APRIL and BAFF inhibitory immunomodulatory proteins, with and without T-cell inhibitory proteins, and methods of use thereof.

Abstract

The present invention provides an improved therapeutic agent that can modulate the immune response, particularly the B-cell immune response, and in some cases the T-cell immune response. [Solution] Immunomodulatory proteins exhibiting neutralizing activity of BAFF and APRIL (or BAFF / APRIL heterotrimers), either alone or in combination with inhibition of T cell costimulation, are provided herein. The immunomodulatory proteins provided herein include variant domains of B cell maturation antigens (BCMAs) alone, or multi-domain immunomodulatory proteins that can inhibit both B cell responses and T cell costimulation. Nucleic acid molecules encoding immunomodulatory proteins are also provided. Immunomodulatory proteins offer therapeutic utility for various immunological diseases or conditions. Compositions and methods for producing and using such proteins are also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application incorporates, by reference, the entire contents of each of the following U.S. Provisional Applications: U.S. Provisional Application No. 63 / 022,373, filed on May 8, 2020, entitled "APRIL AND BAFF INHIBITORY IMMUNOMODULATORY PROTEINS WITH AND WITHOUT AT CELL INHIBITORY PROTEIN AND METHODS OF USE THEREOF"; U.S. Provisional Application No. 63 / 034,361, filed on June 3, 2020, entitled "APRIL AND BAFF INHIBITORY IMMUNOMODULATORY PROTEINS WITH AND WITHOUT AT CELL INHIBITORY PROTEIN AND METHODS OF USE THEREOF"; and September 18, 2020, entitled "APRIL AND BAFF INHIBITORY IMMUNOMODULATORY PROTEINS WITH AND WITHOUT AT CELL INHIBITORY PROTEIN AND We claim priority from U.S. Provisional Application No. 63 / 080,643, titled "METHODS OF USE THEREOF".

[0002] Inclusion by referencing the sequence list This application is filed together with an electronic sequence listing. The sequence listing is provided as a file titled 761612002340SeqList.TXT, created on May 4, 2021, and its size is 992,602 bytes. The electronic information of the sequence listing is incorporated in its entirety by reference.

[0003] field This disclosure provides immunomodulatory proteins that exhibit neutralizing activity against BAFF and APRIL (or BAFF / APRIL heterotrimers), either alone or in combination with inhibition of T cell costimulation. The immunomodulatory proteins include variant domains of B cell maturation antigens (BCMAs) alone, and multi-domains capable of inhibiting both B cell responses and T cell costimulation. This disclosure also provides nucleic acid molecules encoding immunomodulatory proteins. These immunomodulatory proteins offer therapeutic utility for various immunological diseases or conditions. Compositions and methods for producing and using such proteins are provided. [Background technology]

[0004] background There is growing medical interest in modulating immune responses by intervening in processes involving the interaction between soluble ligands and their receptors. Currently, biologics used to enhance or suppress immune responses are generally limited to antibodies (e.g., anti-PD-1 antibodies) or soluble receptors for single cell surface molecules (e.g., Fc-CTLA-4). There is a need for improved therapeutic agents that can modulate immune responses, particularly B-cell immune responses, and possibly T-cell immune responses as well. Embodiments that satisfy such needs are provided. [Overview of the project]

[0005] overview An immunomodulatory protein is provided herein, comprising: at least one T cell inhibitory molecule (TIM) that binds to a T cell stimulating receptor or a ligand of a T cell stimulating receptor and antagonizes the activity of the T cell stimulating receptor; and at least one B cell inhibitory molecule (BIM) that binds to a ligand of a B cell stimulating receptor and / or antagonizes the activity of the B cell stimulating receptor. In some of any embodiments, the immunomodulatory protein comprises: at least one T cell inhibitory molecule (TIM) that binds to a T cell stimulating receptor or a ligand of a T cell stimulating receptor or antagonizes the activity of the T cell stimulating receptor; and at least one B cell inhibitory molecule (BIM) that binds to a ligand of a B cell stimulating receptor and / or antagonizes the activity of the B cell stimulating receptor.

[0006] In some of the various embodiments, the TIM binds to a ligand of the T cell stimulatory receptor. In some of the various embodiments, the T cell stimulatory receptor is CD28, and the ligand of the T cell stimulatory receptor is CD80 or CD86. In some of the various embodiments, the T cell stimulatory receptor is CD28, or the ligand of the T cell stimulatory receptor is CD80 or CD86.

[0007] In some of the embodiments, TIM is the extracellular domain of CTLA-4 or its binding portion that binds to CD80 or CD86. In some of the embodiments, the extracellular domain of CTLA-4 or its binding portion consists of the amino acid sequence described in SEQ ID NO:1 or SEQ ID NO:2, the amino acid sequence of a variant CTLA-4 having at least 85% sequence identity with SEQ ID NO:1 or SEQ ID NO:2, or a portion thereof including an IgV domain. In some of the embodiments, the extracellular domain of CTLA-4 consists of the amino acid sequence described in SEQ ID NO:1. In some of the embodiments, the extracellular domain of CTLA-4 consists of the amino acid sequence of a variant CTLA-4 having at least 85% sequence identity with SEQ ID NO:1, or a portion thereof including an IgV domain, and the variant sequence includes one or more amino acid substitutions in SEQ ID NO:1 or a portion thereof including an IgV domain.

[0008] In some of the embodiments, the variant CTLA-4 sequence contains the amino acid substitution C122S. In some embodiments, the CTLA-4 extracellular domain or its binding portion is described in SEQ ID NO:668.

[0009] In some of the available embodiments, variant CTLA-4 binds to the ectodomains of CD80 and CD86, and optionally, the binding affinity to one or both of CD80 and CD86 is increased compared to the sequence described in SEQ ID NO:1, or a portion thereof containing the IgV domain.

[0010] In some of the various embodiments, one or more amino acid substitutions within the variant CLTA-4 polypeptide include amino acid substitutions selected from L12F, R16H, G29W, T53S, M56T, N58S, L63P, L98Q, or Y105L, or combinations thereof. In some embodiments, one or more amino acid substitutions include G29W, L98Q, and Y105L. In some embodiments, one or more amino acid substitutions are G29W / N58S / L63P / Q82R / L98Q / Y105L. In some embodiments, one or more amino acid substitutions are L12F / R16H / G29W / M56T / L98Q / Y105L. In some embodiments, one or more amino acid substitutions are G29W / L98Q / Y105L.

[0011] In some of the various embodiments, the variant CTLA-4 polypeptide has at least about 85%, at least about 90%, or at least about 95% sequence identity with respect to SEQ ID NO:1 and contains one or more amino acid substitutions described.

[0012] In some of the various embodiments, the variant CTLA-4 polypeptide has at least about 85%, at least about 90%, or at least about 95% sequence identity with respect to SEQ ID NO:2 and contains one or more amino acid substitutions described.

[0013] In some embodiments, the CTLA-4 extracellular domain or its binding portion is described in one of SEQ ID NO:92, SEQ ID NO:112, SEQ ID NO:165, or SEQ ID NO:186, or is a part thereof including the IgV domain.

[0014] In some of the embodiments, variant CTLA-4 consists of the sequence described in SEQ ID NO:92, or a portion thereof including the IgV domain. In some of the embodiments, variant CTLA-4 consists of the sequence described in SEQ ID NO:113, or a portion thereof including the IgV domain. In some of the embodiments, variant CTLA-4 consists of the sequence described in SEQ ID NO:165, or a portion thereof including the IgV domain. In some of the embodiments, variant CTLA-4 consists of the sequence described in SEQ ID NO:186, or a portion thereof including the IgV domain.

[0015] In some of the various embodiments, the ligand for the B cell stimulating receptor is APRIL or BAFF, and the B cell stimulating receptor is a TACI, BCMA, or BAFF receptor.

[0016] In some of the embodiments, BIM is a TACI polypeptide comprising a TACI extracellular domain or its binding site that binds to APRIL, BAFF, or a BAFF / APRIL heterotrimer. In some embodiments, BIM is a TACI extracellular domain or its binding site having an extracellular domain sequence described as (i) the amino acid sequence described as SEQ ID NO:709; (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:709; or (iii) a part of (i) or (ii) that includes one or both of the CRD1 domain and the CRD2 domain that binds to APRIL, BAFF, or a BAFF / APRIL heterotrimer. In some embodiments, BIM is a TACI extracellular domain or its binding site that includes the CRD1 domain and the CRD2 domain.

[0017] In some of the arbitrary embodiments, the BIM, which is a TACI polypeptide, is a cleaved wild-type TACI extracellular domain consisting of the sequence described in SEQ ID NO:516.

[0018] In some of the various embodiments, the BIM, which is a TACI polypeptide, is either a cleaved wild-type TACI extracellular domain or a variant thereof, wherein the cleaved wild-type TACI extracellular domain contains cysteine-rich domain 2 (CRD2) but lacks the entire cysteine-rich domain 1 (CRD1), and / or the variant TACI polypeptide contains one or more amino acid substitutions in the cleaved wild-type TACI extracellular domain.

[0019] In some of the various embodiments, the TACI polypeptide BIM is either a cleaved wild-type TACI extracellular domain or a variant thereof, the cleaved wild-type TACI extracellular domain consisting of a continuous sequence contained within amino acid residues 67-118, including amino acid residues 71-104, relative to the position described in SEQ ID NO:709, and the variant TACI polypeptide contains one or more amino acid substitutions in the cleaved wild-type TACI extracellular domain.

[0020] In some of the embodiments, the TACI polypeptide BIM is one in which the cleaved wild-type TACI extracellular domain is 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 59, 50, or 51 amino acid lengths. In some of the embodiments, the cleaved wild-type TACI extracellular domain consists of amino acid residues 68-110 as described in SEQ ID NO:709. In some of the embodiments, the TACI polypeptide consists of the amino acid sequence described in SEQ ID NO:528; or a variant thereof containing one or more amino acid substitutions to the sequence described in SEQ ID NO:528.

[0021] In some of the arbitrary embodiments, the TACI polypeptide BIM is a cleaved wild-type TACI extracellular domain consisting of the sequence described in SEQ ID NO:528.

[0022] In some of the various embodiments, the BIM, which is a TACI polypeptide, is a cleaved TACI polypeptide or a variant thereof that binds to an APRIL, BAFF, or BAFF / APRIL heterotrimer.

[0023] In some of the arbitrary embodiments, BIM is a TACI polypeptide, the TACI polypeptide is a variant TACI polypeptide, and the variant TACI polypeptide has increased binding affinity to one or both of APRIL and BAFF compared to the cleaved TACI polypeptide.

[0024] In some of the various embodiments, the variant TACI polypeptide contains one or more amino acid substitutions in the extracellular domain (ECD) or specific binding fragment of the reference TACI polypeptide at positions selected from 74, 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 87, 88, 92, 95, 97, 98, 99, 101, 102, and 103, corresponding to the numbering described in SEQ ID NO:709. In some of the various embodiments, one or more amino acid substitutions are selected from E74V, Q75E, Q75R, G76S, K77E, F78Y, Y79F, L82H, L82P, L83S, R84G, R84L, R84Q, D85E, D85V, C86Y, I87L, I87M, S88N, I92V, Q95R, P97S, K98T, Q99E, A101D, Y102D, F103S, F103V, F103Y, or their conserved amino acid substitutions. In some of the various embodiments, one or more amino acid substitutions include at least one of E74V, K77E, Y79F, L82H, L82P, R84G, R84L, R84Q, D85V, or C86Y. In some of the various embodiments, one or more amino acid substitutions are D85E / K98T, I87L / K98T, L82P / I87L, G76S / P97S, K77E / R84L / F103Y, Y79F / Q99E, L83S / F103S, K77E / R84Q, K77E / A101D, K77E / F78Y / Y102D, Q75E / R84Q, Q75R / R84G / I92V, K77E / A101D / Y102D, R84Q / S88N / A101D, R84Q / F103V, K77E / Q95R / A101D, or I87M / A101D. In some embodiments, one or more amino acid substitutions are K77E / F78Y / Y102D. In some embodiments, one or more amino acid substitutions are Q75E / R84Q. In some embodiments, BIM, which is a TACI polypeptide, is the variant TACI polypeptide described in SEQ ID NO:541. In some embodiments, BIM, which is a TACI polypeptide, is the variant TACI polypeptide described in SEQ ID NO:542.

[0025] In some of the arbitrary embodiments, the BIM, which is a TACI polypeptide, is a variant TACI polypeptide in which the TACI polypeptide contains one or more amino acid substitutions in the extracellular domain (ECD) or specific binding fragment of the reference TACI polypeptide at a position selected from 40, 59, 60, 61, 74, 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 87, 88, 92, 95, 97, 98, 99, 101, 102, and 103, corresponding to the position numbering described in SEQ ID NO:709. In some of the arbitrary embodiments, one or more amino acid substitutions are selected from W40R, Q59R, R60G, T61P, E74V, Q75E, Q75R, G76S, K77E, F78Y, Y79F, L82H, L82P, L83S, R84G, R84L, R84Q, D85E, D85V, C86Y, I87L, I87M, S88N, I92V, Q95R, P97S, K98T, Q99E, A101D, Y102D, F103S, F103V, F103Y, or their conserved amino acid substitutions.

[0026] In some of the embodiments, one or more amino acid substitutions include at least one of E74V, K77E, Y79F, L82H, L82P, R84G, R84L, R84Q, D85V, or C86Y. In some of the embodiments, one or more amino acid substitutions include at least the amino acid substitution K77E. In some of the embodiments, one or more amino acid substitutions include at least the amino acid substitution R84G. In some of the embodiments, one or more amino acid substitutions include at least the amino acid substitution R84Q.

[0027] In some of the various embodiments, the reference TACI polypeptide is a cleavable polypeptide comprising the extracellular domain of TACI or its specific binding moiety that binds to APRIL, BAFF, or a BAFF / APRIL heterotrimer.

[0028] In some of the embodiments, the reference TACI polypeptide comprises (i) the amino acid sequence described in SEQ ID NO:709; (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:709; or (iii) a portion of (i) or (ii) comprising one or both of the CRD1 domain and the CRD2 domain that bind to APRIL, BAFF, or the BAFF / APRIL heterotrimer. In some of the embodiments, the reference TACI polypeptide lacks an N-terminal methionine. In some of the embodiments, the reference TACI polypeptide comprises the CRD1 domain and the CRD2 domain. In some of the embodiments, the reference TACI polypeptide comprises the sequence described in SEQ ID NO:516. In some of the embodiments, the reference TACI polypeptide consists of the sequence described in SEQ ID NO:516. In some of the embodiments, the reference TACI polypeptide essentially consists of the CRD2 domain. In some of the embodiments, the reference TACI polypeptide comprises the sequence described in SEQ ID NO:528. In some of the various embodiments, the reference TACI polypeptide consists of the sequence described in SEQ ID NO:528.

[0029] In some of the arbitrary embodiments, one or more amino acid substitutions are D85E / K98T, I87L / K98T, R60G / Q75E / L82P, R60G / C86Y, W40R / L82P / F103Y, W40R / Q59R / T61P / K98T, L82P / I87L, G76S / P97S, K77E / R84L / F103Y, Y79F / Q These are 99E, L83S / F103S, K77E / R84Q, K77E / A101D, K77E / F78Y / Y102D, Q75E / R84Q, Q75R / R84G / I92V, K77E / A101D / Y102D, R84Q / S88N / A101D, R84Q / F103V, K77E / Q95R / A101D, or I87M / A101D. In some of the various embodiments, one or more amino acid substitutions are K77E / F78Y / Y102D. In some of the various embodiments, one or more amino acid substitutions are Q75E / R84Q.

[0030] In some of the arbitrary embodiments, the BIM, which is a TACI polypeptide, is a variant TACI polypeptide having at least about 85%, at least about 90%, or at least about 95% sequence identity with respect to SEQ ID NO:709 and containing one or more amino acid substitutions described.

[0031] In some of the arbitrary embodiments, the BIM, which is a TACI polypeptide, is a variant TACI polypeptide having at least about 85%, at least about 90%, or at least about 95% sequence identity with respect to SEQ ID NO:719 and containing one or more amino acid substitutions described.

[0032] In some of the various embodiments, the BIM, which is a TACI polypeptide, is a variant TACI polypeptide having at least about 85%, at least about 90%, or at least about 95% sequence identity with respect to SEQ ID NO:718 and containing one or more amino acid substitutions described.

[0033] In some of the arbitrary embodiments, the BIM, which is a TACI polypeptide, is a variant TACI polypeptide having at least about 85%, at least about 90%, or at least about 95% sequence identity with respect to SEQ ID NO: 516 and containing one or more amino acid substitutions described.

[0034] In some of the various embodiments, the BIM, which is a TACI polypeptide, is a variant TACI polypeptide having at least about 85%, at least about 90%, or at least about 95% sequence identity with respect to SEQ ID NO:528 and containing one or more amino acid substitutions described.

[0035] In some of the arbitrary embodiments, the BIM, which is a TACI polypeptide, is such that the variant TACI polypeptide has increased binding affinity to one or both APRIL and BAFF compared to the reference TACI polypeptide. In some of the arbitrary embodiments, the variant TACI polypeptide has increased binding affinity to APRIL. In some of the arbitrary embodiments, the variant TACI polypeptide has increased binding affinity to BAFF. In some of the arbitrary embodiments, the variant TACI polypeptide has increased binding affinity to both APRIL and BAFF. In some of the arbitrary embodiments, the increased binding affinity to BAFF or APRIL is independently increased by more than 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, or 60 times.

[0036] In some of the embodiments, a BIM that is a TACI polypeptide is one in which the variant TACI polypeptide contains the sequence described in any one of SEQ ID NO: 517-527, 536, 537, or 682-701. In some of the embodiments, a BIM that is a TACI polypeptide is one in which the variant TACI polypeptide contains the sequence described in any one of SEQ ID NO: 529-535, 538-550, or 673-681. In some of the embodiments, a BIM that is a TACI polypeptide is one in which the variant TACI polypeptide consists of or is essentially derived from the sequence described in any one of SEQ ID NO: 517-527, 536, 537, or 682-701. In some of the embodiments, a BIM that is a TACI polypeptide is one in which the variant TACI polypeptide consists of or is essentially derived from the sequence described in any one of SEQ ID NO: 529-535, 538-550, or 673-681.

[0037] In some of the arbitrary embodiments, the BIM is a variant TACI polypeptide that is described in any one of SEQ ID NO: 535, SEQ ID NO: 541, SEQ ID NO: 542, or SEQ ID NO: 688.

[0038] In some of the embodiments, the BIM, which is a TACI polypeptide, is such that the variant TACI polypeptide consists of or is essentially derived from the sequence described in SEQ ID NO: 535. In some of the embodiments, the BIM, which is a TACI polypeptide, is such that the variant TACI polypeptide consists of or is essentially derived from the sequence described in SEQ ID NO: 541. In some of the embodiments, the BIM, which is a TACI polypeptide, is such that the variant TACI polypeptide consists of or is essentially derived from the sequence described in SEQ ID NO: 542. In some of the embodiments, the BIM, which is a TACI polypeptide, is such that the variant TACI polypeptide consists of or is essentially derived from the sequence described in SEQ ID NO: 688. In some of the embodiments, the BIM, which is a TACI polypeptide, is such that the variant TACI polypeptide consists of or is essentially derived from the sequence described in SEQ ID NO: 535.

[0039] In some of the various embodiments, BIM is a BCMA polypeptide comprising a BCMA extracellular domain or binding moiety that binds to APRIL, BAFF, or a BAFF / APRIL heterotrimer. In some of the various embodiments, the BCMA extracellular domain or binding moiety is an extracellular domain sequence described as (i) the amino acid sequence described as SEQ ID NO:356; (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:356; or (iii) a portion of (i) or (ii) containing a CRD domain.

[0040] In some of the embodiments, the BCMA polypeptide consists of the sequence described in SEQ ID NO:356. In some of the embodiments, the BCMA polypeptide is a variant BCMA polypeptide having one or more amino acid substitutions in the extracellular domain (ECD) or specific binding fragment of the reference BCMA polypeptide at a position selected from 9, 10, 11, 14, 16, 19, 20, 22, 25, 27, 29, 30, 31, 32, 35, 36, 39, 43, 45, 46, 47, and 48, corresponding to the numbering described in SEQ ID NO:710.

[0041] Immunomodulatory proteins comprising variant BCMA polypeptides are provided herein, the variant BCMA polypeptides comprising one or more amino acid substitutions in the extracellular domain (ECD) or specific binding fragment of the reference BCMA polypeptide at positions selected from 9, 10, 11, 14, 16, 19, 20, 22, 25, 27, 29, 30, 31, 32, 35, 36, 39, 43, 45, 46, 47, and 48, corresponding to the position numbering described in SEQ ID NO:710. In some of the arbitrary embodiments, the reference BCMA polypeptide is a polypeptide comprising the extracellular domain or specific binding portion of BCMA that binds to APRIL, BAFF, or a BAFF / APRIL heterotrimer. In some of the various embodiments, the reference BCMA polypeptide comprises (i) the amino acid sequence described in SEQ ID NO:710; (ii) an amino acid sequence having at least 95% 37a sequence identity to SEQ ID NO:710; or (iii) a portion of (i) or (ii) including a CRD. In some of the various embodiments, the reference BCMA lacks an N-terminal methionine.

[0042] In some of the embodiments, the reference BCMA polypeptide comprises (i) the amino acid sequence described in SEQ ID NO:356; (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:356; or (iii) a portion of (i) or (ii) including a CRD. In some of the embodiments, the reference BCMA polypeptide comprises the sequence described in SEQ ID NO:356. In some of the embodiments, the reference BCMA polypeptide comprises the sequence described in SEQ ID NO:356.

[0043] In some of the various embodiments, one or more amino acid substitutions of the BCMA polypeptide are Selected from TIFF2026143547000002.tif37163 or its conserved amino acid substitutions.

[0044] In some of the embodiments, one or more amino acid substitutions of the BCMA polypeptide include at least one substitution at position 19. In some embodiments, at least one substitution is selected from H19A, H19C, H19D, H19E, H19F, H19G, H19I, H19K, H19L, H19M, H19N, H19P, H19Q, H19R, H19S, H19T, H19V, H19W, and H19Y. In some of the embodiments, one or more amino acid substitutions include at least the amino acid substitution H19L. In some of the embodiments, one or more amino acid substitutions include at least the amino acid substitution H19K. In some of the embodiments, one or more amino acid substitutions include at least the amino acid substitution H19R. In some of the embodiments, one or more amino acid substitutions include at least the amino acid substitution H19Y.

[0045] In some of the various embodiments, one or more amino acid substitutions of the BCMA polypeptide include at least one substitution at position 25. In some embodiments, the at least one substitution is selected from Q25E, Q25F, Q25G, Q25H, Q25I, Q25K, Q25L, Q25M, Q25S, Q25V, and Q25Y.

[0046] In some of the various embodiments, one or more amino acid substitutions of the BCMA polypeptide include at least one substitution at position 31. In some embodiments, the at least one substitution is selected from N31D, N31G, N31H, N31K, N31L, N31M, N31P, N31S, N31V, and N31Y.

[0047] In some of the various embodiments, one or more amino acid substitutions of the BCMA polypeptide include at least one substitution at position 35. In some embodiments, the at least one substitution is selected from L35A, L35M, L35P, L35S, L35V, and L35Y.

[0048] In some of the various embodiments, one or more amino acid substitutions of the BCMA polypeptide include at least one substitution at position 36. In some embodiments, the at least one substitution is selected from T36A, T36G, T36N, T36M, T36S, and T36V.

[0049] In some of the various embodiments, one or more amino acid substitutions of the BCMA polypeptide are The filename is TIFF2026143547000003.tif98163.

