below.
• the amino acid sequences of the CLDN18.2, CLDN6, MUC17, and LRRC15 variable regions are provided below in Table 27.
• the panel of agonist LT ⁇ R bispecific binding proteins generated in the AmAb or heteroIgG format was evaluated for cross-linking dependent LT ⁇ R signaling activity in an in vitro co-culture assay.
• chemokine IL-8 release by LT ⁇ R expressing human melanoma cells co-cultured with CHO or Saos-2 cells stably expressing the relevant SAA or TAA is indicative of cross-linking dependent LT ⁇ R signaling activity by the test antibody.
• Table 26 Parental and Bispecific Antibodies Table 27.
• LT ⁇ R expressing human melanoma A375 cells ATCC, CRL-1619
• DMEM 10% FBS, 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose
• the plates were incubated overnight at 37°C/5%CO 2.
• testing molecules were titrated in assay medium (DMEM, 2% FBS, 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose) from 10nM 1:3 down for 11 points.
• the culture supernatant was removed at 40 ⁇ L from each well of the 384-well assay plates.
• the titrated testing molecules were then transferred to the 384-well assay plates pre-seeded with LT ⁇ R expressing A375 cells and were left to incubate at room temperature for 30 minutes.
• Target stable expressing Hamster Ovarian cells (CHOs) or human osteosarcoma Saos-2 cells were harvested, counted, resuspended in assay medium and transferred to the assay plate following plate layout. The assay plates were then incubated at 37°C/5% CO2 overnight. The ratio of LT ⁇ R to either CLDN6, CLDN18.2, MUC17, LRRC15 expressing cells or corresponding non-expressing cells was set to 1:1 based on assay development data. The various target expressing cells provides the cross-linking for the testing molecules and corresponding non-target expressing cells were used to determine cross-linking independency of the test molecules.
• Interpolated EC 50 is inflection point in the dose curve based on each binding protein and transit EC50 is the value at a particular point on the Y axis which is around EC 50 for the majority of molecules.
• the max activity for the bispecific antibodies was calculated by the highest signal of each molecule divided by the average signal of the corresponding neutral control. A summary of individual antibody potency is provided in Table 28 below and shown in FIGs.20-23.
• the majority of the max activity for the LT ⁇ R x MUC17 BsAbs ranged between 2.70 to 3.26-fold over neutral control (FIG.22). All LT ⁇ R x MUC17 BsAb molecules had similar EC 50 values and exhibited cross-linking dependent activity.
• the majority of the max activity for the LT ⁇ R x LRRC15 BsAbs ranged between 4.46 to 5.37-fold over neutral control (FIG.23).
• the LT ⁇ R x LRRC15 BsAb molecules had EC 50 values ranging from 46.42 to 108.12. All LT ⁇ R x LRRC15 BsAb molecules exhibited cross-linking dependent activity.
• Table 28 Summary of LT ⁇ R Bispecific Molecule Potency
• Example 8 Confirmation of LT ⁇ R Receptor-Ligand Non-Blocking Activity Using Cell Based A ssay.
• Human LIGHT-Alexa 647 and human LT ⁇ 1 ⁇ 2-Alexa 647 were prepared by conjugating human LIGHT (R&D Systems, Cat. No.664-LI/CF) and human LT ⁇ 1 ⁇ 2 (R&D Systems, Cat. No.8884- LY/CF) using an Alexa Fluor 647 Microscale Protein Labeling Kit (Thermo Fisher Scientific, Cat. No. A30009) following the protocol set out by the manufacturer.
• Human LT ⁇ R-binding antibodies or bispecific antibodies comprising an LT ⁇ R-binding antibody were assayed for their ability to not block human LIGHT and human LTa1b2 binding to human LT ⁇ R transiently expressed on HEK 293T cells via a FACS-based receptor-ligand assay.
• the samples were co-stained with human IgG to additionally evaluate if the non- blocking of the ligands was due to non-binding of the LT ⁇ R antibodies on human LT ⁇ R transiently expressed on HEK293T cells.
• Human LT ⁇ R was expressed on host human embryonic kidney 293 cells by transfection using an expression vector expressing huLT ⁇ R cDNA [pTT5.2:VK1O2O12::huLT ⁇ R(31-435) C173003 (DNA-38098)] with Opti-MEM® Reduced Serum Medium (Gibco, Cat: 31985-088) and 293FectinTM Reagent (Gibco REF:12347-019, P/N 53020LT) following the protocol set out by the manufacturer.7.5uL of human LT ⁇ R-specific monoclonal antibodies were titrated in a dose titration curve and incubated for 1 hour at 4°C with 7.5uL of transfected hu LT ⁇ R cells in FACS buffer [1XPBS + 2% Fetal Bovine Serum (Sigma-Aldrich, Cat.No.
• FACS plates were topped up with 170uL of FACS buffer, then washed once in FACS buffer by centrifugation for 2 minutes at 2500 RPM. Samples were resuspended in 45 ⁇ L of FACS buffer and were read on an iQue flow cytometry machine with Intellicyt autosampler, according to the manufacturer’s recommendations; data analysis was done on Intellicyt ForeCyt® Enterprise Client Edition 6.2 (R3).