[0050] In some of the various embodiments, one or more amino acid substitutions of the BCMA polypeptide include H19F, H19L, H19K, H19M, H19R, H10Y, N11D / H19Y / N47D, H19Y / R39Q / N47D; S16A / H19Y / R39Q, S9G / H19Y / T32S; H19Y / T36A / N47Y; or Q10E / H19Y / A20T / T36S. In some of the various embodiments, one or more amino acid substitutions of the BCMA polypeptide include S16A / H19Y / R39Q.

[0051] In some of the various embodiments, the variant BCMA polypeptide has at least about 85%, at least about 90%, or at least about 95% sequence identity with respect to SEQ ID NO:710 and contains one or more amino acid substitutions described.

[0052] In some of the various embodiments, the variant BCMA polypeptide has at least about 85%, at least about 90%, or at least about 95% sequence identity with respect to SEQ ID NO:356 and contains one or more amino acid substitutions described.

[0053] In some of the embodiments, the variant BCMA polypeptide has up to 10 amino acid substitutions compared to the reference BCMA polypeptide. In some of the embodiments, the variant BCMA polypeptide has up to 5 amino acid substitutions compared to the reference BCMA polypeptide. In some of the embodiments, the variant BCMA polypeptide has at least 90% sequence identity to SEQ ID NO:356. In some of the embodiments, the variant BCMA polypeptide has at least 95% sequence identity to SEQ ID NO:356.

[0054] In some of the embodiments, the variant BCMA polypeptide has increased binding affinity to one or both APRIL and BAFF compared to the reference BCMA polypeptide. In some of the embodiments, the variant BCMA polypeptide has increased binding affinity to APRIL. In some of the embodiments, the variant BCMA polypeptide has increased binding affinity to BAFF. In some of the embodiments, the variant BCMA polypeptide has increased binding affinity to both APRIL and BAFF. In some of the embodiments, the increased binding affinity to BAFF or APRIL is independently increased by more than 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, or 60 times.

[0055] In some of the embodiments, the variant BCMA polypeptide comprises the sequence described in any one of SEQ ID NO:357-435. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in any one of SEQ ID NO:357-435. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO:357. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO:377. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO:380. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO:381. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO:390. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO:391. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO:396. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO:402. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO:405. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO:406. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO:407. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO:411.In some of the various embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO:405. In some of the various embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO:406.

[0056] In some of the arbitrary embodiments, the immunomodulatory protein comprises a heteromorphic moiety linked to at least one BCMA polypeptide. In some of the arbitrary embodiments, the heteromorphic moiety is a half-life extension moiety, a multimerizing domain, a target-directed moiety that binds to molecules on the cell surface, or a detectable label. In some of the arbitrary embodiments, the half-life extension moiety comprises a multimerizing domain, albumin, albumin-binding polypeptide, Pro / Ala / Ser (PAS), the C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, polyethylene glycol (PEG), a long unstructured hydrophilic sequence of amino acids (XTEN), hydroxyethyl starch (HES), an albumin-binding small molecule, or a combination thereof.

[0057] In some of the various embodiments, the immunomodulatory protein comprises an Fc region of an immunoglobulin linked to at least one BCMA polypeptide.

[0058] In some of the arbitrary embodiments, at least one TIM contains only one TIM. In some of the arbitrary embodiments, at least one TIM contains two, three, four, or five TIMs, and optionally, each TIM is identical. In some of the arbitrary embodiments, each TIM is linked directly or indirectly via a linker, and optionally, the linker is a peptide linker. In some of the arbitrary embodiments, at least one BIM contains only one BIM. In some of the arbitrary embodiments, at least one BIM contains two, three, four, or five BIMs, and optionally, each BIM is identical. In some of the arbitrary embodiments, each BIM is linked directly or indirectly via a linker, and optionally, the linker is a peptide linker.

[0059] In some of the arbitrary embodiments, the linker is a peptide linker, and the peptide linker is Select from TIFF2026143547000004.tif24169, or a combination thereof.

[0060] In some of the arbitrary embodiments, at least one TIM and at least one BIM are linked directly or indirectly via a linker, and optionally the linker comprises a peptide linker and / or a polymerizing moiety. In some of the arbitrary embodiments, the linker comprises a peptide linker, and the peptide linker is TIFF2026143547000005.tif31159, or a combination thereof, is selected. In some of the arbitrary embodiments, the linker comprises a peptide linker, which is selected from SEQ ID NO:711(1xEAAAK), SEQ ID NO:712(2xEAAAK), SEQ ID NO:713(3xEAAAK), SEQ ID NO:714(4xEAAAK), SEQ ID NO:715(5xEAAAK), SEQ ID NO:665(6xEAAAK). In some of the arbitrary embodiments, the immunomodulatory protein is monomeric and / or comprises a single polypeptide chain.

[0061] In some of the various embodiments, at least one TIM is located at the amino terminus relative to at least one BIM within the polypeptide. In some of the various embodiments, at least one TIM is located at the carboxy terminus relative to at least one BIM within the polypeptide.

[0062] In some of the arbitrary embodiments, the immunomodulatory protein further comprises a detectable label, optionally the detectable label being a Flag tag, a His tag, or a myc tag. In some of the arbitrary embodiments, the immunomodulatory protein comprises an amino acid sequence described in any of SEQ ID NO: 618-623, or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto and retaining activity.

[0063] In some of the various embodiments, the immunomodulatory protein includes an amino acid sequence described in any of SEQ ID NO:703-708, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity thereto and retains activity.

[0064] In some embodiments, TIM and BIM are linked by a multimerizing domain. In some of the arbitrary embodiments, the multimerizing domain promotes dimerization, trimerization, tetramerization, or pentamerization. In some of the arbitrary embodiments, the multimerizing domain is an immunoglobulin Fc region. In some of the arbitrary embodiments, the immunomodulatory protein is dimerized. In some of the arbitrary embodiments, the immunoglobulin Fc region is a homodimeric Fc region. In some of the arbitrary embodiments, the immunoglobulin Fc region is a heterodimeric Fc region.

[0065] In some of the various embodiments, the immunomodulatory protein is a homodimer, and each polypeptide in the dimer is identical. In some of the various embodiments, each polypeptide comprises at least one TIM and at least one BIM, wherein at least one TIM is located on the amino-terminal side relative to at least one BIM within each polypeptide. In some of the various embodiments, each polypeptide comprises at least one TIM and at least one BIM, wherein at least one TIM is located on the carboxy-terminal side relative to at least one BIM within each polypeptide.

[0066] In some of the embodiments, the immunoglobulin Fc region is the IgG2 Fc domain. In some embodiments, the IgG2 Fc domain includes the amino acid sequence described in SEQ ID NO: 729 or 853, or an amino acid sequence exhibiting at least 95% sequence identity to SEQ ID NO: 729 or 853. In some embodiments, the IgG2 Fc domain is described in SEQ ID NO: 729. In some embodiments, the IgG2 Fc domain is described in SEQ ID NO: 853.

[0067] In some of the embodiments, the immunoglobulin Fc region is an IgG4 Fc domain. In some of the embodiments, the IgG4 Fc domain is a variant IgG4 Fc domain containing the amino acid substitution S228P. In some embodiments, the IgG4 Fc domain contains an amino acid sequence described in SEQ ID NO: 731 or 854, or an amino acid sequence exhibiting at least 95% sequence identity to SEQ ID NO: 731 or 854. In some embodiments, the IgG4 Fc domain is described in SEQ ID NO: 731. In some embodiments, the IgG4 Fc domain is described in SEQ ID NO: 854.

[0068] In some of the available embodiments, immunoglobulin Fc is either an IgG1 Fc domain or, optionally, a variant Fc exhibiting reduced binding affinity to the Fc receptor and / or reduced effector function compared to a wild-type IgG1 Fc domain. In some embodiments, immunoglobulin Fc is an IgG1 Fc domain, and Fc contains the amino acid sequence described in SEQ ID NO:597. In some of the available embodiments, immunoglobulin Fc is an IgG4 Fc domain, which is either wild-type or modified.

[0069] In some of the embodiments, immunoglobulin Fc is a variant IgG1 Fc domain comprising one or more amino acid substitutions selected from L234A, L234V, L235A, L235E, G237A, S267K, R292C, N297G, and V302C according to EU numbering. In some of the embodiments, the immunoglobulin Fc region comprises amino acid substitutions L234A, L235E, G237A according to EU numbering, or amino acid substitutions R292C, N297G, and V302C according to EU numbering. In some embodiments, Fc is a variant Fc comprising the amino acid sequence described in SEQ ID NO: 589. In some embodiments, Fc is a variant Fc comprising the amino acid sequence described in SEQ ID NO: 855.

[0070] In some embodiments, the immunomodulatory protein is a BCMA-Fc fusion protein. In some of any embodiments, the BCMA-Fc fusion protein has the following structure: BCMA polypeptide (BCMA)-linker-Fc region This includes. In some embodiments, the BCMA-Fc fusion protein is described in SEQ ID NO:629.

[0071] Also provided herein are immunomodulatory BCMA-Fc fusion proteins that are homodimers containing two identical copies of the BCMA-Fc fusion protein described in SEQ ID NO:629, linked by a covalent disulfide bond.

[0072] In some of the various embodiments, immunomodulatory proteins have the following structure: (BCMA)-linker-Fc region-linker-(BCMA) This is a BCMA-Fc fusion protein having [specific characteristics]. In some embodiments, the BCMA-Fc fusion protein is described in SEQ ID NO:809. In some embodiments, the BCMA-Fc fusion protein is described in SEQ ID NO:812.

[0073] In some of the various embodiments, the BCMA-Fc fusion protein has the following structure: (BCMA)-linker-(BCMA)-linker-Fc region It has the following characteristics. In some embodiments, the BCMA-Fc fusion protein is described in SEQ ID NO:813.

[0074] In some of the arbitrary embodiments, an immunomodulatory protein comprising at least one TIM and at least one BIM comprises an amino acid sequence described in any of SEQ ID NO: 610-617, 624-627, 637, 638, 643, 644, 648, 653, and 654, or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto and retaining activity.

[0075] In some embodiments, TIM is the wild-type CTLA-4 extracellular domain or its binding site, and BIM is the TACI extracellular domain or its binding site containing the amino acid substitution K77E / F78Y / Y102D, Q75E / R84Q, or R84G, corresponding to the position described in SEQ ID NO:709. In some embodiments, TIM is described in SEQ ID NO:1, and BIM is described in SEQ ID NO:535, 541, 542, or 688. In some embodiments, the immunomodulatory protein contains the sequence described in SEQ ID NO:611, SEQ ID NO:788, SEQ ID NO:789, SEQ ID NO:790, or SEQ ID NO:792.

[0076] This specification provides an immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:611, linked by a covalent disulfide bond.

[0077] This specification provides an immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:788, linked by a covalent disulfide bond.

[0078] This specification provides an immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:789, linked by a covalent disulfide bond.

[0079] This specification provides an immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:790, linked by a covalent disulfide bond.

[0080] This specification provides an immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:792, linked by a covalent disulfide bond.

[0081] In some of the embodiments, TIM is the wild-type CTLA-4 extracellular domain or its binding portion, and BIM is a cleaved TACI extracellular domain containing the CRD2 domain. In some embodiments, TIM is described in SEQ ID NO:1, and BIM is described in SEQ ID NO:528. In some embodiments, the immunomodulatory protein contains the sequence described in SEQ ID NO:759, SEQ ID NO:853, SEQ ID NO:854, or SEQ ID NO:791.

[0082] This specification provides an immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:759, linked by a covalent disulfide bond.

[0083] This specification provides an immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:853, linked by a covalent disulfide bond.

[0084] This specification provides an immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:854, linked by a covalent disulfide bond.

[0085] This specification provides an immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:791, linked by a covalent disulfide bond.

[0086] In some of the embodiments, TIM is a CTLA-4 extracellular domain or its binding site containing the amino acid substitution G29W / L98Q / Y105L corresponding to the position described in SEQ ID NO:1, and BIM is a TACI extracellular domain or its binding site containing the amino acid substitution K77E / F78Y / Y102D, Q75E / R84Q, or R84G corresponding to the position described in SEQ ID NO:709. In some embodiments, TIM is described in SEQ ID NO:186, and BIM is described in SEQ ID NO:535, 541, 542, or 688. In some embodiments, the immunomodulatory protein contains the sequence described in SEQ ID NO:610.

[0087] An immunomodulatory protein is provided herein that contains two identical copies of the Fc fusion protein described in SEQ ID NO:610, linked by a covalent disulfide bond.

[0088] In some of the arbitrary embodiments, an immunomodulatory protein comprising at least one TIM and at least one BIM comprises an amino acid sequence described in any of SEQ ID NO: 601-609, 631-636, 645-647, 649-652, 655-659, or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity thereto and retaining activity.

[0089] In some of the embodiments, TIM is the wild-type CTLA-4 extracellular domain or its binding site, and BIM is the BCMA extracellular domain or its binding site containing the amino acid substitution H19L corresponding to the position described in SEQ ID NO:710. In some embodiments, TIM is described in SEQ ID NO:1, and BIM is described in SEQ ID NO:406. In some embodiments, the immunomodulatory protein contains the sequence described in SEQ ID NO:602.

[0090] This specification provides an immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:602, linked by a covalent disulfide bond.

[0091] In some of the embodiments, TIM is the CTLA-4 extracellular domain or its binding portion containing the amino acid substitution G29W / L98Q / Y105L corresponding to the position described in SEQ ID NO:1, and BIM is the BCMA extracellular domain or its binding portion containing the amino acid substitution H19L relative to the position described in SEQ ID NO:710. In some embodiments, TIM is described in SEQ ID NO:186, and BIM is described in SEQ ID NO:406. In some embodiments, the immunomodulatory protein contains the sequence described in SEQ ID NO:601.

[0092] This specification provides an immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:601, linked by a covalent disulfide bond.

[0093] In some of the arbitrary embodiments, the immunomodulatory protein is a heterodimer, and each polypeptide of the dimer is linked to an immunoglobulin Fc domain containing one or more amino acid modifications to the wild-type Fc domain, resulting in heterodimerization between polypeptides. In some of the arbitrary embodiments, the wild-type immunoglobulin Fc is the IgG1 Fc domain. In some of the arbitrary embodiments, the other amino acid modification is selected from knob-into-hole modifications and charge mutations to reduce or prevent self-association due to charge repulsion. In some of the arbitrary embodiments, the Fc region optionally further comprises one or more amino acid substitutions to reduce binding affinity to the Fc receptor and / or reduce effector function compared to the wild-type IgG1 Fc domain. In some of the embodiments, one or more amino acid substitutions are selected from L234A, L234V, L235A, L235E, G237A, S267K, R292C, N297G, and V302C according to EU numbering. In some of the embodiments, the immunoglobulin Fc region comprises the amino acid substitutions L234A, L235E, G237A according to EU numbering, or the amino acid substitutions R292C, N297G, and V302C according to EU numbering.

[0094] In some of the arbitrary embodiments, the heterodimer comprises a first polypeptide comprising the amino acid sequence described in SEQ ID NO: 662 or 663; and a second polypeptide comprising the amino acid sequence described in SEQ ID NO: 660.

[0095] In some of the various embodiments, the immunomodulatory protein blocks the binding of APRIL, BAFF, or APRIL / BAFF heterotrimers to BCMA or TACI, or the immunomodulatory protein reduces the levels of circulating APRIL, circulating BAFF, or circulating APRIL / BAFF in the blood after administration to the subject. In some of the various embodiments, the immunomodulatory protein blocks the binding of APRIL, BAFF, or APRIL / BAFF heterotrimers to BCMA or TACI, or the immunomodulatory protein reduces the levels of circulating APRIL, circulating BAFF, or circulating APRIL / BAFF in the blood after administration to the subject. In some of the various embodiments, the immunomodulatory protein reduces or inhibits B cell maturation, differentiation, and proliferation.

[0096] In some of the various embodiments, the immunomodulatory protein blocks the binding of CD80 or CD86 to the costimulatory receptor, optionally the costimulatory receptor is CD28, and the immunomodulatory protein reduces or inhibits T cell costimulation. In some of the various embodiments, the immunomodulatory protein reduces or inhibits B cell maturation, differentiation, or proliferation. In some of the various embodiments, the immunomodulatory protein blocks the binding of CD80 or CD86 to the costimulatory receptor, optionally the costimulatory receptor is CD28, or the immunomodulatory protein reduces or inhibits T cell costimulation.

[0097] Nucleic acid molecules encoding any of the immunomodulatory proteins described herein are provided herein. In some of the embodiments, the nucleic acid molecule is a synthetic nucleic acid. In some of the embodiments, the nucleic acid molecule is a cDNA.

[0098] A vector comprising a nucleic acid molecule in any of the embodiments described herein is provided herein. In some of the embodiments, the vector is an expression vector. In some of the embodiments, the vector is a mammalian expression vector or a viral vector.

[0099] Cells comprising nucleic acids or vectors of any of the embodiments described herein are provided herein. In some of the embodiments, the cells are mammalian cells. In some of the embodiments, the cells are human cells.

[0100] A method for producing an immunomodulatory protein is provided herein, comprising the step of introducing a nucleic acid molecule or a vector, as described herein, into a host cell under conditions that express the protein in the cell. In some of the arbitrary embodiments, the method comprises the step of isolating or purifying the immunomodulatory protein from the cell.

[0101] Immunomodulatory proteins produced by any of the embodiments described herein are provided herein.

[0102] A pharmaceutical composition comprising an immunomodulatory protein in any of the embodiments described herein is provided herein.

[0103] A variant BCMA-Fc fusion protein is provided herein, comprising a variant BCMA polypeptide, an Fc region, and a linker between the BCMA polypeptide and the Fc region, wherein the variant BCMA polypeptide contains one or more amino acid substitutions in the extracellular domain (ECD) or its specific binding fragment of the unmodified BCMA polypeptide, corresponding to a position selected from among 9, 10, 11, 14, 16, 19, 20, 22, 25, 27, 29, 30, 31, 32, 35, 36, 39, 43, 45, 46, 47, and 48, relative to the position described in SEQ ID NO:710.

[0104] In some of the embodiments, the reference BCMA polypeptide is a polypeptide comprising the extracellular domain or specific binding moiety of BCMA that binds to APRIL, BAFF, or a BAFF / APRIL heterotrimer. In some of the embodiments, the reference BCMA polypeptide comprises (i) the amino acid sequence described in SEQ ID NO:710; (ii) an amino acid sequence having at least 95% 37a sequence identity to SEQ ID NO:710; or (iii) a portion of (i) or (ii) including a CRD. In some of the embodiments, the reference BCMA lacks an N-terminal methionine.

[0105] In some of the embodiments, the reference BCMA polypeptide comprises (i) the amino acid sequence described in SEQ ID NO:356; (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:356; or (iii) a portion of (i) or (ii) including a CRD. In some of the embodiments, the reference BCMA polypeptide comprises the sequence described in SEQ ID NO:356. In some of the embodiments, the reference BCMA polypeptide comprises the sequence described in SEQ ID NO:356.

[0106] In some of the various embodiments, one or more amino acid substitutions are Selected from TIFF2026143547000006.tif37165 or its conserved amino acid substitutions. In some of the arbitrary embodiments, one or more amino acid substitutions include at least one substitution at position 19, and optionally, at least one substitution is selected from H19A, H19C, H19D, H19E, H19F, H19G, H19I, H19K, H19L, H19M, H19N, H19P, H19Q, H19R, H19S, H19T, H19V, H19W, H19Y. In some of the arbitrary embodiments, one or more amino acid substitutions include at least the amino acid substitution H19L. In some of the arbitrary embodiments, one or more amino acid substitutions include at least the amino acid substitution H19K. In some of the arbitrary embodiments, one or more amino acid substitutions include at least the amino acid substitution H19R. In some of the various embodiments, one or more amino acid substitutions include at least the amino acid substitution H19Y.

[0107] In some of the various embodiments, one or more amino acid substitutions include at least one substitution at position 25, and optionally at least one substitution is selected from Q25E, Q25F, Q25G, Q25H, Q25I, Q25K, Q25L, Q25M, Q25S, Q25V, and Q25Y. In some of the various embodiments, one or more amino acid substitutions include at least one substitution at position 31, and optionally at least one substitution is selected from N31D, N31G, N31H, N31K, N31L, N31M, N31P, N31S, N31V, and N31Y.

[0108] In some of the embodiments, one or more amino acid substitutions include at least one substitution at position 35, and optionally at least one substitution is selected from L35A, L35M, L35P, L35S, L35V, and L35Y. In some of the embodiments, one or more amino acid substitutions include at least one substitution at position 36, and optionally at least one substitution is selected from T36A, T36G, T36N, T36M, T36S, and T36V. In some of the embodiments, one or more amino acid substitutions are The filename is TIFF2026143547000007.tif97163.

[0109] In some of the embodiments, one or more amino acid substitutions include S16A / H19Y / R39Q. In some of the embodiments, the variant BCMA polypeptide has increased binding affinity to one or both APRIL and BAFF compared to the reference TACI polypeptide.

[0110] In some of the various embodiments, the variant BCMA polypeptide has increased binding affinity to APRIL. In some of the various embodiments, the variant BCMA polypeptide has increased binding affinity to BAFF. In some of the various embodiments, the variant BCMA polypeptide has increased binding affinity to both APRIL and BAFF.

[0111] In some of the embodiments, the increased binding affinity to BAFF or APRIL is independently increased by more than 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 times. In some of the embodiments, the variant BCMA polypeptide contains the sequence described in any one of SEQ ID NO: 357-435. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in any one of SEQ ID NO: 357-435. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO: 381. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO: 411. In some of the embodiments, the variant BCMA polypeptide consists of or is essentially the sequence described in SEQ ID NO: 405. In some of the various embodiments, the variant BCMA polypeptide consists of or is essentially derived from the sequence described in SEQ ID NO:406.

[0112] In some of the various embodiments, the linker includes a peptide linker, and the peptide linker is Select from TIFF2026143547000008.tif24168, or a combination thereof.

[0113] In some of the various embodiments, the Fc fusion protein is a dimer. In some of the various embodiments, the immunoglobulin Fc region is a homodimeric Fc region.

[0114] In some of the embodiments, immunoglobulin Fc is either an IgG1 Fc domain or, optionally, a variant Fc exhibiting reduced binding affinity to the Fc receptor and / or reduced effector function compared to a wild-type IgG1 Fc domain. In some embodiments, immunoglobulin Fc is an IgG1 Fc domain, and Fc comprises the amino acid sequence described in SEQ ID NO:597. In some of the embodiments, immunoglobulin Fc is a variant IgG1 Fc domain comprising one or more amino acid substitutions selected from L234A, L234V, L235A, L235E, G237A, S267K, R292C, N297G, and V302C according to EU numbering. In some of the embodiments, the immunoglobulin Fc region comprises the amino acid substitutions L234A, L235E, G237A according to EU numbering, or the amino acid substitutions R292C, N297G, and V302C according to EU numbering.

[0115] In some of the embodiments, immunoglobulin Fc is described in SEQ ID NO:586. In some embodiments, Fc is variant Fc containing the amino acid sequence described in SEQ ID NO:589. In some embodiments, the Fc fusion protein is a homodimer.

[0116] In some embodiments, the Fc fusion protein neutralizes APRIL and BAFF. In some embodiments, the IC50 for neutralizing APRIL is less than 100 pM, less than 50 pM, less than 40 pM, less than 30 pM, less than 20 pM, less than 10 pM, less than 5 pM, or less than 1 pM, or any value among the above, and / or the IC50 for neutralizing BAFF is less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 75 pM, less than 50 pM, less than 25 pM, or less than 10 pM, or any value among the above.

[0117] In some of the available embodiments, the Fc fusion protein blocks the binding of APRIL, BAFF, or APRIL / BAFF heterotrimers to BCMA or TACI, or the Fc fusion protein reduces the levels of circulating APRIL, circulating BAFF, or circulating APRIL / BAFF in the blood after administration to a subject. In some of the available embodiments, immunoglobulin Fc is described in SEQ ID NO: 586. In some of the available embodiments, the Fc fusion protein blocks the binding of APRIL, BAFF, or APRIL / BAFF heterotrimers to BCMA or TACI, or the Fc fusion protein reduces the levels of circulating APRIL, circulating BAFF, or circulating APRIL / BAFF in the blood after administration to a subject. In some of the available embodiments, the immunomodulatory protein reduces or inhibits B cell maturation, differentiation, and / or proliferation.

[0118] Nucleic acid molecules encoding any of the embodiments described herein are provided herein. In some of the embodiments, the nucleic acid molecule is a synthetic nucleic acid. In some of the embodiments, the nucleic acid molecule is a cDNA.

[0119] A vector comprising a nucleic acid molecule in any of the embodiments described herein is provided herein. In some of the embodiments, the vector is an expression vector. In some of the embodiments, the vector is a mammalian expression vector relative to a viral vector.

[0120] Cells comprising nucleic acids or vectors of any of the embodiments described herein are provided herein. In some of the embodiments, the cells are mammalian cells. In some of the embodiments, the cells are human cells.

[0121] A method for producing an immunomodulatory protein is provided herein, comprising the step of introducing a nucleic acid molecule or a vector of any of the embodiments provided herein into a host cell under conditions that allow the protein to be expressed in the cell. In some of the arbitrary embodiments, the method further comprises the step of isolating or purifying the Fc fusion protein from the cell. A method for producing an Fc fusion protein is provided herein, comprising the step of introducing a nucleic acid molecule or a vector of any of the embodiments provided herein into a host cell under conditions that allow the protein to be expressed in the cell.

[0122] Fc fusion proteins produced by any of the embodiments described herein are provided herein.

[0123] A pharmaceutical composition comprising an Fc fusion protein in any of the embodiments described herein is provided herein. In some of the embodiments, the pharmaceutical composition contains a pharmaceutically acceptable excipient. In some of the embodiments, the pharmaceutical composition is sterile.

[0124] A manufactured article containing a pharmaceutical composition in any of the embodiments described herein, in a vial or container, is provided herein. In some of the embodiments, the vial or container is sealed.

[0125] A kit comprising a pharmaceutical composition of any embodiment provided herein and instructions for use is provided herein. In some of the arbitrary embodiments, the kit comprises a manufactured article of any embodiment described herein and instructions for use.

[0126] A method for reducing the immune response of a target is provided herein, comprising the step of administering an immunomodulatory protein in any form described herein to a target requiring such reduction.

[0127] A method for reducing the immune response of a target is provided herein, comprising the step of administering an Fc fusion protein in any form described herein to a target requiring such reduction.

[0128] A method for reducing the immune response of a subject is provided herein, comprising the step of administering to a subject in need of such reduction a pharmaceutical composition in any of the embodiments described herein. In some of the embodiments, the B-cell immune response is reduced in the subject, thereby reducing or inhibiting the maturation, differentiation, and / or proliferation of B cells. In some of the embodiments, the circulating levels of APRIL, BAFF, or APRIL / BAFF heterotrimer are reduced in the subject.

[0129] A method for reducing the circulating levels of APRIL, BAFF, or APRIL / BAFF heterotrimer in a subject is provided herein, comprising the step of administering a pharmaceutical composition of any embodiment described herein to the subject. In some of the embodiments, the T cell immune response is reduced in the subject, thereby reducing or inhibiting T cell costimulation. In some of the embodiments, a disease or condition in the subject is treated by reducing the immune response.

[0130] A method for treating a disease, disorder, or condition in a subject is provided herein, comprising the step of administering an immunomodulatory protein in any of the embodiments described herein to a subject in need thereof.

[0131] A method for treating a disease, disorder, or condition in a subject is provided herein, comprising the step of administering an Fc fusion protein in any of the embodiments described herein to a subject in need thereof.

[0132] A method for treating a disease, disorder, or condition in a subject is provided herein, comprising the step of administering to a subject in need of such treatment any of the pharmaceutically acceptable compositions described herein.

[0133] Immunomodulatory proteins or pharmaceutical compositions containing them for use in treating diseases, disorders, or conditions in a subject are also provided herein. The use of immunomodulatory proteins or pharmaceutical compositions containing them for the formulation of pharmaceuticals for treating diseases, disorders, or conditions in a subject is also provided herein.

[0134] In some of the various embodiments, the disease, disorder, or condition is an autoimmune disease, an inflammatory condition, a B-cell cancer, an antibody-mediated condition, a renal disease, graft rejection, graft-versus-host disease, or a viral infection. The disease or condition being treated may be any of those described herein. In some of the various embodiments, the disease or condition is an autoimmune disease selected from the group consisting of systemic lupus erythematosus (SLE), Sjögren's syndrome, scleroderma, multiple sclerosis, diabetes mellitus, polymyositis, primary biliary cirrhosis, IgA nephropathy, optic neuritis, amyloidosis, antiphospholipid syndrome (APS), polyglandular autoimmune syndrome type II (APS II), autoimmune thyroid disease (AITD), Graves' disease, autoimmune adrenal nephritis, and pemphigus vulgaris. In some of the various embodiments, the disease or condition is a B-cell cancer, and the cancer is myeloma. In some of the various embodiments, the types of myeloma include multiple myeloma, plasmacytoma, multiple solitary plasmacytoma, and / or extramedullary myeloma. In some of the various embodiments, the types of myeloma include light chain myeloma, nonsecretory myeloma, and / or IgD or IgE myeloma. [Brief explanation of the drawing]

[0135] [Figure 1] A schematic diagram of a functional inhibition assay using BCMA or TACI with recombinant APRIL and BAFF is shown. In this assay, Jurkat cells were transduced to stably express mouse or human TACI on the cell surface, along with a luciferase-based NF-κB reporter. Following activation with recombinant APRIL or BAFF, the endogenous NF-κB transcription factor binds to a DNA response element that regulates the transcription of the firefly luciferase gene. Luciferase expression can be monitored, for example, by detection with Bio-Glo® reagents and measurement using a Cytation 3 reader. [Figure 2]Exemplary human BCMA TD Fc fusion molecules for blocking human APRIL (upper panel) and BAFF (lower panel) mediated signaling are shown. Exemplary BCMA TD Fc fusions were incubated with APRIL (2nM) or BAFF (4nM) for 20 minutes (room temperature with shaking) and then added to wells containing 150,000 Jurkat / TACI / NFκB-luciferase cells for 5 hours. [Figure 3A] Figures 3A and 3B illustrate the function of exemplary BCMA TD Fc fusion molecules, either alone or stacked with CTLA-4 IgD, for blocking APRIL (top panel of each figure) or BAFF (bottom panel of each figure). Figures 3A and 3B show that human BCMA TD retains its function when stacked with CTLA-4. Figures 3C and 3D illustrate the function of exemplary TACI TD Fc fusion molecules, either alone or stacked with CTLA-4 IgD, for blocking APRIL (top panel of each figure) or BAFF (bottom panel of each figure). Figures 3C and 3D show that human TACI TD retains its function when stacked with CTLA-4. [Figure 3B] Refer to the explanation in Figure 3A. [Figure 3C] Refer to the explanation in Figure 3A. [Figure 3D] Refer to the explanation in Figure 3A. [Figure 4] The images show human BCMA fusion molecules alone, or BCMA or TACI TD Fc fusion molecules stacked with CTLA-4 IgD, for blocking mouse APRIL (left panel) and BAFF (right panel) mediated signaling. [Figure 5A] This paper shows the human BCMA TD Fc fusion molecule, either alone or stacked with CTLA-4 IgD, for blocking human APRIL (upper panel) and BAFF (lower panel) mediated signaling, compared to TACI 13-118-Fc, TACI 30-110-Fc, and belimumab. [Figure 5B]Shows human TACI TD Fc fusion molecules when stacked with CTLA-4 IgD for blocking human APRIL (upper panel) and BAFF (lower panel)-mediated signaling, compared with TACI 13-118-Fc, TACI 30-110-Fc and belimumab. [Figure 6] Shows a schematic diagram of a functional inhibition assay for CD80 / CD86-CD28-mediated co-stimulation. Jurkat / IL-2 cells stably express a luciferase reporter driven by the IL-2 promoter when stimulated by anti-CD3 stimulation and anti-CD28 stimulation. Receptor-mediated signaling results in IL-2 promoter-mediated luminescence, and the bioluminescent signal can be detected and quantified, for example, by using a Bio-Glo™ substrate and a luminometer. [Figure 7] Figures 7A and 7B show that when wild-type or CTLA-4 vIgD (IgSF domain) is included in a multispecific (stacked) construct molecule with BCMA converted to TACI TD, the construct maintains the function for blocking CD80 (left panel) or CD86 (right panel). [Figure 8-1] Figures 8A to 8F show the activity of CTLA-4 vIgD, alone or when included in a multispecific (stacked) molecule comprising BCMA or TACI TD, for inhibiting human follicular helper T (TFH) cells and B cells in an autologous TFH-B cell assay. B-Tfh cell cultures were incubated for 7 days in the presence of increasing concentrations (100,000 to 32 pM) of the protein. Cultured cells were surface-stained and analyzed by flow cytometry for: (Figure 8A) recovery of CD4+ T cells, (Figure 8B) recovery of CD4+CD40L+ cells, (Figure 8C) recovery of CD4+ICOS+ cells, (Figure 8D) recovery of CD19+ B cells, and (Figure 8E) B cell activation / upregulation of CD86. The supernatant was collected, and the level of IgM secretion was determined by ELISA (Figure 8F). Data represent the mean (± SEM) of three replicate experiments for each condition. [Figure 8-2] See the description of Figure 8-1. [Figure 9]This shows the serum anti-KLH IgM antibody levels at the end of the study (day 19) in the KLH immunization model. Statistical differences between groups were determined by one-way ANOVA. Only statistically significant differences (p<0.05) are listed. [Figure 10] This table shows the serum anti-KLH IgG1 antibody levels at the end of the study (day 19) in the KLH immunization model. Statistical differences between groups were determined by one-way ANOVA. Only statistically significant differences (p<0.05) are listed. [Figure 11] This shows the spleen weight at the end of the study (day 19) in the KLH model. Statistical differences between the Fc control and other test samples were determined by t-tests. Only significant differences (p<0.05) are listed. [Figure 12-1] Figures 12A–12H show flow cytometry analysis of splenocyte B cells and Tfh subsets at the end of the KLH model (day 19). The spleen was processed and flow cytometry was performed on B220+ B cells (Figures 12A, 12E); marginal zone (MZ) B cells (Figures 12B, 12F); germinal center (GC) B cells (Figures 12C, 12G); and T follicular helper (Tfh) cells (Figures 12D, 12H). "Fc control" = Fc as described in SEQ ID NO: 589. Statistical significance (p<0.05) compared to the Fc control or abatacept was calculated by one-way ANOVA using uncorrected Fisher's LSD test. [Figure 12-2] See the explanation in Figure 12-1. [Figure 13] Figures 13A–13D show flow cytometry analysis of the splenocyte T effector memory subset at the end of the KLH model (day 19). Splenes were processed and CD4+ (Figures 13A, 13C) and CD8+ (Figures 13B, 13D) T effector memory (Tem) cells were analyzed by flow cytometry. "Fc control" = Fc as described in SEQ ID NO: 589. Statistical significance (p<0.05) compared to the Fc control or abatacept was calculated by one-way ANOVA using uncorrected Fisher's LSD. [Figure 14A]Figures 14A to 14I show the analysis of parameters evaluated in the NZB / NZW mouse model of human SLE. Proteinuria score (Figure 14A), mean weight change rate (Figure 14B), and survival rate (Figure 14C) were evaluated from 20 weeks of age. Serum analysis was performed for anti-double-stranded DNA IgG titer (Figure 14D) and blood urea nitrogen (BUN) (Figure 14E) (**Uncorrected Dunn's test, p=0.0047 and p=0.0065 compared to Fc; ***Uncorrected Dunn's test, p=0.0004 compared to Fc). Kidneys were processed and analyzed by histological examination of repeated periodate Schiff (PAS) stained sections. Individual component and total histological scores are shown in Figure 14F. As shown in Figures 14G and 14H, respectively, frozen kidneys were also sectioned and stained for immunohistochemical analysis of glomerular deposition of mouse IgG and complement C3. Figure 14I shows the histological score ± SEM. [Figure 14B] Refer to the explanation in Figure 14A. [Figure 14C] Refer to the explanation in Figure 14A. [Figure 14D] Refer to the explanation in Figure 14A. [Figure 14E] Refer to the explanation in Figure 14A. [Figure 14F] Refer to the explanation in Figure 14A. [Figure 14G] Refer to the explanation in Figure 14A. [Figure 14H] Refer to the explanation in Figure 14A. [Figure 14I] Refer to the explanation in Figure 14A. [Figure 15] A schematic diagram of an exemplary BCMA-Fc fusion protein is shown. [Figure 16] A schematic diagram of an exemplary Fc fusion form of the provided multi-domain (stack) immunomodulatory protein is shown. [Figure 17A]Figures 17A and 17B show exemplary sequence alignments for identifying corresponding residues in a sequence compared to a reference sequence. The symbol "*" between two aligned amino acids indicates that the aligned amino acids are identical. The symbol "-" indicates an alignment gap. Exemplary, non-limiting positions for amino acid substitutions described herein are shown in bold. Based on the alignment of two similar sequences having a common identical residue, those skilled in the art can identify the "corresponding" position in the sequence by comparing it to the reference sequence using a conserved identical amino acid residue as a guide. Figure 17A provides an exemplary alignment of the reference TACI extracellular domain sequence described in SEQ ID NO:709 (including the complete extracellular domain with CRD1 and CRD2 and an initiating methionine residue) and the TACI extracellular domain sequence described in SEQ ID NO:528 (including a single CRD, CRD2 only). By aligning identical residues, for example, it can be shown that amino acid residue E7 in SEQ ID NO:528 corresponds to residue E74 in SEQ ID NO:709, amino acid residue K10 in SEQ ID NO:528 corresponds to residue K77 in SEQ ID NO:709, amino acid residue Y12 in SEQ ID NO:528 corresponds to Y79 in SEQ ID NO:709, amino acid residue L15 in SEQ ID NO:528 corresponds to L82 in SEQ ID NO:709, amino acid residue R17 in SEQ ID NO:528 corresponds to R84 in SEQ ID NO:709, and amino acid residue D16 in SEQ ID NO:528 corresponds to D85 in SEQ ID NO:709. Figure 17B provides an exemplary alignment of the reference BCMA extracellular domain sequence described in SEQ ID NO:710 (including the complete extracellular domain with a CRD and an initiate methionine residue) and the BCMA extracellular domain sequence described in SEQ ID NO:356 (without an initiate methionine).By aligning identical residues, for example, it can be shown that amino acid residue H18 in SEQ ID NO:356 corresponds to residue H19 in SEQ ID NO:710, and amino acid residue R38 in SEQ ID NO:356 corresponds to residue R39 in SEQ ID NO:710. Performing similar alignments between two similar protein sequences to identify corresponding residues is within the scope of the skill of the art, including based on the examples and descriptions herein. [Figure 17B] Refer to the explanation in Figure 17A. [Figure 18] Figures 18A–18D show the analysis of parameters evaluated in the mouse keyhole limpet hemocyanin (KLH) model. Serum -KLH IgM OD levels were evaluated as the primary response (Figure 18A) and secondary response (Figure 18B). Similarly, serum anti-KLH IgG1 OD levels were evaluated as both the primary response (Figure 18C) and secondary response (Figure 18D). [Figure 19] Figures 19A and 19B show the analysis of spleens harvested from a mouse keyhole limpet hemocyanin (KLH) immunization model. The spleens were processed and analyzed by weight (Figure 19A) and total cell count (Figure 19B). [Figure 20] This paper presents an analysis of the spleen to evaluate the cell subtype population composition derived from a mouse keyhole limpet hemocyanin (KLH) model, and shows the results of comparing the number of B cell subsets with the group mean. [Figure 21] This paper presents an analysis of the spleen to evaluate the cellular subtype phenotypic composition derived from a mouse keyhole limpet hemocyanin (KLH) model, showing the results regarding the number of germinal center B cells and plasma cells (Figure 21). [Figure 22] Figures 22A to 22D show the number of T cells in the mouse keyhole limpet hemocyanin (KLH) model. Spleen CD3+ T cells, CD8+ T cells, CD4+ T cells, and follicular helper T cells are shown in Figures 22A, 22B, 22C, and 22D, respectively. [Figure 23]Shows Tcm and Tem cell populations in a mouse keyhole limpet hemocyanin (KLH) model. [Figure 24] Figures 24A to 24B and 25A to 25B show the overall incidence and extent of sialadenitis (Figures 24A to 24B) and insulitis (Figures 25A to 25B) in diabetes-prone mice after treatment with 186-CTLA-4 Fc and the test molecule CTLA4 186-GSG4S-Fc-(G4S)4-TACI 541. [Figure 25] See the description of Figure 24. [Figure 26] Shows the mean blood glucose concentration (mg / dL) measured in blood on days 7, 8, 9 and 10 in diabetes-prone mice after treatment with 186-CTLA-4 Fc and the test molecule CTLA4 186-GSG4S-Fc-(G4S)4-TACI 541. [Figure 27] Figures 27 and 28A to 28C show results obtained from an in vivo mouse bm12-induced SLE model tested with an exemplary CTLA4 186-GSG4S-Fc-(G4S)4-TACI 541 multi-domain molecule, as compared with WT-TACI Fc alone. BUN concentration from serum collected on day 82 (end of study) is shown in Figure 27. Figures 28A to 28C show levels of IgG2b (Figure 28A), IgG2c (Figure 28B) and IgG3 (Figure 28C) from serum collected on days 14, 42 and 82. [Figure 28] See the description of Figure 27. [Figure 29] Figure 29 shows the level of anti-dsDNA antibody levels from serum collected in an in vivo mouse bm12-induced SLE model tested with an exemplary CTLA4 186-GSG4S-Fc-(G4S)4-TACI 541 multi-domain molecule. MODE FOR CARRYING OUT THE INVENTION

[0136] Detailed Description This specification provides immunomodulatory proteins involved in one or more other immune receptors or ligands, produced, for example, on antigen-presenting cells or as soluble factors, to suppress or reduce B cell response or activity, and possibly T cell response as well. Among the immunomodulatory proteins provided are proteins that bind to BAFF ligand or APRIL ligand to neutralize their activity and block or antagonize the activity of B cell stimulating receptors, such as TACI or BCMA. The immunomodulatory proteins provided may be fusion proteins of the BCMA extracellular domain or its binding site (hereinafter, BCMA ECD) and a multimerizing domain such as immunoglobulin Fc. For example, BCMA-Fc fusion proteins are provided herein. Furthermore, a multi-domain immunomodulatory protein (also called a “stack” immunomodulatory protein) is provided, comprising at least one first binding domain that binds to a BAFF ligand or an APRIL ligand to neutralize their activity and blocks or antagonizes the activity of B cell stimulating receptors such as TACI or BCMA, and at least one second binding domain that, for example, binds to a CD80 ligand or a CD86 ligand to block or antagonize the activity of T cell stimulating receptors, neutralizing their activity through interaction with the T cell stimulating receptor CD28 or the negative regulatory protein CTLA-4. In some embodiments, the immunomodulatory proteins provided herein may be used to treat diseases, disorders or conditions associated with dysregulated immune responses, such as inflammatory diseases or autoimmune diseases.

[0137] The immune system relies on immune checkpoints to prevent autoimmunity (i.e., self-tolerance) and protect tissues from excessive damage during immune responses, such as during attacks against pathogenic infections. However, in some cases, the immune system can become dysregulated, leading to abnormal immune responses against normal body parts or tissues, resulting in autoimmune diseases, autoimmune conditions, or autoimmune symptoms. In other cases, an undesirable immune response can occur against foreign tissues, such as grafts, potentially leading to graft rejection.

[0138] In some aspects, immunotherapies that alter immune cell activity, such as B cell activity and / or T cell activity, can treat certain diseases, disorders, and conditions in which the immune response is dysregulated. In particular, inhibition or attenuation of immune responses, such as B cell responses and / or T cell responses, may be desirable to reduce or prevent undesirable inflammation, autoimmune symptoms, and / or graft rejection. However, therapeutic approaches that attempt to modulate the interaction between ligands and their receptors, for example, mediating immune responses within immune synapses, are not entirely satisfactory. In some cases, therapeutics that intervene in and alter the immunomodulatory effects of immune cells, such as T cells or B cells, are constrained by the requirements of spatial orientation and the size limitations imposed by the extent of immune synapses. In some aspects, existing therapeutic agents, including antibody drugs, may not be able to interact simultaneously with multiple target proteins involved in modulating these interactions. For example, soluble receptors and soluble antibodies generally bind competitively (e.g., they do not bind to multiple target species at once) and therefore lack the ability to bind to multiple targets simultaneously. Furthermore, pharmacokinetic differences between drugs that independently target one of these receptors may make it difficult to adequately maintain the desired blood concentrations of a combination of drugs targeting two different receptors throughout the therapeutic course.

[0139] BAFF and APRIL are TNF superfamily members that bind to both TACI and BCMA on B cells. BAFF also binds to a third receptor, BAFF-R. Together, BAFF and APRIL support B cell development, differentiation, and survival. Their co-neutralization dramatically reduces B cell function, including antibody production, while inhibition of either BAFF or APRIL alone mediates relatively mild effects. CTLA-4-based therapies, which block T cell costimulation, provide safe and moderately effective T cell inhibition in many disease situations, whereas B cell targeted therapies, while showing promising therapeutic potential, are not entirely satisfactory.

[0140] Some embodiments of the immunomodulatory proteins provided result in improved neutralizing activity and suppression or reduction of the B cell response. In some embodiments, the improved activity is mediated by increased or improved binding or interaction between the provided immunomodulatory proteins and BAFF and / or APRIL. For example, variant BCMA polypeptides comprising one or more amino acid substitutions (replacement or mutation) resulting in improved protein binding affinity to BAFF and / or APRIL are provided herein. In particular, some embodiments of the immunomodulatory proteins provided result in improved combined inhibition of BAFF and APRIL. Furthermore, the provided immunomodulatory proteins include those that suppress BAFF and / or APRIL-mediated activity, either alone (e.g., BCMA-Fc) or in combination with inhibition of T cell costimulation. For example, among the embodiments provided is a multi-domain immunomodulatory protein, a B-cell inhibitor molecule (BIM), which is an extracellular domain portion (e.g., BCMA ECD) that binds to BAFF and / or APRIL, fused to a T-cell inhibitor molecule (TIM), which is another domain that binds to the T-cell inhibitor molecule (TIM) and / or the T-cell inhibitor molecule (TIM), which antagonizes or blocks the T-cell response. The immunomodulatory proteins provided are intended to result in improved activity that modulates the B-cell response alone or in conjunction with the modulation of the T-cell response. Thus, the immunomodulatory proteins provided result in effective and sustained disease suppression in treating autoimmune or inflammatory diseases, including severe B-cell-associated autoimmune diseases such as SLE.