• the amount of human LIGHT-Alexa 647 or human LT ⁇ 1 ⁇ 2-Alexa 647 was quantified by GeoMean fluorescence signal by flow cytometry analysis, and the signal was converted to a percent inhibition of receptor-ligand (R-L) interaction.
• Loss of ligand binding signal in the presence of LT ⁇ R antibodies was calculated as percent inhibition of R-L binding.
• the maximum GeoMean signal for human LIGHT-Alexa 647 or human LT ⁇ 1 ⁇ 2-Alexa 647 was determined by averaging the GeoMean signals of the ligand-Alexa 647 signal over 4 replicates that were incubated with human LT ⁇ R transiently expressed on HEK 293T cells.
• the minimum GeoMean signal for human LIGHT-Alexa 647 or human LT ⁇ 1 ⁇ 2-Alexa 647 was determined from GeoMean signal of the ligand-Alexa 647 signal incubated with HEK 293T cells transfected with a mock vector.
• FIG.24A and 24B are graphs plotting inhibition of LIGHT (FIG.24A) and LT ⁇ 1 ⁇ 2 (FIG.
• FIG. 24B shows concentration dependent LT ⁇ R binding by the indicated binder molecules. Table 29. Summary of LT ⁇ R Ligand Blocking Activity of Select Agonist LT ⁇ R Binding Proteins [0437] LT ⁇ R ligand blocking activity of bispecific LT ⁇ R binding proteins was also assessed.
• FIGs. 25A and 26A are graphs showing inhibition of LT ⁇ 1 ⁇ 2 binding to cell expressed LT ⁇ R by the indicated LT ⁇ R-LRRC15 bispecific binders, and FIGs. 25B and 26B show inhibition LIGHT binding to cell expressed LT ⁇ R by the same LT ⁇ R-LRRC15 bispecific binders.
• a summary of the ligand blocking activity of the tested LT ⁇ R bispecific binding proteins is provided above in Table 29.
• FIGs.25C and 26C shows concentration dependent LT ⁇ R binding by the indicated bispecific binding proteins.
• Culture medium was changed to BDQY Hybridoma Medium a day before FACS enrichment sorting.
• Cells were washed in 10 mL sterile FACS buffer and then incubated with biotin-labelled recombinant human LT ⁇ R (R&D Systems, Cat: 7538LR) at 3 ⁇ g/mL concentration and human LT ⁇ 1 ⁇ 2- Alexa 647 (R&D Systems, Cat. No.8884-LY/CF) at 20 ⁇ g/mL concentration in 2 mL reaction volume for 30 minutes at 4 degrees Celsius.
• the cells were washed in 10 mL FACS buffer, resuspended in 2 mL of BDQY Hybridoma Medium containing 5 ⁇ L of 7-AAD (BD Pharmingen, Cat: 559925) then put through a 40 micron cell strainer to remove any clumps.
• Cells were bulk sorted on BD FACSAria by gating on live cell population, then on dual positive for Alexa Fluor 488 IgG and BV421 LT ⁇ R fluorescence signals, and finally on Alexa Fluor 647 LT ⁇ 1 ⁇ 2 and BV421 LT ⁇ R fluorescence signals.
• the target cells were then single cell sorted onto 384-well microtiter plates containing BDQY hybridoma medium and cultured for up to 2 weeks b efore the supernatants were collected for screening.
• the target cells were then single cell sorted onto 384-well microtiter plates containing BDQY hybridoma medium and cultured for up to 2 weeks b efore the supernatants were collected for screening.
• Biotinylated goat anti human IgG Fc capture antibody (Jackson ImmunoResearch, Cat: 109-065-098) was coupled at 2ug/mL concentration streptavidin coated, uniquely barcoded LumAvidin Beads® (LumAvidin Microspheres, Luminex Corp., Austin,Texas, U.S.A.) for 30 minutes in the dark at room temperature and washed twice.
• the reference antibody hybridoma supernatant samples at 4 ⁇ g/mL concentration were incubated with the goat anti human IgG Fc- coated beads for 30 minutes in the dark at room temperature and washed twice.
• His-tagged human LT ⁇ R antigen (VK1O2O12::huLT ⁇ R(31-227)::8xHis, 46449-2) made up at 60nM and combined with Chrompure human IgG, whole molecule (Jackson ImmunoResearch, Cat: 009-000-003) at 5ug/mL were incubated with the coated beads for 30 minutes in the dak at room temperature and washed twice. Beads were resuspended in FACS buffer containing Stabilguard® (SurModics SG01).
• the antigen-coated, reference antibody-bound beads were pooled and then divided into individual sample wells containing the ZenonTM labeled test antibody (hybridoma supernatant) sample (or negative control), incubated for 30 minutes in the dark at room temperature and washed twice. The final concentration of the test antibody (hybridoma supernatant) was at 1ug/mL. The samples were washed twice to remove unbound detection reagent and resuspended in FACS buffer. Samples were analyzed using an Intellicyt iQueTM Screener Platform.