[0141] For example, the provided embodiment is based on the finding that directional evolution by affinity modification of the TNFR domain (TD) of the ectodomain of a particular molecule (e.g., BCMA) has facilitated the development of a molecule with improved affinity for APRIL and / or BAFF. Thus, the affinity modification produces a variant BCMA containing a variant TNFR domain (vTD). Fusion of such a molecule with immunoglobulin Fc results in an immunomodulatory protein that suppresses B cell activity and response. Similarly, the provided embodiment is also based on the finding that further inclusion of a TD domain, e.g., wild-type (WT) TD or vTD, as a multidomain fusion with the immunoglobulin superfamily (IgSF) domain of a T cell inhibitory molecule, e.g., the extracellular domain of CTLA-4, further enhances immunosuppressive activity. Such activity may be further enhanced by directional evolution by affinity modification of the IgSF domain of CTLA-4 to produce a variant IgSF domain (vIgD) that further enhances affinity for the CD28 costimulatory receptor and the CD80 and / or CD86 ligands, which are ligands for the inhibitory CTLA-4 receptor. The findings herein demonstrate that these immunomodulatory proteins consistently exhibit potent immunosuppressive activity and efficacy in vitro and in vivo, and appear superior to existing and / or approved immunomodulatory agents such as belimumab, abatacept, atacicept, or teritacicept. Therefore, such biologics may be attractive development candidates for the treatment of B-cell related diseases, including severe autoimmune and / or inflammatory diseases such as SLE, Sjögren's syndrome, and other connective tissue diseases.

[0142] All publications referenced in this application, including patent documents, scientific papers, and databases, are incorporated by reference in whole for any purpose, as if each individual publication were incorporated by reference individually. If any definition provided herein conflicts with or otherwise contradicts any definition provided in a patent, patent application, published patent application, or other publication incorporated herein by reference, the definition provided herein shall prevail over the definition incorporated herein by reference.

[0143] The section headings used in this specification are for organizational purposes only and should not be interpreted as limiting the subject matter described.

[0144] I. Definition Unless otherwise defined, all technical terms, notations, and other technical and scientific or specialized terms used herein are intended to have the same meaning as those generally understood by those skilled in the art in which the claimed subject matter pertains. In some cases, terms that have a generally understood meaning are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from those generally understood in the art.

[0145] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise.

[0146] As used herein, the term “about” refers to the normal range of error for each value, which is readily known to those skilled in the art. Any reference to any value or parameter “about” herein includes (and describes) aspects relating to the value or parameter itself. For example, a statement referring to “about X” includes a statement “X”.

[0147] The term "affinity-modified," as used in relation to protein domains, refers to a mammalian protein having an amino acid sequence modified in its extracellular domain or specific binding portion (compared to the corresponding wild-type parent domain or unmodified domain) to increase or decrease binding activity, such as binding affinity to at least one of its binding partners (or "counter-structures"), compared to the parent wild-type or unmodified (i.e., unaffinity-modified domain) protein. In some embodiments, an affinity-modified domain may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid differences, e.g., amino acid substitutions, compared to the wild-type or unmodified domain. Binding activity, e.g., increased or decreased binding affinity, can be determined using well-known binding assays, including flow cytometry. See also Larsen et al., American Journal of Transplantation, Vol 5:443-453 (2005). Linsley et al., Immunity, 1:7930801 (1994). The increased protein binding activity, e.g., affinity, to its binding partner is at least 10% greater than the wild-type control value, and in some embodiments, at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000% greater than the wild-type control value. The decreased protein binding activity, e.g., affinity, to at least one of its binding partners is 90% or less of the control value, but 10% or more of the wild-type control value, and in some embodiments, 80%, 70%, 60%, 50%, 40%, 30%, or 20% or less of the wild-type control value, but 10% or more. Affinity-modified proteins have altered primary amino acid sequences in their extracellular domain or specific binding sites, resulting from the substitution, addition, or deletion of amino acid residues. The term "affinity-modified" is not construed as imposing any conditions on any particular starting composition or method by which an affinity-modified protein was produced.Therefore, affinity-modified proteins are not limited to wild-type protein domains transformed into affinity-modified domains by any particular process of affinity modification. Affinity-modified domain polypeptides can be generated, for example, starting from wild-type mammalian domain sequence information, then modeled in silico for binding to their binding partners, and finally synthesized recombinantly or chemically, in order to obtain affinity-modified domain compositions of a substance. However, in an alternative example, affinity-modified domains can be produced by site-directed mutagenesis of wild-type domains. Thus, an affinity-modified IgSF domain or an affinity-modified TD domain represents a certain product and does not necessarily represent a product produced by any given process. A variety of techniques, including recombinant methods, chemical synthesis, or combinations thereof, can be used.

[0148] The term "affinity-modified IgSF domain" refers to an affinity-modified domain of an immunoglobulin superfamily (IgSF) protein member that has a modified amino acid sequence of the immunoglobulin domain (e.g., IgV) within the extracellular domain or specific binding region of the IgSF protein (compared to the corresponding wild-type parent domain or unmodified domain) such that binding activity, such as binding affinity to at least one of its binding partners (or "partner structures"), is increased or decreased compared to the parent wild-type or unmodified protein containing a non-affinity-modified IgSF domain or an unmodified IgSF domain.

[0149] The term "affinity-modified TD domain" refers to the affinity-modified domain of a member of the tumor necrosis receptor superfamily (TNFRSF) protein or its TNF ligand, which has a modified amino acid sequence of either the TNFR domain or the TNF domain, respectively. For example, the affinity-modified TD domain of a TNFRSF protein has a modified amino acid sequence of the TNFR domain, which consists of at least one cysteine-rich domain (CRD) within the extracellular domain or specific binding region of the TNFRSF protein, so that binding activity, such as binding affinity to at least one of its binding partners (or "partner structures"), is increased or decreased compared to the parent wild-type or unmodified protein containing a non-affinity-modified TD domain or an unmodified TD domain.

[0150] The term “B cell inhibitor molecule” or BIM refers to a protein molecule that antagonizes or blocks the activity of B cell stimulatory receptors. BIMs antagonize the activity of B cell stimulatory receptors by directly binding to their congener ligands, thereby blocking or reducing the binding between the ligand and the B cell stimulatory receptor. For example, BIMs bind to APRIL and / or BAFF, which are ligands for B cell maturation antigen (BCMA), B cell activator receptor (BAFF-R), and transmembrane activators and calcium modulators and cyclophylline ligand-interactors (TACI). In certain embodiments, the BIMs provided herein include an extracellular domain or a portion thereof containing the TNF receptor superfamily domain (TD, e.g., CRD) of the B cell stimulatory receptor, which binds to the congener ligand APRIL and / or BAFF, as well as to a heterotrimer of APRIL and BAFF. For example, BIM includes the extracellular domain of TACI, or a portion of the extracellular domain of TACI, including the TD domain, which binds to the congeneral ligand APRIL and / or BAFF, and the heterotrimer of APRIL and BAFF. In other examples, BIM includes the extracellular domain of BCMA, or a portion of the extracellular domain of BCMA, including the TD domain, which binds to the congeneral ligand APRIL and / or BAFF, and the heterotrimer of APRIL and BAFF. BIM may also include affinity-modified variants of the extracellular domain or portion of TACI or BCMA, which have another amino acid modification (e.g., amino acid substitution) in the TD domain that increases the binding affinity to the congeneral ligand (e.g., APRIL and / or BAFF, and the heterotrimer of APRIL and BAFF).

[0151] As used herein, “B cell stimulating receptor” refers to one or more of the related tumor necrosis factor (TNFR) superfamily receptors expressed on B cells, including B cell maturation antigens (BCMAs), B cell activator receptors (BAFF-Rs), and transmembrane activators and calcium-modulating and cyclophylline ligand-interactors (TACIs). The involvement or ligation of these related receptors by their congener ligands, BAFF and / or APRIL, or heterotrimers of APRIL and BAFF, modulates B cell homeostasis, including B cell survival, B cell maturation and differentiation, and immunoglobulin class switching. B cell stimulating receptors generally comprise an extracellular portion, a transmembrane domain, and a cytoplasmic domain, the cytoplasmic domain containing one or more TNF receptor-related factor (TRAF) binding sites. Recruitment of various TRAF molecules to the cytoplasmic domain can activate various transcription factors, such as NF-κB (e.g., NF-κB1 or NF-κB2), to mediate B cell signaling pathways that modulate B cell homeostasis.

[0152] As used herein, “to bind,” “bound,” or its grammatical variations thereof, refers to a situation in which one molecule engages in any attractive interaction with another molecule, resulting in a stable association in close proximity to one another. Binding includes, but is not limited to, non-covalent bonds and covalent bonds (such as reversible and irreversible covalent bonds), and includes, but is not limited to, intermolecular interactions of proteins, nucleic acids, carbohydrates, lipids, and small molecules, such as compounds including drugs.

[0153] As used herein, binding activity refers to the characteristics of a molecule, such as a polypeptide, relating to whether it binds to one or more binding partners and how it binds. Binding activity may include any measure of a molecule's binding to a binding partner. Binding activity may include the ability to bind to a binding partner, affinity to the binding partner (e.g., high affinity), avidity to the binding partner, strength of binding to the binding partner, and / or specificity or selectivity for binding to the binding partner.

[0154] As used herein, the term “binding affinity” refers to the specific binding affinity of a protein to its binding partner (i.e., its counterpart structure) under specific binding conditions. Binding affinity refers to the strength of the interaction between two or more molecules, such as binding partners, typically the strength of a non-covalent interaction between two binding partners. The increase or decrease in binding affinity of an affinity-modified domain or an immunomodulatory protein containing an affinity-modified domain is determined by comparison with the binding affinity of the unmodified domain (e.g., a native or wild-type IgSF domain, or a native or wild-type TD domain). Methods for determining binding affinity or relative binding affinity are known in the art and include solid-phase ELISA immunoassays, ForteBio Octet, Biacore measurements, or flow cytometry. See, for example, Larsen et al., American Journal of Transplantation, vol. 5: 443-453 (2005); Linsley et al., Immunity, Vol 1(9): 793-801 (1994). In some embodiments, binding affinity can be measured by flow cytometry, for example, based on mean fluorescence intensity (MFI) in a flow-binding assay.

[0155] As used herein, the term "binding avidity" refers to the specific binding avidity of a protein to its binding partner (i.e., its counterpart structure) under specific binding conditions. In biochemical dynamics, avidity refers to the cumulative strength of multiple affinities between proteins to their respective non-covalent interactions, such as their binding partners (i.e., their counterpart structures). Therefore, avidity is different from affinity, which represents the strength of a single interaction.

[0156] The term “biological half-life” refers to the amount of time it takes for a substance, such as an immunomodulatory protein, to lose half of its pharmacological or physiological activity or concentration. Biological half-life can be affected by the substance’s elimination, excretion, breakdown (e.g., enzymatic breakdown / digestion), or absorption and concentration within a particular organ or tissue of the body. In some embodiments, biological half-life can be assessed by determining the time it takes for the plasma concentration of a substance to reach half of its steady-state level (“plasma half-life”). Conjugates that can be used to derivatize proteins and extend their biological half-lives are known in the art and include, but are not limited to, multimerizing domains (e.g., Fc immunoglobulin domains), polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (elongated recombinant peptide; see International Publication No. 2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylated), and poly-Pro-Ala-Ser (PAS), polyglutamic acid (glutamylated).

[0157] As used herein, the term “cell surface partner” (or “cell surface binding partner”) refers to a partner (or binding partner) expressed on a mammalian cell. Typically, a cell surface binding partner is a transmembrane protein. In some embodiments, a cell surface binding partner is a receptor.

[0158] With respect to proteins such as receptors and soluble ligands, or extracellular domains or parts thereof or affinity-modified variants thereof, the terms “binding partner” or “mutant structure” refer to at least one molecule (typically a native mammalian protein) to which the referenced protein specifically binds under specific binding conditions. In some aspects, an affinity-modified domain, or an immunomodulatory protein containing an affinity-modified domain, specifically binds to the binding partner of the corresponding domain of a native or wild-type protein, but with increased or decreased affinity. A “cell surface binding partner” is a binding partner expressed on a mammalian cell. Typically, a cell surface binding partner is a transmembrane protein. In some aspects, a cell surface binding partner is on cells such as mammalian cells that form immune synapses, e.g., immune cells, and is a receptor or ligand of a receptor expressed by immune cells.

[0159] In relation to binding to cell surface molecules, the term "cis" refers to the binding to two or more different cell surface molecules, each present on the surface of the same cell. In some aspects, cis means that two or more cell surface molecules are exclusively present on one of two mammalian cells forming an IS, or exclusively on the other (but not both).

[0160] As used herein, the term “conservative amino acid substitution” means an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Examples of amino acids with side chains having similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. The conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

[0161] With respect to protein locations, such as when a nucleotide or amino acid position "corresponds" to a nucleotide or amino acid position in a disclosed sequence as listed in a sequence listing, the term "corresponds" refers to a nucleotide or amino acid position identified when aligned with the disclosed sequence based on structural sequence alignment or using a standard alignment algorithm such as the GAP algorithm. By aligning sequences, those skilled in the art can identify corresponding residues, for example, by using conserved identical amino acid residues as guides. Figures 17A and 17B illustrate the identification of corresponding residues by aligning two sequences.

[0162] As used herein, a “domain” (typically a sequence of three or more amino acids, generally five or seven or more, e.g., 10 to 200 amino acid residues) refers to a portion of a molecule, such as a protein or coding nucleic acid, that is structurally and / or functionally distinct from the rest of the molecule and is identifiable. For example, a domain includes a portion of a polypeptide chain composed of one or more structural motifs, capable of forming independently folded structures within a protein, and / or recognized by functional activity such as binding activity. A protein may have one or more distinct domains. For example, a domain may be identified, defined, or distinguished by its primary sequence or structural homology to a related family member, e.g., homology to a motif. In another example, a domain may be distinguished by its function, e.g., its ability to interact with biomolecules such as congenital binding partners. Domains may independently exhibit biological function or activity, such as being able to perform activity such as binding, either independently or when fused to another molecule. A domain may be a linear or nonlinear amino acid sequence. Many polypeptides contain multiple domains. Such domains are known and can be identified by those skilled in the art. Definitions are provided for illustrative purposes in this specification, but it will be understood that recognizing specific domains by name is well within the scope of those skilled in the art. Domains can be identified using appropriate software as needed. References to amino acids containing specific sequences, listed as SEQ ID NOs used to describe domain configurations (e.g., IgSF domain or TD domain), are for illustrative purposes only and are not intended to limit the scope of the embodiments provided. Descriptions of polypeptides and their domains are understood to be theoretically derived based on homology analysis and alignment with similar molecules. Additionally, in some cases, adjacent N-terminal and / or C-terminal amino acids of a given domain (e.g., IgSF domain or TD) may also be included in the sequence, for example, to ensure proper domain folding when expressed.Therefore, the exact gene locus can vary and is not necessarily the same for each protein. For example, a particular IgSF domain, such as a particular IgV or IgC domain, may be several amino acids longer or shorter (1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids). Similarly, a particular TD domain, such as a particular CRD domain, may be several amino acids longer or shorter (1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).

[0163] In this specification, the terms “ectodomain,” “extracellular domain,” or “ECD” are used without distinction and refer to the region of a membrane protein, such as a transmembrane protein, that lies outside the vesicle membrane (e.g., in the space outside the cell) when the full-length form of the membrane protein is expressed from a cell. For the purposes of this specification, references to ECD are understood to refer to sequences and domains that constitute this region and do not require the protein containing the ECD to be a membrane protein or that the domain is located outside the cell. For example, soluble immunomodulatory proteins may contain an ECD sequence of a membrane protein fused to another part, e.g., a multimerization domain, e.g., an Fc region. Ectodomains often interact with specific ligands or specific cell surface receptors, for example, via binding domains that specifically bind to ligands or cell surface receptors. Examples of binding domains include immunoglobulin domains (also called IgD, IgSF domains) or cysteine-rich domains (CRDs). Ectodomains of members of the immunoglobulin superfamily include IgD (e.g., IgV domain). The ectodomains of members of the TNFR superfamily include a TD domain (e.g., a CRD domain). Therefore, references to ECD in this specification include the full-length sequence of the ECD of a membrane protein and its specific binding fragment containing IgD or CRD that binds to a ligand or congenital binding partner.

[0164] The term “effective dose” or “therapeutic effective dose” refers to the amount and / or concentration of a therapeutic composition containing an immunomodulatory protein or Fc fusion protein that, when administered ex vivo (by contact with patient-derived cells) or in vivo (by administration to the patient), for example, by improving or eliminating the symptoms and / or causes of the disease, either alone (i.e., as monotherapy) or in combination with additional therapeutic agents, results in a statistically significant inhibition of disease progression. An effective dose for treating a disease, condition, or disorder, such as an immune system disorder or immune system disorder, may be an amount that reduces, mitigates, or alleviates at least one symptom or biological response or action associated with the disease, condition, or disorder, prevents the progression of the disease, condition, or disorder, or improves the patient’s physical function. In the case of cell therapy, the effective dose is an effective dose or number of cells administered to the patient. In some embodiments, the patient is a human patient.

[0165] As used herein, a fusion protein refers to a polypeptide encoded by a nucleic acid sequence comprising the coding sequences of two or more proteins, possibly two, three, four, five, or more proteins, such that the coding sequences are in the same reading frame so that when the fusion construct is transcribed and translated in a host cell, a protein comprising two or more proteins is produced. Each of the two or more proteins may be adjacent to another protein in the construct, or separated by a linker polypeptide comprising one, two, three, or more, but typically fewer than 20, 15, 10, 9, 8, 7, or 6 amino acids. The protein product encoded by the fusion construct is called a fusion polypeptide. An example of a fusion protein according to the provided embodiment is an Fc fusion protein comprising an affinity modification domain (e.g., a variant or part of BCMA containing a CRD) linked to an immunoglobulin Fc domain.

[0166] The term “half-life extension portion” refers to a portion of a polypeptide fusion or chemical conjugate that extends the half-life of a protein circulating in mammalian serum compared to the half-life of the protein not conjugated with that portion. In some embodiments, the half-life is extended by more than 1.2 times, or about 1.2 times, about 1.5 times, about 2.0 times, about 3.0 times, about 4.0 times, about 5.0 times, or about 6.0 times. In some embodiments, the half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours, or more than 1 week after in vivo administration compared to a protein without a half-life extension portion. Half-life refers to the amount of time it takes for a protein to lose half of its concentration, volume, or activity. Half-life can be determined, for example, by using an ELISA assay or an activity assay. Exemplary half-life extension regions include the Fc domain, the polymerization domain, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptide; see International Publication No. 2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylated), and poly-Pro-Ala-Ser (PAS), and polyglutamic acid (glutamylated).

[0167] The Fc (crystalline fragment) region or Fc (crystalline fragment) domain (also called Fc polypeptide) of an immunoglobulin molecule primarily corresponds to the constant region of the immunoglobulin heavy chain and, in some cases, is responsible for a variety of functions, including antibody effector function. The Fc domain comprises some or all of the hinge domain of the immunoglobulin molecule, along with CH2 and CH3 domains. In some cases, all or part of the Fc hinge sequence may be deleted for inclusion in the provided fusion protein. The Fc domain can form a dimer of two polypeptide chains linked by one or more disulfide bonds. In some embodiments, Fc is a variant Fc exhibiting reduced activity (e.g., reduced by about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) to promote effector function. In some embodiments, references to amino acid substitutions within the Fc region are by the EU numbering system unless otherwise stated based on a specific SEQ ID NO. EU numbering follows the publicly known and most recently updated IMGT Scientific Chart (IMGT®, International ImMunoGeneTics Information System®, http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created: May 17, 2001, last updated: January 10, 2013) and the EU index reported in Kabat, EA et al. Sequences of Proteins of Immunological Interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0168] An immunoglobulin Fc fusion ("Fc fusion"), such as an immunomodulatory Fc fusion protein, is a molecule comprising one or more polypeptides functionally linked to the Fc region of an immunoglobulin. An Fc fusion may, for example, comprise an Fc region functionally linked to the TIM or BIM of a provided immunomodulatory protein. An Fc fusion may, for example, comprise an Fc region functionally linked to the extracellular domain or portion thereof of a BCMA containing a CRD, including one of the affinity variants provided. The immunoglobulin Fc region may be linked indirectly or directly to one or more polypeptides. Various linkers, known in the art, may optionally be used to link Fc to a fusion partner to produce an Fc fusion. Fc fusions of the same species can be dimerized to form an Fc fusion homodimer. Fc fusions of different species (e.g., knob-into-hole operation) may be used to form an Fc fusion heterodimer. In some embodiments, Fc is a mammalian Fc, such as mouse Fc or human Fc.

[0169] The term “host cell” refers to any cell that can be used to express a protein encoded by a recombinant expression vector. Host cells can be prokaryotes, such as Escherichia coli (E. coli), or eukaryotes, such as unicellular eukaryotes (e.g., yeast or other fungi), plant cells (e.g., tobacco or tomato plant cells), animal cells (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells or insect cells), or hybridomas. Examples of host cells include Chinese hamster ovary (CHO) cells or their derivatives growing in serum-free medium, such as the Veggie CHO cell line and related cell lines, or the DHFR-deficient CHO strain DX-B11.

[0170] As used herein, the term “immunological synapse” or “immune synapse” (abbreviated as “IS”) means the interface between mammalian cells expressing MHC I (major histocompatibility complex) or MHC II, such as antigen-presenting cells or tumor cells, and mammalian lymphocytes, such as effector T cells or natural killer (NK) cells.

[0171] As used herein, the term “immunoglobulin” (abbreviated as “Ig”) is synonymous with the term “antibody” (abbreviated as “Ab”) and refers to mammalian immunoglobulin proteins, including any of the five human classes: IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. The term also includes immunoglobulins of less than full length, whether fully or partially synthesized (e.g., recombinant or chemosynthetic) or naturally produced, including any fragment thereof that includes at least a portion of the variable heavy (VH) chain region and / or variable light (VL) chain region of the immunoglobulin molecule, sufficient to form an antigen-binding site and to bind specifically to an antigen when constructed. Antibodies may also include all or part of the constant region. Such fragments include antigen-binding fragments (Fab), variable fragments (Fv) containing VH and VL, single-stranded variable fragments (scFv) containing linked VH and VL on a single chain, and other antibody V-region fragments, such as Fab', F(ab)2, F(ab')2, dsFv diabodies, Fc, and Fd polypeptide fragments. Therefore, references to antibodies in this specification are understood to include full-length antibodies and antigen-binding fragments. The term antibody also includes antibody compositions having polyepitope specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies, and single-stranded molecules. Bispecific antibodies, homo-bispecificity, and hetero-bispecificity are included within the scope of the term. Antibodies include polyclonal antibodies or monoclonal antibodies. Antibodies also include synthetic antibodies or recombinantly produced antibodies. For information on the structures and properties of various classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0172] The terms “full-length antibody,” “intact antibody,” or “whole antibody” are used interchangeably to refer to an antibody in substantially intact form, as opposed to an antibody fragment. A full-length antibody is typically an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4), two full-length light chains (VL-CL), and a hinge region, such as antibodies produced by antibody-secreting B cells from mammalian species (e.g., humans, mice, rats, rabbits, non-human primates, etc.) and synthetically produced antibodies with the same domains. Specifically, whole antibodies include those having heavy and light chains containing an Fc region. The constant domain may be a native sequence constant domain (e.g., the human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

[0173] "Antibody fragment" includes a portion of an intact antibody, the antigen-binding region of an intact antibody, and / or the variable region. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv(dsFv), Fd fragments, Fd' fragments; diabodies; linear antibodies (U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng.). 8(10) See 1057-1062

[1995] ); single-chain antibody molecules containing single-chain Fv (scFv) or single-chain Fab (scFab); containing either of the above antigen-binding fragments and multispecific antibodies derived from the antibody fragment.

[0174] "Fv" consists of one heavy chain variable domain and one light chain variable domain linked by non-covalent bonds. The folding of these two domains yields six complementarity-determining regions (CDRs) (three in each of the heavy and light chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of Fv containing only the three antigen-specific CDRs) can, in some cases, recognize and bind to the antigen, albeit with lower affinity than the entire binding site.

[0175] "dsFv" is V H -V L This refers to Fv having manipulated intermolecular disulfide bonds that stabilize the pair.

[0176] "Fd fragment" is the variable domain (V) of the antibody heavy chain. H ) and one constant region domain (C H This is a fragment of an antibody containing (1).

[0177] A "Fab fragment" is an antibody fragment resulting from the digestion of full-length immunoglobulin by papain, or a fragment with the same structure that is synthetically produced, for example, by recombinant methods. Fab fragments have a light chain (V L and C L (including) and the variable domain of the heavy chain (V H ) and one constant region domain of the heavy chain (C H Includes another chain containing 1).

[0178] An "F(ab')2 fragment" is an antibody fragment resulting from the digestion of immunoglobulins with pepsin at pH 4.0-4.5, or a fragment with the same structure that is synthetically produced, for example, by recombinant methods. An F(ab')2 fragment essentially contains two Fab fragments, each heavy chain portion containing several additional amino acids, including cysteine ​​residues that form a disulfide bond connecting the two fragments.

[0179] A "Fab' fragment" is a fragment that contains half of an F(ab')2 fragment (one heavy chain and one light chain).

[0180] An "Fd' fragment" is an antibody fragment comprising one heavy chain portion of an F(ab')₂ fragment.

[0181] An "Fv' fragment" is the V H domain and V L domain of an antibody molecule, and is a fragment comprising only said domains.

[0182] A "scFv fragment" refers to an antibody fragment comprising a variable light chain (V L ) and a variable heavy chain (V H ) covalently linked by a polypeptide linker in any order. The linker has a length such that the two variable domains can be cross-linked without substantial interference. Exemplary linkers include (Gly-Ser) n residues having several Glu residues or Lys residues dispersed throughout to increase solubility.

[0183] A "diabody" is a dimeric scFv. Diabodies typically have a shorter peptide linker than scFv and preferentially dimerize.

[0184] As used herein, the term “immunoglobulin superfamily” or “IgSF” refers to a group of cell surface proteins and soluble proteins involved in cell recognition, binding, or adhesion processes. Molecules are classified as members of this superfamily based on shared structural features with immunoglobulins (i.e., antibodies). They all possess a domain known as an immunoglobulin domain or immunoglobulin fold. Many “non-antibody IgSF” members include cell surface proteins or receptors that are not antibodies. Members of IgSF include cell surface antigen receptors, co-receptors and costimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules, certain cytokine receptors, and intracellular muscle proteins. They are generally associated with their roles within the immune system. Proteins within immune synapses are often members of IgSF. IgSF can also be classified into “subfamilies” based on shared characteristics such as function. Such subfamilies typically contain 4 to 30 IgSF members.

[0185] As used herein, the terms “IgSF domain,” “immunoglobulin domain,” “Ig domain,” or “IgD” refer to one or more structural domains of the IgSF protein. Ig domains are named after immunoglobulin molecules. They contain approximately 70–110 amino acids and are classified according to their size and function. Ig domains have a characteristic Ig fold with a sandwich-like structure formed by two sheets of antiparallel beta strands. The interaction between the hydrophobic amino acids inside the sandwich and the highly conserved disulfide bond formed between the cysteine ​​residues of the B and F strands stabilizes the Ig fold. In some cases, one end of the Ig domain has a region called a complementarity-determining region, which in some aspects is involved in the specificity of antibodies against their ligands. Ig-like domains can be classified (into classes) as IgV, IgC1, IgC2, or IgI. Most Ig domains are either variable (IgV) or constant (IgC). An IgV domain with nine beta strands is generally longer than an IgC domain with seven beta strands. The Ig domains of some IgSF members are similar to IgV domains in amino acid sequence but similar to IgC domains in size. These are called IgC2 domains, while the standard IgC domain is called an IgC1 domain. A T cell receptor (TCR) chain contains two Ig domains in its extracellular portion: one IgV domain at the N-terminus and one IgC1 domain adjacent to the cell membrane. "Non-antibody IgSF domains" refer to one or more IgSF domains present in non-antibody proteins, typically within the extracellular portion or extracellular domain of a particular cell surface protein. Therefore, the extracellular domain (ECD) of an IgSF family member contains one or more Ig domains. For this reason, the term Ig domain is also used in reference to the ECD of such protein molecules. A reference to a variant IgSF domain (vIgD) refers to a variant or modified sequence of IgD.

[0186] As used herein, the term “immunological activity” refers to the activity of one or more immune cells, such as T cells or B cells, including, for example, activation, cell survival, cell proliferation, cytokine production (e.g., interferon-gamma), cytotoxic activity, or the ability to activate the NF-κB pathway or other signaling cascades resulting in the activation of transcription factors within immune cells. Assays for evaluating the immunomodulatory activity of immunomodulatory proteins may be compared to control proteins with known activity.

[0187] An “immunomodulatory protein” or “immunomodulatory polypeptide” is a protein that modulates immunological activity. To “modulate” an immune response, or to “modulate” an immune response, means that immunological activity is enhanced or suppressed. Such modulation includes any induction of immunological activity of immune cells such as B cells or T cells, or a change in the degree or extent of immunological activity of immune cells such as B cells or T cells, or the suppression of immunological activity of immune cells such as B cells or T cells. For example, a soluble Fc fusion protein as used herein may suppress the immunological activity of B cells, T cells, or both B cells and T cells. Immunomodulatory proteins can be a single polypeptide chain or a multimer (dimer or higher-order multimer) of at least two polypeptide chains covalently linked to each other, for example, by interchain disulfide bonds. Thus, monomeric proteins, dimeric proteins, and higher-order multimeric proteins are within the scope of this defined term. A multimeric protein can be a homomultimer (of the same polypeptide chain) or a heteromultimer (of different polypeptide chains).

[0188] As used herein, modification refers to alteration of the amino acid sequence of a polypeptide or the nucleotide sequence within a nucleic acid molecule, and includes changes in the amino acids or nucleotides of the sequence, respectively. Amino acid modification or change may be a deletion, insertion, or substitution (substitution) of an amino acid or nucleotide, respectively. Methods for modifying polypeptides are common to those skilled in the art, such as by using recombinant DNA methods.

[0189] The term "multimerizing domain" refers to an amino acid sequence that facilitates the formation of a multimer of two or more polypeptides. A multimerizing domain includes sequences that facilitate stable interactions between a polypeptide molecule and one or more additional polypeptide molecules, each containing complementary multimerizing domains (e.g., a first multimerizing domain and a second multimerizing domain), which may be the same or different multimerizing domains. Interactions between complementary multimerizing domains, for example, between the first and second multimerizing domains, form a stable protein-protein interaction to produce a multimer of the polypeptide molecule and the additional polypeptide molecule. In some cases, the multimerizing domains are the same and interact with themselves to form a stable protein-protein interaction between two polypeptide chains. Generally, polypeptides can be directly or indirectly linked to multimerizing domains. Exemplary multimerizing domains include immunoglobulin sequences or parts thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. The multimerization domain may be, for example, an immunoglobulin constant region or domain, such as an IgG-derived Fc domain or a part thereof containing an IgG1 subtype, IgG2 subtype, IgG3 subtype, or IgG4 subtype, IgA, IgE, IgD, and IgM, or modified forms thereof.

[0190] The terms “nucleic acid” and “polynucleotide” are used interchangeably to refer to polymers of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form. Unless specifically limited, the terms encompass nucleic acids that include known analogues of natural nucleotides, possess similar binding properties to natural nucleotides, and are metabolized in a similar manner to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions) and complementary nucleotide sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue. The terms nucleic acid or polynucleotide encompass cDNA or mRNA encoded by genes.

[0191] As used herein, the terms “functional combination,” “functional order,” and “functionally linked” refer to the linking of nucleic acid sequences in a manner or orientation such that the segments are arranged to function in coordination for an intended purpose. In some embodiments, the terms refer to the linking of nucleic acids to produce a nucleic acid molecule capable of leading the transcription of a given gene and / or to produce a functionally desired protein molecule. For example, segments of a DNA sequence, e.g., coding sequences and regulatory sequences, are linked in such a way that gene expression is enabled when an appropriate molecule (e.g., a transcription-activating protein) is bound to the regulatory sequence.

[0192] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in mammals, often in humans. A pharmaceutical composition typically comprises an effective amount of an active substance (e.g., an immunomodulatory protein) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively.

[0193] The terms “polypeptide” and “protein” are used herein without distinction and refer to molecular chains of two or more amino acids linked by peptide bonds. The terms do not refer to a specific length of the product. Therefore, “peptides” and “oligopeptides” are included within the definition of polypeptide. The terms include post-translational modifications of polypeptides, such as glycosylation, acetylation, and phosphorylation. The terms also include molecules that can be synthesized or recombinantly expressed using known protein manipulation techniques, which may include one or more amino acid analogs, or non-canonical or non-natural amino acids. Furthermore, proteins may be derivatized by known organic chemical techniques as described herein.

[0194] For example, the term “purified” applied to nucleic acids or proteins (e.g., immunomodulatory proteins) encoding immunomodulatory proteins generally indicates a nucleic acid or polypeptide that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., purified polypeptides or polynucleotides form distinct bands in electrophoretic gels, chromatographic eluents, and / or media subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that essentially produces one band in an electrophoretic gel is “purified.” Purified nucleic acids or proteins are at least about 50% pure, and typically at least about 75%, 80%, 85%, 90%, 95%, 96%, 99%, or more pure (e.g., by weight percentage or molar basis).

[0195] The term “recombinant” indicates that a material (e.g., nucleic acid or polypeptide) is artificially (i.e., unnaturally) modified by human intervention. The modification may be made to the material within its natural environment or state, or removed from its natural environment or state. For example, “recombinant nucleic acid” is produced, for example, by recombining nucleic acids during cloning, affinity modification, DNA shuffling, or other well-known molecular biological procedures. “Recombinant DNA molecule” consists of DNA segments linked together by such molecular biological techniques. As used herein, the terms “recombinant protein” or “recombinant polypeptide” refer to protein molecules (e.g., immunomodulatory proteins) expressed using recombinant DNA molecules. “Recombinant host cell” is a cell that contains and / or expresses recombinant nucleic acid, or has been genetically modified, such as by introducing nucleic acid molecules encoding recombinant proteins, such as immunomodulatory proteins provided herein. In eukaryotes, transcriptional regulatory signals include “promoter” elements and “enhancer” elements. Promoter and enhancer consist of short arrays of DNA sequences that specifically interact with cellular proteins involved in transcription. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including genes in yeast, insect, and mammalian cells, as well as viruses (similar regulatory elements, i.e., promoters, are also found in prokaryotes). The choice of specific promoters and enhancers depends on which cell type is used to express the protein of interest.

[0196] As used herein, the term “recombinant expression vector” refers to a DNA molecule comprising a desired coding sequence (e.g., encoding an immunomodulatory protein) and appropriate nucleic acid sequences necessary for the expression of the functionally linked coding sequence within a particular cell. In prokaryotes, the nucleic acid sequences necessary for expression include a promoter, optionally an operator sequence, a ribosome binding site, and optionally other sequences. In eukaryotic cells, it is known that promoters, enhancers, and termination and polyadenylation signals are utilized. Secretory signal peptide sequences may also be optionally encoded by a recombinant expression vector and functionally linked to the coding sequence, for example, for their expression as a secretible protein if desired, or for easier isolation or purification of the immunomodulatory protein from the cell, so that the expressed protein can be secreted by a recombinant host cell. The term includes vectors as self-replicating nucleic acid structures, as well as vectors that are integrated into the genome of the host cell into which they are introduced. Among vectors are viral vectors, such as lentiviral vectors.

[0197] As used herein, the term “sequence identity” refers to sequence identity between genes or proteins at the nucleotide level or amino acid level, respectively. “Sequence identity” is a measure of identity between proteins at the amino acid level and between nucleic acids at the nucleotide level. Protein sequence identity can be determined by comparing the amino acid sequences at a given position within each sequence when the sequences are aligned. Similarly, nucleic acid sequence identity can be determined by comparing the nucleotide sequences at a given position within each sequence when the sequences are aligned. Methods for aligning sequences for comparison are well known in the art, and such methods include GAP, BESTFIT, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software, FASTA, and TFASTA. The BLAST algorithm calculates percent sequence identity and performs a statistical analysis of the similarity between two sequences. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website. In some cases, percentage sequence identity may be determined as the percentage of amino acid residues (or nucleotide residues) in a candidate sequence that are identical to amino acid residues (or nucleotide residues) in a reference sequence, after the sequences have been aligned and gaps introduced as necessary to achieve maximum percentage sequence identity. References to sequence identity include sequence identity across the entire length of each sequence being compared. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve maximum alignment across the entire length of the sequences being compared.

[0198] As used herein with respect to proteins, the term "soluble" means that a protein is not a membrane protein or is not fixed to the cell membrane. A protein may be constructed as a soluble protein by containing only an extracellular domain or a portion thereof and not a transmembrane domain. In some cases, the solubility of a protein may be improved by linking or binding, directly or indirectly via linkers, to an Fc domain or other half-life-extending molecules, which may also improve the stability and / or half-life of the protein. In some aspects, a soluble protein is an Fc-fusion protein.

[0199] As used herein, the term “specifically binds” means the ability of a protein to bind to a target protein such that, under specific binding conditions, its affinity or avidity is at least 10 times, but optionally 50, 100, 250, or 500 times, or even at least 1000 times, the average affinity or avidity of the same protein to a sufficiently statistically sized aggregate of random peptides or polypeptides. A specifically binding protein does not need to bind exclusively to a single target molecule, but may bind specifically to multiple target molecules. In some cases, a specifically binding protein may bind to a protein (e.g., a paralog or ortholog) that has a structural conformation similar to that of the target protein. Those skilled in the art will recognize that specific binding to molecules with the same function in different animal species (i.e., orthologs) or to molecules with substantially similar epitopes to the target molecule (e.g., paralogs) is possible without compromising the specificity of the binding determined compared to a unique non-target, statistically effective aggregate (e.g., random polypeptide). Therefore, the immunomodulatory proteins of the present invention or their BIM or TIM can specifically bind to multiple different species of target molecules due to cross-reactivity. Specific binding between two proteins can be determined using solid-phase ELISA immunoassay, ForteBio Octet, or Biacore measurements. Generally, the interaction between two binding proteins is approximately 1 × 10⁻⁶. -5Less than M, often about 1 × 10⁻⁶ -12 It has a low dissociation constant (Kd) of M. In certain aspects of this disclosure, the interaction between the two binding proteins is approximately 1 × 10⁻⁶. -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M or 1x10 -11 It has a dissociation constant of M or less.

[0200] As used herein with respect to proteins, the terms “specific binding fragment” or “fragment” mean a polypeptide that is shorter than the full-length protein or its specific domain or region, and that specifically binds in vitro and / or in vivo to the binding partner of the full-length protein or its specific domain or region. A specific binding fragment relates to a fragment of the full-length extracellular domain of a polypeptide or the binding domain of a polypeptide, but still binds to the binding partner of the binding domain. For example, a specific binding fragment relates to a fragment of the full-length extracellular domain of an IgSF family member or its full-length IgSF domain (e.g., IgV or IgC), but still binds to the binding partner of an IgSF family member, or the binding partner of an IgSF domain of an IgSF family member. In another example, a specific binding fragment relates to a fragment of the extracellular domain of a full-length TNFR family member or its full-length TNFR domain (TD) (e.g., CRD), but still binds to the binding partner of a TNFR family member, or the binding partner of a CRD of a TNFR family member. In some embodiments, the specific binding fragment is the full-length sequence of the extracellular domain, or at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the domain or region of the extracellular domain. In some embodiments, the specific binding fragment may have an amino acid length of at least 50 amino acids, for example, at least 60, 70, 80, 90, 100, or 110 amino acids.

[0201] As used herein, “Subject” refers to a mammal such as a human or other animal, typically a human. A subject may be male or female and may be of any suitable age, including infants, young people, adolescents, adults, and elderly subjects.

[0202] As used herein, for example, with respect to synthetic nucleic acid molecules, synthetic genes, or synthetic peptides, “synthetic” refers to nucleic acid molecules or polypeptide molecules produced by recombinant and / or chemical synthesis methods.

[0203] As used herein, the term “TNF receptor superfamily” or “TNFRSF” refers to a group of cell surface cytokine receptors, all of which are type I (N-terminal extracellular) transmembrane glycoproteins, containing 1 to 6 cysteine-rich domains (CRDs) in their extracellular domains. Molecules are classified as members of this superfamily based on a shared structural feature that includes one or more cysteine-rich domains (CRDs) present in their N-terminal extracellular region, often playing some role in the protein binding of their congeneral binding partners or ligands. TNFRSF proteins may have one or several CRDs (e.g., CRD1, CRD2, etc.). Typically, the ECD or ectodomain of a TNFRSF member contains 1 to 6 pseudo-repeats of a CRD. For example, the BAFF receptor and BCMA each contain one CRD, while TACI contains two CRDs (CRD1 and CRD2). TNFRSF members are usually trimer complexes or multimer complexes stabilized by their cysteine-disulfide bonds. When TNFRSF proteins bind to their ligands, they promote various intracellular biological activities, such as apoptosis (programmed cell death) or the induction of cell survival and proliferation.

[0204] The term "TD" refers to one or more structural domains of a TNFRSF protein or TNF family ligand. For example, the TD of a TNFRSF protein is a cysteine-rich domain (CRD) module of approximately 40 amino acids containing six conserved cysteines. Therefore, references to CRDs can also be used interchangeably with the term TD in relation to the TD of a TNFRSF protein. The six cysteines are involved in the formation of intrachain disulfide bonds. The extracellular domain (ECD) of a TNFRSF member contains one or more CRD domains. Therefore, the term TD is also used in relation to the ECD of such a protein molecule. A reference to variant TD (vTD) refers to a variant or modified sequence of TD.

[0205] The term “T cell inhibitory molecule” or TIM refers to a protein molecule that antagonizes or blocks the activity of a T cell stimulating receptor. A TIM can antagonize the activity of a T cell stimulating receptor by directly binding to the T cell stimulating receptor or a ligand of the T cell stimulating receptor, thereby blocking or reducing the binding between the ligand and the T cell stimulating receptor. For example, a TIM can antagonize or inhibit the activity of a T cell costimulatory receptor such as CD28. In certain embodiments, the TIMs provided herein include an extracellular domain or a portion thereof containing an immunoglobulin superfamily (IgSF) domain, e.g., an IgV domain, of a T cell stimulating receptor homologous ligand. For example, a TIM includes the extracellular domain of CTLA-4, or a portion of the extracellular domain of CTLA-4 containing an IgSF domain (e.g., an IgV domain) that binds to a T cell stimulating receptor (e.g., CD28). TIM may also include affinity-modified variants of the extracellular domain or part thereof of a T cell-stimulating receptor, such as CTLA-4, which have another amino acid modification (e.g., amino acid substitution) in the IgSF domain that increases the binding affinity to the T cell-stimulating receptor (e.g., CD28).

[0206] As used herein, “T cell-stimulating receptor” refers to a cell surface molecule expressed on a T cell whose involvement or ligation results in the direct or indirect activation of one or more intracellular tyrosine kinases and / or the induction or enhancement of the effector cell function of one or more T cells on which it is expressed. A T cell-stimulating receptor generally comprises an extracellular component, a transmembrane domain, and a cytoplasmic domain. In some embodiments, the cytoplasmic domain includes an immunoreceptor-activated tyrosine motif (ITAM; defined by the sequence YXX(L / I)X6-8YXX(L / I)) or, otherwise, an intracellular signaling domain that can interact with or associate with one or more accessory proteins, e.g., one or more adapter proteins, that are involved in or modulate tyrosine phosphorylation in signaling pathways. In some cases, T cell-stimulating receptors interact with or associate with adapter proteins containing ITAMs, or with one or more protein-binding domains that bind to specific amino acid sequences within proteins in the signaling pathway, such as phosphotyrosine residues, e.g., Src homology 2 (SH2) and SH3 domains. Examples of adapter proteins include, but are not limited to, Lck, Fyn, ZAP70, SLP76, PI3K, Grb2, PKCθ, and SHC1. Thus, it is understood that T cell-stimulating receptors themselves do not need to possess intrinsic enzymatic activity, but can indirectly mediate enzymatic activity via accessory or adapter proteins. Typically, the involvement of T cell-stimulating receptors initiates, mediates, or enhances T cell activation, resulting in measurable morphological, phenotypic, and / or functional changes in T cells, including cell proliferation, cytolytic activity, cytokine production or secretion, or the expression of cell surface molecules such as receptors or adhesion molecules. In some embodiments, T cell stimulating receptors include T cell receptors (TCRs), CD3, CD4, CD8, CD28, ICOS, or CD2. For example, the T cell stimulating receptor is a co-stimulatory receptor such as CD28.

[0207] In relation to binding to cell surface molecules, the term "trans" refers to binding to two different cell surface molecules, each present on the surface of a different cell. In some embodiments, trans means that, with respect to two different cell surface molecules, the first is exclusively present on one of two mammalian cells forming an IS, and the second is exclusively present on the other of the two mammalian cells forming the IS.

[0208] As used herein, the term “transmembrane protein” refers to a membrane protein that substantially or completely penetrates a lipid bilayer, such as a biological membrane, such as a mammalian cell membrane, or a lipid bilayer, such as those found in artificial constructs like liposomes. A transmembrane protein contains a transmembrane domain ("transmembrane domain") that incorporates the transmembrane protein into the lipid bilayer and makes this integration thermodynamically stable under physiological conditions. The transmembrane domain is generally predictable from its amino acid sequence via any number of commercially available bioinformatics software applications, based on its higher hydrophobicity compared to the protein region that interacts with aqueous environments (e.g., cytosol, extracellular fluid). The transmembrane domain is often a hydrophobic α-helix that penetrates the membrane. A transmembrane protein can penetrate both layers of a lipid bilayer once or multiple times.

[0209] As used herein, the terms “treating,” “treatment,” or “therapy” of a disease, condition, or disorder mean slowing, stopping, or reversing the progression of a disease or disorder by administering an immunomodulatory protein or the manipulated cells of the present invention alone or in combination with another compound described herein, as evidenced by a reduction, cessation, or elimination of any clinical or diagnostic symptom. “Treatment,” “treatment,” or “therapy” also means a reduction in the severity of symptoms in an acute or chronic disease, condition, or disorder, or a reduction in the relapse rate, such as in the course of an autoimmune disease or inflammatory state that relapses or goes into remission, or a reduction in inflammation, such as in the inflammatory phase of an autoimmune disease or inflammatory state. As used in the context of the present invention, “preventing,” “prophylaxis,” or “prevention” of a disease or disorder refers to administering an immunomodulatory protein alone or in combination with another compound to prevent the onset or development of some or all of the symptoms of a disease or disorder, or to reduce the likelihood of the development of a disease, condition, or disorder.

[0210] The term “variant” (and may also be used interchangeably with “modified” or “mutant”) as used in relation to variant proteins or variant polypeptides refers to proteins, such as mammalian (e.g., human or mouse) proteins, that have been produced by human intervention. A variant is a polypeptide having an altered or modified amino acid sequence compared to an unmodified or wild-type protein or its domain, such as by one or more amino acid substitutions, deletions, additions, or combinations thereof. Variant polypeptides may include differences of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids, such as amino acid substitutions. Variant polypeptides generally exhibit at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with respect to the corresponding form of the wild-type or unmodified protein, e.g., its mature sequence (lacking a signal sequence) including the extracellular domain or its binding domain, or a portion thereof. Amino acids that do not exist naturally and naturally occurring amino acids are included within a range of acceptable substitutions or additions. Variant proteins are not limited to any particular method of production, including, for example, chemical synthesis, recombinant DNA technology, or a combination thereof. The variant proteins of the present invention specifically bind to at least one or more binding partners. In some embodiments, the modified amino acid sequence results in modified (i.e., increased or decreased) binding activity, e.g., binding affinity or binding affinity, to one or more binding partners. Thus, variant proteins may be "affinity-modified" proteins as described herein.