• the reference-only antibody binding signal was subtracted from the reference plus test antibody signal for each competition/binding reaction (i.e., across the entire reference antibody set).
• An individual antibody binding profile was defined as the collection of net binding values for each competition/binding reaction.
• the degree of similarity between individual profiles was then assessed by calculating the coefficient of determination between each of the test antibody profiles.
• Test antibodies showing high degrees of similarity (R2 > 0.7) to each other were then grouped into common binning profiles. Using this method, the LT ⁇ R binding antibodies were sub-divided into 7 unique binning profiles (A, B, C, D, F, G and H).
• Hybridoma supernatants containing human LT ⁇ R-binding antibodies were assayed for their ability to not block human LIGHT and human LT ⁇ 1 ⁇ 2 binding to human LT ⁇ R transiently expressed on HEK 293T cells via the FACS-based receptor-ligand assay described in Example 8.
• Each hybridoma supernatant containing a human LT ⁇ R-binding antibody was tested for ligand blocking activity in duplicate.
• the percent inhibition for each LT ⁇ R-binding antibody that shares an epitope binning profile with the LT ⁇ R antibodies identified herein as 19325 (41B2) and 19319 (23E9), which bind to CRD4 of LT ⁇ R, is provided in the Table of FIG.27.
• hybridoma supernatants containing LT ⁇ R-binding antibodies were assayed for their ability to block human LIGHT (R&D Systems, Cat. No. 664-LI/CF) and human LT ⁇ 1 ⁇ 2 (R&D Systems, Cat. No. 8884-LY/CF) binding to human LT ⁇ R-His via a Carterra-based receptor-ligand assay.
• LT ⁇ R antibody prevented human LIGHT or human LT ⁇ 1 ⁇ 2 from binding to human LT ⁇ R-His
• response (RU) from injection of ligand after exposing receptor to antibodies would not increase relative to the RU achieved in the presence of LT ⁇ R receptor alone, thus preventing the formation of a three-component sandwich complex due to competition.
• the Table of FIG.27 shows the receptor-ligand interaction blocking ability of select hybridoma supernatant samples as measured by this Caterra method.
• the panel of human LT ⁇ R-binding antibodies was captured on a streptavidin (SA) chip (SAHC30M, Carterra Part # 4294) with pre-loaded biotinylated anti-huFc VHH (Chromotek, Cat#: shurbGB-1) in HBSTE buffer [10 mM HEPES, pH7.4, 150 mM NaCl, 0.05% tween-20 and 1 mM EDTA; made in house] at 25°C on a Carterra ® LSA.
• the antibodies were normalized and diluted to 2 ⁇ g/mL and printed on spots of chip by 96-channel printhead for 15 mins.
• the chip was then injected with human LT ⁇ R- His at 300 nM using single flow cell, with association time of 5 mins to saturate the receptor binding and dissociation time of 1 min which is the minimal value allowed by software to minimize the dissociation.
• Human LIGHT or human LT ⁇ 1 ⁇ 2 ligand was injected immediately after that at 150 nM with 5 mins association and 1 min dissociation.10 mM Glycine, pH 1.5 (Carterra Part # 3639) was used for regeneration before the re-printing of antibodies for the other ligand assay.
• LT ⁇ R antibody loading human LT ⁇ R-His receptor binding, and human LIGHT or human LT ⁇ 1 ⁇ 2 ligand sandwiching were quantified by RU using Kinetics software (Carterra).
• the sandwich level was calculated as a ratio of ligand RU to receptor RU.
• the minimum sandwich level was determined with an irrelevant human IgG1 control measured in the same experimental setting.
• sandwich level values were low, possibly due to low concentration of antibody, injection bulk shift effect, weak binder towards LT ⁇ R or fast LT ⁇ R dissociation rate. Thus, these antibodies, which cannot be decided whether it is a non-blocker or blocker, were indicated as “NA” in result table.
• Affinity was measured on a Carterra LSA at 25°C in HBSTE running buffer [10 mM HEPES, pH7.4, 150 mM NaCl, 0.05% tween-20 and 1 mM EDTA]. 10 mM Glycine, pH 1.5 (Carterra Part # 3639) was used for regeneration. Hybridoma supernatants containing LT ⁇ R -binding antibodies were normalized and diluted to 0.3 ⁇ g/mL and printed for 10 mins onto the spots of a streptavidin (SA) chip (SAHC30M, Carterra Part # 4294) with pre-loaded biotinylated anti-huFc VHH (Chromotek, Cat#: shurbGB-1) using 96-channel printhead.
• SA streptavidin
• Buffer wells and irrelevant human IgG1 sample were used as negative controls.
• the target, human or cyno LT ⁇ R was injected as analyte at 243 nM, 81 nM, 27 nM, 9 nM, 3 nM and 1 nM.
• the association time was 5 mins and dissociation time was 30 minutes.
• the data was double background referenced in that both the reference spot on the chip and a 0 nM analyte concentration were subtracted from the data.
• a 1:1 Langmuir binding model was used to analyze the data in the Kinetics software (Carterra). The results for human and cyno affinity gaps are shown in the Table of FIG.27.

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