[0211] In this specification, the terms “wild-type” or “natural” or “native” are used interchangeably and are applied to biological materials such as nucleic acid molecules, proteins, and host cells that are found naturally and have not been modified by human intervention.

[0212] II. BCMA Immunomodulatory Proteins and Variant BCMA Polypeptides BCMA immunomodulatory proteins are provided herein, comprising a portion of the extracellular domain (ECD) or a variant thereof of the BCMA receptor that binds to at least one BCMA congenital binding partner. Variant BCMA polypeptides exhibiting modified (e.g., increased) binding activity or affinity for one or more of the BCMA congenital binding partners are also provided herein. In some embodiments, the BCMA congenital binding partners are one or more of BAFF or APRIL, or a BAFF / APRIL heterotrimer. The BCMA immunomodulatory proteins and BCMA immunomodulatory polypeptides provided comprise a soluble fusion protein in which the BCMA portion of the extracellular domain or a variant thereof is linked to another portion, e.g., immunoglobulin Fc or other multimerizing domain or half-life extension portion. Thus, in some embodiments, the immunomodulatory protein is a BCMA-Fc fusion protein. In some embodiments, a BCMA-Fc fusion protein is provided, comprising (1) a BCMA polypeptide or a variant BCMA polypeptide consisting of the extracellular domain or a portion thereof of a BCMA receptor, which binds to at least one BCMA homologous binding partner, and (2) an Fc domain. The BCMA polypeptide or variant BCMA polypeptide may be linked directly or indirectly (e.g., via a peptide linker) to the Fc domain.

[0213] BCMA is a tumor necrosis factor receptor family member characterized by having an extracellular domain (ECD) containing a cysteine-rich pseudo-repeating domain (CRD). BCMA is a membrane-bound receptor having an extracellular domain containing a single CRD, a transmembrane domain, and a cytoplasmic domain containing a TRAF-binding site for binding to TRAF signaling molecules. BCMA binds to its congener ligands APRIL and BAFF, but its affinity for BAFF is weaker. BCMA has been reported to bind to BAFF with a binding strength 2-3 orders of magnitude weaker than its binding to other congener receptors, BAFF-R and TACI (Bossen and Schneider et al. 2006 Seminars in Immunology, 18:263-75).

[0214] The amino acid sequence of full-length BCMA is described in SEQ ID NO:667. This protein is a type II membrane protein and lacks a signal peptide. After expression in eukaryotic cells, the N-terminal methionine is removed. In some embodiments, the mature BCMA protein does not contain the N-terminal methionine described in SEQ ID NO:667. The extracellular domain of BCMA (amino acid residues 1-54 of SEQ ID NO:667; ECD described in SEQ ID NO:710) contains a single cysteine-rich domain (CRD, also called the tumor necrosis family receptor domain or TD) that exhibits affinity for binding to APRIL and, to a lesser extent, affinity for binding to BAFF. The CRD contains amino acid residues 7-41 of the sequence described in SEQ ID NO:710.

[0215] In some embodiments, the variant BCMA polypeptides provided herein include one or more amino acid modifications, such as substitutions (or "mutations" or "replacements"), deletions, or additions, within the extracellular domain of a reference BCMA polypeptide, e.g., a wild-type BCMA polypeptide containing a CRD (hereinafter also referred to as TD) or an unmodified BCMA polypeptide. Thus, the provided variant BCMA polypeptides are or include a variant TD ("vTD") in which one or more amino acid modifications (e.g., substitutions) are located in the CRD.

[0216] In some embodiments, the reference (e.g., unmodified) BCMA sequence is either a wild-type BCMA sequence or a portion thereof including the CRD. In some embodiments, the reference (e.g., unmodified) BCMA is either the extracellular domain (ECD) of BCMA or a portion thereof including the CRD, or includes it. In some embodiments, the variant BCMA polypeptide includes or is essentially derived from the CRD or its specific binding fragment. In some embodiments, the variant BCMA is a soluble polypeptide lacking a transmembrane domain. In some embodiments, the variant BCMA polypeptide further includes a transmembrane domain and, possibly, a cytoplasmic domain.

[0217] In some embodiments, the reference (e.g., unmodified) BCMA sequence is a mammalian BCMA sequence. In some embodiments, the reference (e.g., unmodified) BCMA sequence may be a mammalian BCMA including, but not limited to, human, mouse, cynomolgus monkey, or rat. In some embodiments, the reference (e.g., unmodified) BCMA sequence is human. An example of the extracellular domain of a human BCMA sequence is described in SEQ ID NO:710.

[0218] In some embodiments, the reference (e.g., unmodified) BCMA sequence has (i) the amino acid sequence described in SEQ ID NO:710, or the same sequence lacking the N-terminal methionine; (ii) an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with respect to SEQ ID NO:710, and which binds to APRIL or BAFF; or (iii) is a fragment or portion of (i) or (ii) containing a CRD, the fragment or portion of (i) or (ii) which binds to APRIL or BAFF. In some embodiments, the reference (e.g., unmodified) BCMA sequence lacks the N-terminal methionine described in SEQ ID NO:710. BCMA extracellular domain (ECD): SEQ ID NO: 710 TIFF2026143547000009.tif4139

[0219] In some embodiments, the reference (e.g., unmodified) BCMA sequence lacks the N-terminal methionine described in SEQ ID NO:710. In some embodiments, the reference (e.g., unmodified) BCMA sequence has (i) the amino acid sequence described in SEQ ID NO:356; (ii) an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with respect to SEQ ID NO:356 and binds to APRIL or BAFF; or (iii) is a fragment or portion of (i) or (ii) containing a CRD, the portion of which binds to APRIL or BAFF. BCMA extracellular domain (ECD): SEQ ID NO: 356 TIFF2026143547000010.tif4135

[0220] Among the BCMA polypeptides provided are variant BCMA polypeptides. Immunomodulatory proteins containing the provided variant BCMA polypeptide, such as BCMA-Fc fusion proteins, are also provided. In any of the embodiments provided, the variant BCMA sequence has the sequence of a reference (e.g., unmodified) BCMA sequence, e.g., one of the above, but further comprises another amino acid modification, e.g., one or more amino acid substitutions. In particular, variant BCMA polypeptides are provided herein that include at least one affinity-modified TD domain (CRD) or specific binding fragment containing one or more amino acid substitutions in the TD domain of a reference (e.g., unmodified or wild-type) BCMA polypeptide, such that the variant BCMA polypeptide exhibits modified (e.g., increased) binding activity or binding affinity to one or both APRIL or BAFF compared to the reference (e.g., unmodified or wild-type) BCMA polypeptide. In some embodiments, the variant BCMA polypeptide has a binding affinity to APRIL and / or BAFF that differs from the binding affinity to a reference (e.g., unmodified or wild-type) BCMA polypeptide control sequence, as determined, for example, by solid-phase ELISA immunoassay, flow cytometry, or Biacore assay. The binding affinity to each of the congeneral binding partners is independent. That is, in some embodiments, the variant BCMA polypeptide has an increased binding affinity to one or both APRIL and BAFF, and a decreased or unchanged binding affinity to the other of APRIL or BAFF, compared to the reference (e.g., unmodified or wild-type) BCMA polypeptide.

[0221] In some embodiments, the variant BCMA polypeptide has increased binding affinity to BAFF compared to the reference (unmodified or wild-type) BCMA polypeptide. In some embodiments, the variant BCMA polypeptide has increased binding affinity to APRIL compared to the reference (unmodified or wild-type) BCMA polypeptide. In some embodiments, the variant BCMA polypeptide has increased binding affinity to both APRIL and BAFF compared to the reference (unmodified or wild-type) BCMA polypeptide. The congener ligands BAFF and / or APRIL may be mammalian proteins, e.g., human or mouse proteins. In some embodiments, the variant BCMA polypeptide having increased or enhanced binding affinity to APRIL and / or BAFF has an increase in binding affinity of at least about 5%, e.g., at least about 10%, 15%, 20%, 25%, 35%, or 50%, compared to the reference (e.g., unmodified or wild-type) BCMA polypeptide control. In some embodiments, the increase in binding affinity compared to a reference (e.g., unmodified or wild-type) BCMA polypeptide is greater than 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 times. In any of the examples, the reference (e.g., unmodified or wild-type) BCMA polypeptide has the same sequence as the variant BCMA polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0222] In some embodiments, the equilibrium dissociation constant (K) of any of the embodiments described above is used for BAFF. d ) is 1 × 10 -5 M, 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M or 1×10 -11 M, or 1 × 10 -12 M may be less than M. In some embodiments, K of any of the above embodiments relative to BAFF d is 1 × 10 -9 M, 1×10-10 M or 1x10 -11 M, or 1 x 10 -12 Less than M, or approximately 1 × 10 -9 M, 1×10 -10 M or 1x10 -11 M, or 1 x 10 -12 It is less than M. In some embodiments, K of any of the above embodiments relative to BAFF d is 1 × 10 -9 From M, 1 x 10 -12 M or approximately 1 x 10 -12 M is M. In some embodiments, K is one of the aforementioned embodiments relative to BAFF. d is 1 × 10 -9 M or approximately 1 x 10 -9 M, 2×10 -9 M or approximately 2 x 10 -9 M, 4×10 -9 M or approximately 4 x 10 -9 M, 6×10 -9 M or approximately 6 x 10 -9 M, 8×10 -9 M or approximately 8 x 10 -9 M, 1×10 -10 M or approximately 1 x 10 -10 M, 2×10 -10 M or approximately 2 x 10 -10 M, 4×10 -10 M or approximately 4 x 10 -10 M, 6×10 -10 M or approximately 6 x 10 -10 M, 8×10 -10 M or approximately 8 x 10 -10 M, 1×10 -11 M or approximately 1 x 10 -11 M, 2×10 -11 M or approximately 2 x 10 -11 M, 4×10 -11 M or approximately 4 x 10 -11 M, 6×10 -11 M or approximately 6 x 10 -11 M, 8×10 -11 M or approximately 8 x 10 -11 M, or 1 x 10 -12 M or approximately 1 x 10 -12M, or any value between any of the foregoing. In some aspects, the provided aspects comprise the variant BCMA polypeptide described above, and K for BAFF d is decreased (increased binding affinity) by more than 1.5-fold, or more than about 1.5-fold, for example 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more, or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more.

[0223] In some aspects, the equilibrium dissociation constant (K d ) for APRIL according to any of the foregoing aspects is 1×10 -5 M, 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M or 1×10 -11 M, or may be less than 1×10 -12 M. In some aspects, K for APRIL according to any of the foregoing aspects d is 1×10 -9 M, 1×10 -10 M or 1×10 -11 M, or less than 1×10 -12 M, or about 1×10 -9 M, 1×10 -10 M or 1×10 -11 M, or less than 1×10 -12 M. In some aspects, K for APRIL according to any of the foregoing aspects d is from 1×10 -9 M to 1×10 -12 M or about 1×10 -12 M. In some aspects, K for APRIL according to any of the foregoing aspects d is 1×10 -9 M or about 1×10 -9 M, 2×10 -9 M or about 2×10 -9 M, 4×10 -9 M or about 4×10 -9 M, 6×10 -9 M or about 6×10-9 M, 8×10 -9 M or approximately 8 x 10 -9 M, 1×10 -10 M or approximately 1 x 10 -10 M, 2×10 -10 M or approximately 2 x 10 -10 M, 4×10 -10 M or approximately 4 x 10 -10 M, 6×10 -10 M or approximately 6 x 10 -10 M, 8×10 -10 M or approximately 8 x 10 -10 M, 1×10 -11 M or approximately 1 x 10 -11 M, 2×10 -11 M or approximately 2 x 10 -11 M, 4×10 -11 M or approximately 4 x 10 -11 M, 6×10 -11 M or approximately 6 x 10 -11 M, 8×10 -11 M or approximately 8 x 10 -11 M, or 1 x 10 -12 M or approximately 1 x 10 -12 M is any value between any of the above. In some embodiments, the provided embodiment comprises the above variant BCMA polypeptide and K relative to APRIL. d The binding affinity decreases by more than 1.5 times, or more than approximately 1.5 times, for example, more than 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more, or by approximately 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more (increase in binding affinity).

[0224] A reference (e.g., unmodified or wild-type) BCMA sequence does not necessarily have to be used as a starting composition for generating the variant BCMA polypeptides described herein. Therefore, the use of the term “modified,” e.g., “substitution,” does not imply that this embodiment is limited to a specific method for producing variant BCMA polypeptides or immunomodulatory proteins containing them. Variant BCMA polypeptides can be produced, for example, by de novo peptide synthesis, and therefore do not necessarily require modifications such as “substitution,” meaning altering codons to encode substitutions. This principle also extends to the terms “addition” and “deletion” of amino acid residues, which similarly do not imply a specific method of production. The means for designing or producing variant BCMA polypeptides are not limited to a specific method. However, in some embodiments, a reference (e.g., unmodified or wild-type) BCMA coding nucleic acid is mutagenicated from a reference (e.g., unmodified or wild-type) BCMA genetic material and screened for desired specific binding affinity or other functional activity. In some embodiments, variant BCMA polypeptides are synthesized de novo using protein or nucleic acid sequences available in any number of publicly available databases and then screened. The National Center for Biotechnology Information provides such information, and its website is publicly accessible via the internet, as is the UniProtKB database mentioned above.

[0225] Unless otherwise specified, as shown throughout this disclosure, amino acid modifications within variant BCMA polypeptides are designated by amino acid position numbers corresponding to the position numbering of the reference ECD sequence listed in SEQ ID NO:710. Identifying the corresponding positions of modifications within a BCMA polypeptide, including its TD(CRD), such as amino acid substitutions, by, for example, alignment of a reference sequence (e.g., SEQ ID NO:356) with SEQ ID NO:710, is within the scope of the art. An example of an alignment for identifying corresponding residues is shown in Figure 17B. In the list of modifications throughout this disclosure, amino acid positions are shown in the center, with the corresponding reference (e.g., unmodified or wild-type) amino acids listed before the number, and the identified variant amino acid substitutions listed after the number. If the modification is a deletion at that position, "del" is indicated; if the modification is an insertion at that position, "ins" is indicated. In some cases, insertions are listed with their centrally located amino acid positions, the corresponding reference amino acids are listed before and after the numbers, and identified variant amino acid insertions are listed after the unmodified (e.g., wild-type) amino acids.

[0226] In some embodiments, the variant BCMA polypeptide has one or more amino acid modifications, e.g., substitutions within a reference (e.g., unmodified or wild-type) BCMA sequence, such as any of those described. The one or more amino acid modifications, e.g., substitutions, may be located within the ectodomain (extracellular domain) of the reference (e.g., unmodified or wild-type) BCMA sequence. In some embodiments, the one or more amino acid modifications, e.g., substitutions, are located within the CRD domain or its specific binding fragment.

[0227] In some embodiments, the variant BCMA polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, e.g., substitutions. The modifications, e.g., substitutions, may be present within the CRD. In some embodiments, the variant BCMA polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions within the CRD or its specific binding fragment. In some embodiments, a variant BCMA polypeptide comprising one or more described amino acid modifications (e.g., amino acid substitutions) has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a reference (e.g., unmodified or wild-type) BCMA polypeptide or its specific binding fragment having the amino acid sequence of SEQ ID NO: 710 or 356. In some embodiments, a variant BCMA polypeptide comprising one or more described amino acid modifications (e.g., amino acid substitutions) has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO:356.

[0228] In some embodiments, the variant BCMA polypeptide has one or more amino acid modifications, such as substitutions, on a reference BCMA polypeptide or its specific binding fragment corresponding to positions 9, 10, 11, 14, 16, 19, 20, 22, 25, 27, 29, 30, 31, 32, 35, 36, 39, 43, 45, 46, 47, or 48, based on the numbering of SEQ ID NO:710. In some embodiments, the variant BCMA polypeptide has one or more amino acid modifications, for example, It has substitutions selected from TIFF2026143547000011.tif44164, or the conserved amino acid substitutions thereof. In some embodiments, the reference BCMA polypeptide is described in SEQ ID NO:356.

[0229] Conservative amino acid modifications, such as substitutions, are any amino acid in the same class as the substituted amino acid, other than the reference (e.g., unmodified) or wild-type amino acid. The classes of amino acids are aliphatic (glycine, alanine, valine, leucine, and isoleucine), hydroxyl or sulfur-containing (serine, cysteine, threonine, and methionine), cyclic (proline), aromatic (phenylalanine, tyrosine, and tryptophan), basic (histidine, lysine, and arginine), and acidic / amide (aspartate, glutamate, asparagine, and glutamine).

[0230] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 9. In some embodiments, the at least one amino acid substitution is S9G, S9N, or S9Y.

[0231] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 10. In some embodiments, the at least one amino acid substitution is Q10E, Q10P.

[0232] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 11. In some embodiments, the at least one amino acid substitution is N11D, N11S.

[0233] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 19. In some embodiments, the at least one amino acid substitution is H19A, H19C, H19D, H19E, H19F, H19G, H19I, H19K, H19L, H19M, H19N, H19P, H19Q, H19R, H19S, H19T, H19V, H19W, or H19Y. In some embodiments, the at least one amino acid substitution is H19L. In some embodiments, the at least one amino acid substitution is H19K. In some embodiments, the at least one amino acid substitution is H19Q. In some embodiments, the at least one amino acid substitution is H19R. In some embodiments, the at least one amino acid substitution is H19Y.

[0234] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 22. In some embodiments, the at least one amino acid substitution is I22L, I22V.

[0235] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 25. In some embodiments, the at least one amino acid substitution is Q25E, Q25F, Q25G, Q25H, Q25I, Q25K, Q25L, Q25M, Q25S, Q25V, or Q25Y.

[0236] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 27. In some embodiments, the at least one amino acid substitution is R27H, R27L.

[0237] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 30. In some embodiments, the at least one amino acid substitution is S30G, S30Y.

[0238] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 31. In some embodiments, the at least one amino acid substitution is N31D, N31G, N31H, N31K, N31L, N31M, N31P, N31S, N31V, or N31Y.

[0239] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 32. In some embodiments, the at least one amino acid substitution is T32I, T32S.

[0240] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 35. In some embodiments, the at least one amino acid substitution is L35A, L35M, L35P, L35S, L35V, or L35Y.

[0241] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 36. In some embodiments, the at least one amino acid substitution is T36A, T36G, T36N, T36M, T36S, or T36V.

[0242] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 39. In some embodiments, the at least one amino acid substitution is R39L, R39Q.

[0243] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 43. In some embodiments, the at least one amino acid substitution is A43E, A43S.

[0244] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 45. In some embodiments, the at least one amino acid substitution is V45A, V45D, or V45I.

[0245] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 46. In some embodiments, the at least one amino acid substitution is T46A, T46I.

[0246] In some embodiments, the variant BCMA polypeptide contains at least one amino acid substitution at position 47. In some embodiments, the at least one amino acid substitution is N47D, N47Y.

[0247] In some embodiments, one or more amino acid substitutions include S16A / H19Y / R39Q.

[0248] In some embodiments, variant BCMA polypeptides contain any of the mutations listed in Table 1. Table 1 also presents exemplary sequences by referencing the SEQ ID NOs of the reference (e.g., unmodified) BCMA polypeptide and the exemplary variant BCMA polypeptide. As shown, the exact locus or residues corresponding to a given domain may vary, for example, depending on the method used to identify or classify that domain. Also, in some cases, the adjacent N-terminal and / or C-terminal amino acids of a given domain (e.g., CRD) may also be included in the sequence of the variant BCMA polypeptide, for example, to ensure proper folding of the domain when expressed. It is understood that the examples of SEQ ID NOs in Table 1 should not be interpreted as limiting. For example, a particular domain of a variant BCMA polypeptide, e.g., the ECD domain, or a portion thereof containing CRD1 / CRD2, or CRD2 only, may be several amino acids longer or shorter than the amino acid sequence described in their respective SEQ ID NOs, e.g., 1 to 10, e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids longer or shorter.

[0249] In some embodiments, the variant BCMA polypeptide contains one of the mutations listed in Table 1. In some examples, the mutation is created within a reference BCMA containing the amino acid sequence described in SEQ ID NO: 710. In some examples, the mutation is created within a reference BCMA containing the amino acid sequence described in SEQ ID NO: 356.

[0250] The use of the term “modification,” e.g., “substitution” or “mutation,” does not mean that this embodiment is limited to a specific method for producing immunomodulatory proteins. Variant BCMA polypeptides can be produced, for example, by de novo peptide synthesis, and therefore do not necessarily require modifications such as “substitution,” meaning altering codons to encode substitutions. This principle also extends to the terms “addition” and “deletion” of amino acid residues, which similarly do not imply a specific method of production. The means for designing or producing vTDs are not limited to a specific method. However, in some embodiments, wild-type TD-coding nucleic acids or unmodified TD-coding nucleic acids are mutagenicated from wild-type TD genetic material or unmodified TD genetic material and screened for changes in desired specific binding activity, e.g., binding affinity, and / or NF-κB regulation or other functional activity. In some embodiments, vTDs are synthesized de novo using protein or nucleic acid sequences available in any number of publicly available databases and then screened. The National Center for Biotechnology Information provides such information, and its website is publicly accessible via the internet, such as the UniProtKB database.

[0251] In some embodiments, the variant BCMA polypeptide comprises an extracellular domain (ECD) sequence described in any one of SEQ ID NO:357-435. In some embodiments, the variant BCMA polypeptide comprises a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NO:357-435, and retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) BCMA. In some embodiments, the variant BCMA polypeptide comprises a specific binding fragment of any one of SEQ ID NO:357-435, the specific binding fragment binding to BAFF and / or APRIL, and comprising a continuous sequence containing amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) BCMA.

[0252] In some embodiments, the variant BCMA polypeptide comprises the sequence described in SEQ ID NO:381. In some embodiments, the variant BCMA polypeptide is essentially derived from the sequence described in SEQ ID NO:381. In some embodiments, the variant BCMA polypeptide consists of the sequence described in SEQ ID NO:381.

[0253] In some embodiments, the variant BCMA polypeptide comprises the sequence described in SEQ ID NO:405. In some embodiments, the variant BCMA polypeptide is essentially composed of the sequence described in SEQ ID NO:405. In some embodiments, the variant BCMA polypeptide consists of the sequence described in SEQ ID NO:405.

[0254] In some embodiments, the variant BCMA polypeptide comprises the sequence described in SEQ ID NO:406. In some embodiments, the variant BCMA polypeptide is essentially composed of the sequence described in SEQ ID NO:406. In some embodiments, the variant BCMA polypeptide consists of the sequence described in SEQ ID NO:406.

[0255] In some embodiments, the variant BCMA polypeptide comprises the sequence described in SEQ ID NO:410. In some embodiments, the variant BCMA polypeptide is essentially derived from the sequence described in SEQ ID NO:410. In some embodiments, the variant BCMA polypeptide consists of the sequence described in SEQ ID NO:410.

[0256] In some embodiments, the variant BCMA polypeptide comprises the sequence described in SEQ ID NO:411. In some embodiments, the variant BCMA polypeptide is essentially composed of the sequence described in SEQ ID NO:411. In some embodiments, the variant BCMA polypeptide consists of the sequence described in SEQ ID NO:411.

[0257] In some embodiments, the variant BCMA polypeptide is encoded by a nucleotide sequence described in any of SEQ ID NO:437-515. In some embodiments, the variant BCMA polypeptide is encoded by a nucleotide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NO:437-515, and retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) BCMA. Nucleic acids are also provided herein that include sequences that exhibit at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, for example, at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NO: 437-515.

[0258] (Table 1) Exemplary variants of BCMA within BCMA immunomodulatory proteins (e.g., BCMA-Fc) or as BIMs. TIFF2026143547000012.tif150165TIFF2026143547000013.tif226165TIFF2026143547000014.tif115165

[0259] In some embodiments, BCMA-ECD fusion sequences are also provided herein in which any of the above BCMA sequences is linked to or fused to a multimerizing domain, for example, any of those described herein. Exemplary multimerizing domains are described in Section IV.C. In some embodiments, the multimerizing domain is an immunoglobulin (e.g., IgG1) Fc region, and the fusion protein is a BCMA-Fc comprising (1) a BCMA sequence comprising any of the provided BCMA-ECD sequences, and (2) an immunoglobulin Fc region. Thus, in some embodiments provided, there is a BCMA-Fc fusion protein comprising (1) a BCMA sequence comprising or consisting of any of the above BCMA-ECD polypeptide sequences, such as a variant BCMA polypeptide, and (2) an immunoglobulin Fc region. In some embodiments, the BCMA-Fc fusion is a variant BCMA-Fc fusion comprising or consisting of any of the above variant BCMA polypeptides and an immunoglobulin Fc region.

[0260] In some embodiments, variant BCMA-Fc fusion sequences comprising (1) a BCMA ECD sequence comprising any one of SEQ ID NO: 357 to 435, and (2) an immunoglobulin Fc region are provided herein. In some embodiments, variant BCMA-Fc fusion sequences comprising (1) a BCMA ECD sequence consisting of or essentially comprising any one of SEQ ID NO: 357 to 435, and (2) an immunoglobulin Fc region are provided herein.

[0261] In some embodiments, variant BCMA-Fc fusion sequences comprising (1) a BCMA ECD sequence comprising any one of SEQ ID NO: 357 to 435, and (2) an immunoglobulin Fc region are provided herein. In some embodiments, variant BCMA-Fc fusion sequences comprising (1) a BCMA ECD sequence consisting of or essentially comprising any one of SEQ ID NO: 357 to 435, and (2) an immunoglobulin Fc region are provided herein.

[0262] In the provided embodiment of BCMA-Fc, the immunoglobulin Fc region may be the wild-type Fc of an immunoglobulin, e.g., IgG1 Fc. In some cases, the Fc region may be a variant Fc lacking effector function (also called "effectorless Fc"). Exemplary Fc regions and their variants within the provided BCMA-Fc fusion protein are described in Section IV.C below.

[0263] In some embodiments, Fc is mouse Fc or human Fc. In some embodiments, Fc is mammalian IgG1 Fc region, IgG2 Fc region, IgG3 Fc region or IgG4 Fc region, or human IgG1 Fc region, IgG2 Fc region, IgG3 Fc region or IgG4 Fc region.

[0264] In some embodiments, Fc is derived from IgG1, such as human IgG1. In some embodiments, Fc is IgG1 Fc as described in SEQ ID NO:586, having an allotype containing residues Glu(E) and Met(M) at positions 356 and 358 according to EU numbering. In some embodiments, Fc contains the amino acid sequence described in SEQ ID NO:586, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with respect to SEQ ID NO:586. In other embodiments, Fc is IgG1 Fc containing amino acids of the human G1m1 allotype, such as residues containing Asp(D) and Leu(L) at positions 356 and 358, as described in SEQ ID NO:597. Accordingly, in some cases, Fc provided herein may include amino acid substitutions E356D and M358L for reconstitution of allotype G1 m1 residues. In some embodiments, Fc includes the amino acid sequence described in SEQ ID NO:597, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with respect to SEQ ID NO:597.

[0265] In some embodiments, the Fc region has the amino acid sequence described in SEQ ID NO:597.

[0266] In some embodiments, variant Fc comprises the sequence described in SEQ ID NO:755. In some embodiments, variant Fc comprises the sequence described in SEQ ID NO:756. In some embodiments, the Fc region used in the constructs provided herein may further lack a C-terminal lysine residue.

[0267] In some embodiments, Fc is derived from IgG2 such as human IgG2. In some embodiments, Fc includes the amino acid sequence described in SEQ ID NO:726, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with respect to SEQ ID NO:726.

[0268] In some embodiments, Fc is derived from IgG4, such as human IgG4. In some embodiments, Fc includes the amino acid sequence described in SEQ ID NO:727, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with respect to SEQ ID NO:727. In some embodiments, IgG4 Fc is a stabilized Fc in which the CH3 domain of human IgG4 is replaced by the CH3 domain of human IgG1, exhibiting inhibition of aggregate formation; an antibody in which the CH3 and CH2 domains of human IgG4 are replaced by the CH3 and CH2 domains of human IgG1, respectively; or an antibody in which the arginine at position 409, as indicated by the EU index proposed by Kabat et al., is replaced by lysine, exhibiting inhibition of aggregate formation (see, for example, U.S. Patent No. 8,911,726). In some embodiments, Fc is IgG4 containing the S228P mutation, which has been shown to prevent recombination between therapeutic antibodies and endogenous IgG4 by Fab arm exchange (see, for example, Labrijin et al. (2009) Nat. Biotechnol., 27(8):767-71). In some embodiments, Fc is the amino acid sequence described in SEQ ID NO:728, or SEQ ID Contains amino acid sequences exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with NO:728.

[0269] In some embodiments, the Fc region is a variant Fc region in which the wild-type Fc is modified by one or more amino acid substitutions to reduce effector activity or to inactivate Fc for Fc effector function. Exemplary effectorless or inactivating mutations include those described herein, including in Section IV.C. In some embodiments, the Fc region of an immunomodulatory protein has an Fc region in which one or more of the amino acids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, and 329 (indicated by EU numbering) are substituted with different amino acids compared to the native Fc region. Such alterations of the Fc region include, for example, Current Opinion in Deglycosylated chains (N297A and N297Q), IgG1-N297G, IgG1-L234A / L235A, IgG1-L234A / L235E / G237A, IgG1-A325A / A330S / P331S, IgG1-C226S / C229S, IgG1-C226S / C229S / E233P / L234V / L235A, IgG1-E233P / L234V / L235A / G236del / S267K, IgG1-L234F / L235E / P331S, IgG1-S267E / L3 This includes variations such as 28F, IgG2-V234A / G237A, IgG2-H268Q / V309L / A330S / A331S, IgG4-L235A / G237A / E318A, and IgG4-L236E; variations such as G236R / L328R, L235G / G236R, N325A / L328R, and N325LL328R described in International Publication No. 2008 / 092117; amino acid insertions at positions 233, 234, 235, and 237 (indicated by EU numbering); and variations at sites described in International Publication No. 2000 / 042072.

[0270] In some embodiments, the variant Fc region comprises one or more amino acid modifications (e.g., amino acid substitutions) derived from wild-type IgG1, e.g., wild-type human IgG1. In some embodiments, the wild-type IgG1 Fc may be the Fc described in SEQ ID NO: 586, having an allotype containing residues Glu(E) and Met(M) at positions 356 and 358 according to EU numbering. In some embodiments, the variant Fc region is derived from the amino acid sequence described in SEQ ID NO: 586. In other embodiments, the wild-type IgG1 Fc comprises amino acids from the human G1m1 allotype, e.g., residues containing Asp(D) and Leu(L) at positions 356 and 358, as described in SEQ ID NO: 597. Thus, in some cases, the variant Fc is derived from the amino acid sequence described in SEQ ID NO: 597.

[0271] In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of wild-type Fc or unmodified Fc as described in SEQ ID NO: 586 or 597 (corresponding to K447del in EU numbering).

[0272] In some embodiments, the variant Fc region includes a C5S amino acid modification of the wild-type Fc region or the unmodified Fc region, numbered SEQ ID NO:586 (corresponding to C220S in EU numbering).

[0273] In some embodiments, the Fc region is a variant Fc comprising at least one amino acid substitution, which is N82G according to the numbering of SEQ ID NO: 586 (corresponding to N297G according to EU numbering). In some embodiments, Fc further comprises at least one amino acid substitution, which is R77C or V87C according to the numbering of SEQ ID NO: 586 (corresponding to R292C or V302C according to EU numbering). In some embodiments, the variant Fc region further comprises a C5S amino acid modification according to the numbering of SEQ ID NO: 586 (corresponding to C220S according to EU numbering). For example, in some embodiments, the variant Fc region includes the following amino acid modification, i.e., N297G according to EU numbering, and one or more of the following amino acid modifications, i.e., C220S, R292C, or V302C (corresponding to N82G and one or more of the following amino acid modifications, i.e., C5S, R77C, or V87C, based on SEQ ID NO: 586), for example, the Fc region includes the sequence described in SEQ ID NO: 598.

[0274] In some embodiments, variant Fc contains the amino acid substitution L234A / L235E / G237A according to EU numbering. In some embodiments, variant Fc contains the amino acid substitution A330S / P331S according to EU numbering. In some embodiments, variant Fc contains the amino acid substitution L234A / L235E / G237A / A330S / P331S (Gross et al. (2001) Immunity 15:289). In some embodiments, variant Fc contains the sequence described in SEQ ID NO:757. In some embodiments, variant Fc contains the sequence described in SEQ ID NO:758. In some embodiments, the Fc region used in the constructs provided herein may further lack a C-terminal lysine residue.

[0275] In some embodiments, the Fc region is a variant Fc containing variants L234A, L235E, and G237A, as indicated by EU numbering. In some embodiments, the wild-type Fc is further modified by the removal of one or more cysteine ​​residues, for example, by the substitution of a cysteine ​​residue with respect to the serine residue at position 220 (C220S), as indicated by EU numbering. Exemplary inactive Fc regions with reduced effector function are described in SEQ ID NO: 599 and SEQ ID NO: 591, based on the allotypes described in SEQ ID NO: 586 or SEQ ID NO: 597, respectively. In some embodiments, the Fc region may further lack a C-terminal lysine residue. In some embodiments, the variant Fc region contains one or more of the amino acid modifications C220S, L234A, L235E, or G237A, for example, the Fc region contains the sequences described in SEQ ID NO: 589, 591, 599, or 724. In some embodiments, variant Fc has the sequence described in SEQ ID NO: 589. In some embodiments, variant Fc has the sequence described in SEQ ID NO: 591. In some embodiments, variant Fc has the sequence described in SEQ ID NO: 599. In some embodiments, variant Fc has the sequence described in SEQ ID NO: 724.

[0276] In some embodiments, the Fc region is variant Fc having the sequence described in SEQ ID NO:589.

[0277] In some embodiments, the Fc region is a variant Fc region containing one or more amino acid modifications C220S, L235P, L234V, L235A, G236del, or S267K, for example, the Fc region contains the sequence described in SEQ ID NO:722. In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type Fc or unmodified Fc described in SEQ ID NO:586 (corresponding to K447del in EU numbering).

[0278] In some embodiments, the Fc region is a variant Fc region containing one or more of the amino acid modifications C220S, R292C, N297G, and V302C. In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc as described in SEQ ID NO:586 (corresponding to K447del in EU numbering). An exemplary variant Fc region is described in SEQ ID NO:723.

[0279] In some embodiments, the variant Fc region includes one or more of the amino acid modifications C220S / E233P / L234V / L235A / G236del / S267K. In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc as described in SEQ ID NO:586 (corresponding to K447del in EU numbering). An exemplary variant Fc region is described in SEQ ID NO:725.

[0280] In some embodiments, the Fc region is a variant Fc region containing any combination of the Fc mutations in Table 4. In some embodiments, the Fc region is a variant Fc region having the sequence listed in any one of the SEQ ID NOs in Table 4.

[0281] For example, the variant Fc region may be an effectorless Fc exhibiting reduced effector activity compared to wild-type IgG1 described in SEQ ID NO: 586 or SEQ ID NO: 597. In some embodiments, the variant Fc includes an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with any of SEQ ID NO: 591, 598, 599, 722, 589, 723, 724, or 725. In some embodiments, the variant Fc has the sequence described in SEQ ID NO: 589.

[0282] In some embodiments, a BCMA polypeptide, such as a variant BCMA polypeptide, is directly linked to the Fc sequence. In some embodiments, a BCMA polypeptide, such as a variant BCMA polypeptide, is indirectly linked to the Fc sequence via a linker or the like. In some embodiments, one or more "peptide linkers" link the BCMA polypeptide (e.g., a variant BCMA polypeptide) to the Fc region. In some embodiments, the peptide linker may be a single amino acid residue or longer. In some embodiments, the peptide linker has at least one amino acid residue, but its length is 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid residues. Exemplary linkers are described in the subsection "Linkers".

[0283] In some embodiments, the linker is (in a single-letter amino acid code): GGGGS ("4GS"; SEQ ID NO: 593), or a polymer of the 4GS linker, e.g., a repeat of 2, 3, 4, or 5 4GS linkers. In some embodiments, the peptide linker is (GGGGS)2 (SEQ ID NO: 594), (GGGGS)3 (SEQ ID NO: 595), (GGGGS)4 (SEQ ID NO: 600), or (GGGGS)5 (SEQ ID NO: 671). In some embodiments, the linker may also contain a series of alanine residues, either alone or in addition to another peptide linker (such as a 4GS linker or a polymer thereof). In some embodiments, the linker (in a single-letter amino acid code) is GSGGGGS (SEQ ID NO: 590) or GGGGSSA (SEQ ID NO: 596). In some examples, the linker is 2xGGGGS followed by 3 alanines (GGGGSGGGGSAAA; SEQ ID NO: 721).

[0284] In some embodiments, a BCMA-Fc fusion protein is provided that is a dimer formed by two identical BCMA Fc polypeptides (e.g., variant BCMA polypeptides) linked to an Fc domain. In some embodiments, to produce a homodimer, either identical species of the provided BCMA-Fc fusion polypeptides, e.g., variant BCMA-Fc fusion polypeptides, is dimerized. In some embodiments, the dimer is a homodimer in which two BCMA Fc polypeptides, e.g., variant BCMA Fc polypeptides, are the same. To generate a homodimer Fc molecule, the Fc region can form a homodimer having a matched Fc region by co-expression of individual Fc regions within the cell.

[0285] Nucleic acid molecules encoding BCMA-Fc fusion proteins, such as variant BCMA-Fc fusion proteins, are also provided. In some embodiments, for the production of Fc fusion proteins, the nucleic acid molecules encoding BCMA-Fc fusion proteins, such as variant BCMA-Fc fusion proteins, are inserted into a suitable expression vector. The resulting BCMA-Fc fusion protein, such as variant BCMA-Fc fusion protein, can be expressed in host cells transformed by the expression, where inter-domain construction occurs via interchain disulfide bonds formed between Fc moieties, resulting in a dimer, such as a bivalent BCMA-Fc fusion protein. The resulting Fc fusion protein can be readily purified by affinity chromatography using a protein A column or a protein G column.

[0286] In some embodiments, the immunomodulatory protein provided, for example BCMA-Fc, is a homodimer containing two identical polypeptide chains when produced and expressed from cells. Figure 15 shows the structure of an exemplary BCMA-Fc fusion protein provided herein.

[0287] III. Multidomain immunomodulatory proteins A multi-domain immunomodulatory protein is provided herein, comprising (1) one or more B cell inhibitory molecules (BIMs) bound to a ligand of a B cell stimulating receptor, and (2) one or more T cell inhibitory molecules (BIMs) bound to a T cell stimulating receptor, or a ligand of a T cell stimulating receptor. Among the multi-domain immunomodulatory proteins provided, the BIMs antagonize the activity of the B cell stimulating receptor, e.g., reduce or inhibit the activity of the B cell stimulating receptor, and the TIMs antagonize the activity of the T cell stimulating receptor, e.g., reduce or inhibit the activity of the T cell stimulating receptor. Thus, the immunomodulatory proteins provided combine the B cell inhibitors and T cell inhibitors to form a single molecule. In some embodiments, the BIMs and TIMs are linked directly or indirectly. The immunomodulatory proteins provided may be fusion proteins in which the multi-domain BIM component and the multi-domain TIM component are further linked to another part, e.g., a multimerizing domain or a half-life extension molecule. In certain embodiments, the multi-domain immunomodulatory protein is a BIM / TIM Fc fusion protein. The provided molecules modulate B cell pathways and T cell pathways, and thereby can be used to treat autoimmune diseases, particularly those whose pathogenesis is related to B cell and T cell responses.

[0288] In some embodiments, B cell stimulating receptors (B-cell receptors) are receptors expressed on B cells that stimulate B cell responses, such as B cell maturation and differentiation. B cell stimulating receptors can be BAFF-R, BCMA, and / or TACI. In certain embodiments, the provided multi-domain immunomodulatory protein antagonizes the activity of one or more of BAFF-R, BCMA, or TACI. In some embodiments, BIM binds to ligands of BAFF-R, BCMA, or TACI. The ligand can be BAFF or APRIL, homotrimeric molecules common to members of the TNF superfamily. Both BAFF and APRIL are primarily expressed by myeloid cells and have been reported to act as co-stimulatory B-cell factors. BAFF and APRIL share two receptors, TACI and BCMA. BAFF can also bind to and stimulate BAFF-R. In some cases, the ligand can be a heterotrimer of BAFF and APRIL. For example, heterotrimeric complexes of APRIL and BAFF are found in serum, particularly in subjects with autoimmune diseases, such as those with immune-based systemic rheumatic diseases.

[0289] In some embodiments, the T cell stimulatory receptor (T-cell receptor) contains an immunoreceptor activation tyrosine motif (ITAM) or interacts with adapter proteins involved in the T cell signaling pathway to transmit an activation signal. The T cell stimulatory receptor may be a costimulatory receptor expressed on T cells, e.g., CD28 or ICOS. In certain embodiments, the multi-domain immunomodulatory protein provided antagonizes the activity of the T cell stimulatory receptor, e.g., a T cell costimulatory receptor, e.g., CD28 or ICOS. The TIM may bind to the costimulatory receptor or to a ligand of the costimulatory receptor. In some embodiments, the multi-domain immunomodulatory protein provided may be generated so that the TIM binds directly to the T cell stimulatory receptor. For example, the TIM may be a CD28-binding molecule or an ICOS-binding molecule. In other embodiments, the multi-domain immunomodulatory protein provided may be generated so that the TIM binds to a ligand of the T cell stimulatory receptor, thereby indirectly antagonizing or indirectly inhibiting the T cell stimulatory receptor. For example, the TIM binds to CD80 or CD86, which are ligands of CD28.

[0290] In some embodiments, one or more TIMs and / or BIMs independently comprise an antibody or an antigen-binding antibody fragment. In some aspects, the TIMs and / or BIMs may be antibodies that bind to human antibodies and / or human proteins.

[0291] In some embodiments, at least one of the TIM or BIM is not an antibody or antigen-binding fragment. In some embodiments, at least one of the TIM or BIM is or includes the extracellular domain of a cell surface molecule expressed on an immune cell. For example, certain members of the non-antibody immunoglobulin superfamily (IgSF) are expressed on T cells or modulate T cell activity. These include, for example, certain T cell costimulatory molecules or their ligands. In some cases, the TIM includes the (IgSF) domain (IgD) of an IgSF member (e.g., wild-type IgD), or variant IgD (hereinafter referred to as "vIgD") which contains one or more amino acid modifications (e.g., substitutions) to IgD. Similarly, certain members of the TNF receptor superfamily are expressed on B cells or modulate B cell activity. These include, for example, certain B cell stimulating receptors such as TACI or BCMA. In some cases, BIM may contain a TNF receptor domain (TD) of a TNFR superfamily member (e.g., wild-type TD), or a variant TD (hereinafter referred to as "vTD") that includes another amino acid modification (e.g., substitution) to the TD.

[0292] In some embodiments, BIM can bind to a ligand of a B cell stimulatory receptor with at least a certain binding activity, such as binding affinity, as measured by one of several known methods. In some embodiments, TIM can bind to a T cell stimulatory receptor, or a ligand of a T cell stimulatory receptor, with at least a certain binding activity, such as binding affinity, as measured by one of several known methods. In some embodiments, affinity is the equilibrium dissociation constant (K D ) represented by or EC 50This is represented by [expression]. Various assays are known to evaluate binding activity, including binding affinity, and / or to determine whether a binding molecule (e.g., TIM or BIM) specifically binds to a particular binding partner. In some embodiments, surface plasmon resonance (SPR) analysis can be used to determine the binding kinetics and binding constant of a complex between two proteins using the BIAcore® instrument (see, e.g., Scatchard et al., Ann. NYAcad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Patents 5,283,173, 5,468,614, or equivalents). In other embodiments, biolayer interferometry (BLI) using the ForteBio Octet system, for example, with a streptavidin-coated sensor and a biotinylated recombinant protein domain, may be used. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), or determination of binding by monitoring changes in the spectroscopic or optical properties of the protein by fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays, without limitation, include Western blotting, ELISA, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing, and other methods for detecting the binding of expressed nucleic acids or proteins.

[0293] In some aspects, BIM and TIM are used independently, -5 K below M D This indicates the binding affinity for the binding partner (i.e., the equilibrium dissociation constant of a specific binding interaction in M ​​units; assuming a bimolecular interaction, the binding rate (on-rate) of this association reaction [k on or k a Dissociation rate (off-rate) [k off or k d(Equal to the ratio of ). For example, the equilibrium dissociation constant K D is 10 -6 M~10 -12 M, for example, 10 -7 M~10 -11 M, 10 -8 M~10 -10 M, or 10 -9 M~10 -10 It is in the range of M. The coupling rate (association rate constant; k) on or k a (in units of 1 / Ms) and dissociation rate (dissociation rate constant; k off or k d The coefficient (in units of 1 / s) can be determined using any assay method known in the art, such as surface plasmon resonance (SPR).

[0294] In some aspects, BIM can range from 0.001 nM or about 0.001 nM to 1000 nM, for example, from 0.01 nM or about 0.01 nM to about 500 nM, from 0.01 nM or about 0.01 nM to about 400 nM, from 0.01 nM or about 0.01 nM to about 100 nM, from 0.01 nM or about 0.01 nM to about 50 nM, 0.01 nM or about 0.01 From nM, approximately 10nM; from 0.01nM or approximately 0.01nM, approximately 1nM; from 0.01nM or approximately 0.01nM, approximately 0.1nM; from 0.1nM or approximately 0.1nM, approximately 500nM; from 0.1nM or approximately 0.1nM, approximately 400nM; from 0.1nM or approximately 0.1nM, approximately 100nM; from 0.1nM or approximately 0.1nM, approximately 50nM; if 0.1nM From approximately 0.1nM to approximately 10nM, from 0.1nM or approximately 0.1nM to approximately 1nM, from 0.5nM or approximately 0.5nM to approximately 200nM, from 1nM or approximately 1nM to approximately 500nM, from 1nM or approximately 1nM to approximately 100nM, from 1nM or approximately 1nM to approximately 50nM, from 1nM or approximately 1nM to approximately 10nM, from 2nM or approximately 2nM to approximately 50nM, 10 The binding affinity for the ligand of the B cell stimulating receptor is as follows: nM or approximately 10nM to approximately 500nM, 10nM or approximately 10nM to approximately 100nM, 10nM or approximately 10nM to approximately 50nM, 50nM or approximately 50nM to approximately 500nM, 50nM or approximately 50nM to approximately 100nM, or 100nM or approximately 100nM to approximately 500nM.In certain embodiments, the binding affinity of BIM to the inhibitory receptor is 400nM, 300nM, 200nM, 100nM, 50nM, 40nM, 30nM, 25nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or 1nM or less, or 400nM, 300nM, 200nM, 100nM, 50nM, 40nM, 30nM, 25nM, 20nM, 19nM, 18nM, 17 nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or 1nM or less, or approximately 400nM, 300nM, 200nM, 100nM, 50nM, 40nM, 30nM, 25nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or 1nM or less.

[0295] In some embodiments, the TIM ranges from 0.001 nM or about 0.001 nM to about 1000 nM, for example, from 0.01 nM or about 0.01 nM to about 500 nM, from 0.01 nM or about 0.01 nM to about 400 nM, from 0.01 nM or about 0.01 nM to about 100 nM, from 0.01 nM or about 0.01 nM to about 50 nM, 0.01 nM or about 0.01 nM From approximately 10 nM, 0.01 nM, or approximately 0.01 nM, from approximately 1 nM, 0.01 nM, or approximately 0.01 nM, from approximately 0.1 nM, 0.1 nM, or approximately 0.1 nM, from approximately 500 nM, 0.1 nM, or approximately 0.1 nM, from approximately 400 nM, 0.1 nM, or approximately 0.1 nM, from approximately 100 nM, 0.1 nM, or approximately 0.1 nM, from approximately 50 nM, 0.1 nM, or approximately 0. From 1 nM to approximately 10 nM, from 0.1 nM or approximately 0.1 nM to approximately 1 nM, from 0.5 nM or approximately 0.5 nM to approximately 200 nM, from 1 nM or approximately 1 nM to approximately 500 nM, from 1 nM or approximately 1 nM to approximately 100 nM, from 1 nM or approximately 1 nM to approximately 50 nM, from 1 nM or approximately 1 nM to approximately 10 nM, from 2 nM or approximately 2 nM to approximately 50 nM, 10 nM or approximately 1 It exhibits binding affinity to T cell-stimulating receptors or ligands of T cell-stimulating receptors, ranging from 0 nM to approximately 500 nM, 10 nM or approximately 10 nM to approximately 100 nM, 10 nM or approximately 10 nM to approximately 50 nM, 50 nM or approximately 50 nM to approximately 500 nM, 50 nM or approximately 50 nM to approximately 100 nM, or 100 nM or approximately 100 nM to approximately 500 nM.In certain embodiments, the binding affinity of the TIM to the stimulating receptor or T cell stimulating receptor ligand is 400nM, 300nM, 200nM, 100nM, 50nM, 40nM, 30nM, 25nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or 1nM or less, or 400nM, 300nM, 200nM, 100nM, 50nM, 40nM, 30nM, 25nM, 20nM, 19n M, 18nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or 1nM or less, or approximately 400nM, 300nM, 200nM, 100nM, 50nM, 40nM, 30nM, 25nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or 1nM or less.

[0296] In some embodiments, TIM, or multi-domain immunomodulatory proteins containing TIM, are not agonists of T cell stimulatory (e.g., costimulatory) receptors. In some embodiments, TIM, or multi-domain immunomodulatory proteins containing TIM, bind to T cell costimulatory receptors, e.g., CD28 or ICOS, but exhibit relatively low affinity for T cell costimulatory receptors. In some embodiments, TIM is 1 × 10⁻¹⁶ -9 For example, exceeding M, such as 1 × 10⁻⁶. -7 M or approximately 1 x 10 -7 From M, 1 x 10 -9 M or approximately 1 x 10 -9 M has binding affinity to T cell costimulatory receptors. In some embodiments, TIM has 1 × 10 -7 M or approximately 1 x 10 -7 M, 2.5 x 10 -7 M or approximately 2.5 x 10 -7 M, 5×10 -7 M or approximately 5 x 10 -7 M, 7.5×10-7 M or approximately 7.5 x 10 -7 M, 1×10 -8 M or approximately 1 x 10 -8 M, 2.5 x 10 -8 M or approximately 2.5 x 10 -8 M, 5×10 -8 M or approximately 5 x 10 -8 M, 7.5×10 -8 M or approximately 7.5 x 10 -8 M, or 1 x 10 -9 M or approximately 1 x 10 -9 It has a binding affinity for the T cell costimulatory receptor of M, or any value between any of the aforementioned values.

[0297] In some embodiments, the TIM of the multi-domain immunomodulatory protein provided does not directly bind to the T cell costimulatory receptor. In some embodiments, the TIM of the multi-domain immunomodulatory protein provided binds to the ligand of the T cell costimulatory receptor.

[0298] In some embodiments, the provided multi-domain immunomodulatory protein may contain BIMs and TIMs in various configurations or forms, including forms having one or more further parts. In some embodiments, the provided immunomodulatory protein contains one or more BIMs, each being a polypeptide located N-terminal to the TIM. In some embodiments, one or more BIMs are located C-terminal to the TIM. One or more BIMs and one or more TIMs may be linked directly or indirectly via a linker. In some embodiments, the immunomodulatory protein may be formalized as a multimeric molecule via fusion with a multimerizing domain such as an Fc protein. In some embodiments, the multi-domain immunomodulatory protein may be formalized as a multimeric molecule, e.g., a dimeric, trimer, tetramer, or pentamer. In some embodiments, the immunomodulatory protein is formalized as a monomeric molecule containing a single polypeptide fusion of one or more BIMs and one or more TIMs. Figure 16 shows exemplary forms and configurations that may be included in the provided multi-domain immunomodulatory protein.

[0299] The following subsections describe exemplary BIM and TIM components of the multi-domain immunomodulatory proteins provided, as well as exemplary forms of such immunomodulatory proteins.

[0300] A. B-cell inhibitor molecules (BIMs) In some embodiments, the immunomodulatory protein provided comprises a BIM that binds to one or more ligands of a B cell stimulating receptor. In some embodiments, the B cell stimulating receptor is a member of TNFRSF. In some embodiments, the one or more B cell stimulating receptors are TACI and BCMA. In some embodiments, the ligand of the B cell stimulating receptor is BAFF or APRIL. In some embodiments, the BIM binds to BAFF, APRIL, and / or a BAFF / APRIL heterotrimer. In some embodiments, the BIM can bind to BAFF, APRIL, and a BAFF / APRIL heterotrimer.

[0301] In some embodiments, the BIM is an antibody or antigen-binding fragment that binds to a ligand of a B cell stimulating receptor. In some embodiments, the BIM is an antibody or antigen-binding fragment that binds to BAFF and / or APRIL, for example, human BAFF and / or human APRIL.

[0302] In some embodiments, the BIM is a ligand-binding partner of a B cell stimulating receptor, or includes it. In some embodiments, the multi-domain immunomodulatory proteins provided herein are soluble proteins and / or do not include a portion containing a transmembrane domain. Those skilled in the art will understand that cell surface proteins, including B cell stimulating receptors, e.g., TNFRSF proteins such as BCMA and TACI, typically have an intracellular domain, a transmembrane domain, and an extracellular domain (ECD), and that soluble forms of such proteins can be made using the extracellular domain or an immunoactive sub-domain thereof. Therefore, in some embodiments, the BIM lacks the transmembrane domain or a portion of the transmembrane domain of a B cell stimulating receptor, e.g., BCMA or TACI. In some embodiments, the BIM lacks the intracellular (cytoplasmic) domain or a portion of the intracellular domain of a B cell stimulating receptor, e.g., BCMA or TACI. In some embodiments, the BIM includes only the ECD domain containing a TD such as a CRD, or a portion thereof, or its specific binding fragment.

[0303] For example, in some aspects, the BIM is or includes the ECD of the B cell stimulating receptor, or its specific binding portion or specific binding fragment, which includes at least one TD (e.g., at least one CRD) that binds to the ligand of the B cell stimulating receptor. For example, the BIM may include the ECD of TACI or BCMA, or the specific binding portion or specific binding fragment of TACI or BCMA, which includes at least one TD (e.g., at least one CRD) that binds to APRIL, BAFF, and / or APRIL / BAFF heterotrimer. In some embodiments, the BIM consists of or is essentially the ECD of the B cell stimulating receptor, or its specific binding portion or specific binding fragment, which includes at least one TD (e.g., at least one CRD), for example, the BIM consists of or is essentially the ECD of TACI or BCMA, or the specific binding portion or specific binding fragment of TACI or BCMA, which includes at least one TD (e.g., at least one CRD). In some embodiments, the BIM is shorter than the full-length sequence of the ECD of the B cell stimulating receptor. In some embodiments, the BIM is a single CRD, or a specific binding fragment of a CRD, or comprises only that. In some embodiments, the BIM consists of or is essentially a CRD of a B cell stimulating receptor, for example, consisting of or being essentially a single CRD of TACI or BCMA. In some embodiments, the BIM sequence, which comprises an ECD containing a TD (e.g., at least one CRD) or its binding portion or binding fragment, is a mammalian sequence, including, but not limited to, human, mouse, cynomolgus monkey, or rat. In some embodiments, the BIM sequence is human and / or binds to a human protein.

[0304] In some aspects, the BIM is an affinity-modified domain (vTD) that exhibits increased binding activity, e.g., increased binding affinity to B cell-stimulating receptor ligands, compared to the binding activity of an unmodified or wild-type TD to the same molecule. In some embodiments, the BIM comprises a vTD having one or more amino acid substitutions compared to the TD of a TNFRSF member, e.g., BCMA or TACI, where the one or more amino acid substitutions confuse or result in increased binding affinity to a congener ligand of the B cell-stimulating receptor.

[0305] In some aspects, the BIM is a vTD containing one or more amino acid modifications, e.g., one or more substitutions (or "mutations" or "replacements"), deletions, or additions, compared to the wild-type or unmodified TD of the B cell stimulatory receptor ligand binding partner. In some aspects, the vTD contains up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, e.g., amino acid substitutions, deletions, or additions, in the TD domain of the TNFRSF binding partner of the B cell stimulatory receptor. The modifications (e.g., substitutions) may be present in the CRD. In some embodiments, the vTD has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions) in the CRD or its specific binding fragment. In some embodiments, the vTD has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the wild-type TD or the unmodified TD or its specific binding fragment.

[0306] Non-exclusive examples of BIMs in the multi-domain immunomodulatory proteins provided are described in the following subsections. Any of the BIMs described herein may be combined with the TIMs described in Section III.B.

[0307] 1. TACI In some embodiments, BIM is a wild-type TACI ECD containing at least one TD (e.g., at least one CRD) that binds to APRIL, BAFF, and / or APRIL / BAFF heterotrimer, or a specific binding moiety or specific binding fragment thereof, or includes the same. In some embodiments, BIM is a variant TACI ECD containing at least one TD (e.g., at least one CRD) that binds to APRIL, BAFF, and / or APRIL / BAFF heterotrimer, or a specific binding moiety or specific binding fragment thereof, or includes the same. In some embodiments, BIM is a TACI polypeptide or a variant thereof having any of the sequences described herein.

[0308] TACI is a member of the tumor necrosis factor receptor family characterized by having an extracellular domain (ECD) containing cysteine-rich pseudo-repeat domains (CRDs). TACI is a membrane-bound receptor having an extracellular domain containing two cysteine-rich pseudo-repeats (CRD1 and CRD2), a transmembrane domain, and a cytoplasmic domain that interacts with CAML (calcium modulator and cyclophylline ligand), an endogenous membrane protein located in intracellular vesicles, which is a co-inducer of NF-AT activation when overexpressed in Jurkat cells. TACI is associated with B cells and subsets of T cells. The TACI receptor binds to two members of the tumor necrosis factor (TNF) ligand family. One of the ligands is called BAFF (B cell Activating Factor of the TNF Family), as well as various other names such as ZTNF4, "Nutrokine-α", "BLyS", "TALL-1", and "THANK" (Yu et al., International Publication No. WO98 / 18921 (1998), Moore et al., Science 285:269 (1999); Mukhopadhyay et al., J. Biol. Chem. 274:15978 (1999); Schneider et al., J. Exp. Med. 189:1747 (1999); Shu et al., J. Leukoc. Biol. 65:680 (1999)). The other ligand is called APRIL, as well as various other names such as "ZTNF2" and "TNRF death ligand-1" (Hahne et al., J.Exp.Med.188:1185(1998); Kelly et al., Cancer Res.60:1021(2000)). Both ligands are also bound by the B cell maturation receptor (BCMA) (Gross et al., Nature 404:995(2000)). Binding of the TACI receptor to its ligand, BAFF or APRIL, stimulates B cell responses, including T cell-independent B cell antibody responses, isotype switching, and B cell homeostasis.

[0309] The amino acid sequence of full-length TACI is described in SEQ ID NO:666. This protein is a type III membrane protein and lacks a signal peptide. After expression in eukaryotic cells, the N-terminal methionine is removed. In some embodiments, the mature TACI protein does not contain the N-terminal methionine described in SEQ ID NO:666. The extracellular domain of TACI (amino acid residues 1-166 of SEQ ID NO:666; ECD described in SEQ ID NO:709) contains two cysteine-rich domains (CRDs, also called tumor necrosis family receptor domains or TDs) that exhibit affinity for binding to BAFF and APRIL, respectively. The first cysteine-rich domain (CRD1) contains amino acid residues 34-66 of the sequence described in SEQ ID NO:709. The second cysteine-rich domain (CRD2) corresponds to amino acids 71-104 of the sequence described in SEQ ID NO:709. TACI also includes a stalk region of approximately 60 amino acids following the second cysteine ​​repeat within the extracellular domain, corresponding to amino acid residues 105-165 of the sequence described in SEQ ID NO:709.

[0310] In some embodiments, the BIM is a variant TACI polypeptide comprising one or more amino acid modifications, e.g., one or more substitutions (or "mutations" or "replacements"), deletions or additions) within the extracellular domain of a wild-type TACI polypeptide or an unmodified TACI polypeptide, e.g., a CRD (hereinafter also referred to as TD). Thus, a BIM provided that is a variant TACI polypeptide is or comprises a variant TD ("vTD") in which one or more amino acid modifications (e.g., substitutions) are located in the CRD. In some embodiments, the one or more amino acid modifications, e.g., one or more substitutions (or "mutations" or "replacements"), deletions or additions are located in the CRD1 region. In some embodiments, the one or more amino acid modifications, e.g., one or more substitutions (or "mutations" or "replacements"), deletions or additions are located in the CRD2 region. In some embodiments, one or more amino acid modifications, such as one or more substitutions (or "mutations" or "replacements"), deletions, or additions, are located in amino acids within the CRD1 and CRD2 regions.

[0311] In some embodiments, the reference (e.g., unmodified) TACI sequence is either a wild-type TACI sequence or a portion thereof containing one or both CRDs. In some embodiments, the reference (e.g., unmodified) TACI is either a portion thereof containing the extracellular domain (ECD) of TACI, or one or both CRD domains, or includes them. In some embodiments, the extracellular domain of the reference (e.g., unmodified) TACI polypeptide contains CRD1 and CRD2; however, the variant TACI polypeptide does not need to contain both CRD1 and CRD2. In some embodiments, the variant TACI polypeptide contains or is essentially derived from CRD1 or its specific binding fragment. In some embodiments, the variant TACI polypeptide contains or is essentially derived from CRD2 or its specific binding fragment. In some embodiments, the variant TACI is a soluble polypeptide and lacks a transmembrane domain. In some embodiments, the variant TACI polypeptide further contains a transmembrane domain and, possibly, a cytoplasmic domain.

[0312] In some embodiments, the reference (e.g., unmodified) TACI sequence is a mammalian TACI sequence. In some embodiments, the reference (e.g., unmodified) TACI sequence may be a mammalian TACI including, but not limited to, human, mouse, cynomolgus monkey, or rat. In some embodiments, the reference (e.g., unmodified) TACI sequence is human. An example of the extracellular domain of a human TACI sequence is described in SEQ ID NO:709.

[0313] In some embodiments, the reference (e.g., unmodified) TACI sequence has (i) the amino acid sequence described in SEQ ID NO:709, or the sequence lacking the N-terminal methionine; (ii) an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with respect to SEQ ID NO:709, and which binds to APRIL, BAFF, or the APRIL / BAFF heterotrimer; or (iii) is a fragment or portion of (i) or (ii) containing CRD1 and / or CRD2, wherein the portion binds to APRIL, BAFF, or the APRIL / BAFF heterotrimer. In some embodiments, the reference (e.g., unmodified) TACI sequence lacks the N-terminal methionine described in SEQ ID NO:709. TACI extracellular domain (ECD): SEQ ID NO: 709 TIFF2026143547000015.tif17154

[0314] In some embodiments, the reference (e.g., unmodified) TACI sequence is the extracellular domain sequence of TACI, which is part of the ECD including an N-terminal deletion compared to the amino acid sequence described in SEQ ID NO:709. In some embodiments, the N-terminal deletion is the deletion of N-terminal amino acid residues 1-28 corresponding to the residues described in SEQ ID NO:709. In some embodiments, the N-terminal deletion is the deletion of N-terminal amino acid residues 1-29 corresponding to the residues described in SEQ ID NO:709. In some embodiments, the N-terminal deletion is the deletion of N-terminal amino acid residues 1-30 corresponding to the residues described in SEQ ID NO:709. In some embodiments, the N-terminal deletion is the deletion of N-terminal amino acid residues 1-31 corresponding to the residues described in SEQ ID NO:709. In some embodiments, the N-terminal deletion is the deletion of N-terminal amino acid residues 1-32 corresponding to the residues described in SEQ ID NO:709. In some embodiments, the N-terminal deletion is the deletion of N-terminal amino acid residues 1-33, which correspond to the residues described in SEQ ID NO:709.

[0315] In some of the embodiments provided, the reference (e.g., unmodified) TACI sequence is an ECD portion comprising the deletion of one or more residues in the stalk portion of the TACI extracellular domain. In some embodiments, the reference (e.g., unmodified) TACI sequence is an ECD portion beginning at residue 105, corresponding to the residue of the ECD sequence described in SEQ ID NO:709, up to amino acid residue 166, or lacking one or more consecutive C-terminal amino acid residues including amino acid residue 166. In some embodiments, the ECD sequence is deleted at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62.

[0316] In some embodiments, the reference (e.g., unmodified) TACI sequence includes an ECD portion having a continuous amino acid sequence, comprising CRD1 and / or CRD2 (e.g., CRD1 and CRD2, or CRD2 only) and only a segment or portion of the stalk sequence. A preferred stalk segment comprises one or more amino acids from amino acid residues 105-154 of SEQ ID NO:709. For example, the stalk segment, relative to SEQ ID NO:709, comprises amino acid residues 105, 105-106, 105-107, 105-108, 105-109, 105-110, 105-111, 105-112, 105-113, 105-114, 105-115, 105-116, amino acid residues 105-117, amino acid residues 105-118, amino acid residues 105-119, amino acid residues 105-120, amino acid residues 105-121, amino acid residues 105-122, amino acid residues 105-123, amino acid residues 105-124, amino acid residues 105-125, amino acid residues 105-126, amino acid residues 105-127, amino acid residues 105-128, amino acid residues 105-129, Amino acid residues 105-130, 105-131, 105-132, 105-133, 105-134, 105-135, 105-136, 105-137, 105-138, 105-139, 105-140, 105-141, 105-1 It may consist of 42, amino acid residues 105-143, 105-144, 105-145, 105-146, 105-147, 105-148, 105-149, 105-150, 105-151, 105-152, 105-153, and 105-154.

[0317] In some embodiments, the reference (e.g., unmodified) TACI sequence lacks one or more potential furin cleavage sites, or has one or more mutated potential furin cleavage sites. In some cases, the reference (e.g., unmodified) TACI sequence is an ECD or moiety with a mutated arginine residue at position 119 (e.g., R119G). In some cases, the reference (e.g., unmodified) TACI sequence is an ECD or moiety with a mutated glutamine residue at position 121 (e.g., Q121P). In some cases, the reference (e.g., unmodified) TACI sequence is an ECD or moiety with a mutated arginine residue at position 122 (e.g., R122Q).

[0318] In some embodiments, the reference TACI sequence is a TACI ECD sequence described in International PCT Publication Numbers WO2000 / 067034, WO2002 / 094852, or WO2008 / 154814.

[0319] In some embodiments, the reference TACI sequence is a TACI ECD sequence having or consisting of the sequence described in SEQ ID NO:719. TACI ECD(CRD1 / CRD2):SEQ ID NO:719 TIFF2026143547000016.tif11153

[0320] In some embodiments, the reference TACI sequence is a TACI ECD sequence having or consisting of the sequence described in SEQ ID NO:718. TACI ECD(CRD1 / CRD2):SEQ ID NO:718 TIFF2026143547000017.tif11153

[0321] In some embodiments, the reference TACI sequence is a TACI ECD sequence having or consisting of the sequence described in SEQ ID NO:516 (encoded by the nucleotide sequence described in SEQ ID NO:551). TACI ECD(CRD1 / CRD2):SEQ ID NO:516 TIFF2026143547000018.tif11159

[0322] In some embodiments, the reference TACI sequence is essentially composed solely of the CRD2 sequence, with the entire CRD1 sequence and substantially the entire stalk region being deleted or absent, and this is the extracellular domain region of TACI. Previous studies have shown that residues within the stalk region may contain protease cleavage sites, but at least CRD1 and CRD2 were considered necessary for sufficient expression and / or binding activity of TACI to its homologous ligands. For example, in international PCT publication number WO2002 / 094852, it was demonstrated that a TACI molecule containing CRD1 and CRD2 but lacking the entire amino-terminal region and a partial sequence of the stalk region exhibited reduced proteolysis when expressed. Other studies have shown that at least a portion of the preceding N-terminal region of CRD1 is necessary for sufficient binding activity of TACI to its homologous ligand; see, for example, international publication number WO2008 / 154814, where residues 13-118 or 13-108 of the TACI extracellular region were determined to be necessary for biological activity while minimizing degradation of TACI during expression. Surprisingly, it is found herein that the TACI extracellular region consisting essentially of only CRD2 with a small portion of the stalk region exhibits substantially improved homologous binding activity compared to the longer TACI molecule containing both CRD1 and CRD2 (e.g., Example 8).

[0323] In some embodiments, BIM is a TACI polypeptide that is part of the TACI extracellular domain (ECD) region, containing CRD2, deleting the N-terminal region and CRD1, and deleting one or more residues in the stalk portion of the TACI extracellular domain, compared to the amino acid sequence described, for example, SEQ ID NO:709. In some embodiments, the part of the TACI extracellular domain containing CRD2 contains amino acid residues 71-104, corresponding to the residues described in SEQ ID NO:709. In the embodiments provided, the immunomodulatory protein BIM is a TACI polypeptide containing deletions of N-terminal amino acid residues 1-66, corresponding to the residues described in SEQ ID NO:709. In the embodiments provided, the immunomodulatory protein BIM is a TACI polypeptide containing deletions of N-terminal amino acid residues 1-67, corresponding to the residues described in SEQ ID NO:709. In the embodiments provided, the immunomodulatory protein BIM is a TACI polypeptide containing deletions of N-terminal amino acid residues 1-68, corresponding to the residues described in SEQ ID NO:709. In the embodiments provided, the immunomodulatory protein BIM is a TACI polypeptide containing deletions of N-terminal amino acid residues 1-69 corresponding to the residue described in SEQ ID NO:709. In the embodiments provided, the immunomodulatory protein BIM is a TACI polypeptide containing deletions of N-terminal amino acid residues 1-70 corresponding to the residue described in SEQ ID NO:709. In some of any such embodiments, the immunomodulatory protein BIM is a TACI polypeptide lacking one or more consecutive C-terminal amino acid residues, starting from residue 105 corresponding to the residue of the ECD sequence described in SEQ ID NO:709, up to amino acid residue 166, or containing amino acid residue 166. In some embodiments, the ECD sequence is deleted at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62.

[0324] In some embodiments, the BIM of the immunomodulatory protein provided herein is a TACI polypeptide having a sequence that includes an ECD portion having a continuous amino acid sequence of TACI ECD, which includes CRD2 (e.g., residues 71-104 relative to SEQ ID NO:709) but lacks the N-terminal region and CRD1, and lacks one or more residues of the stalk portion of the TACI extracellular domain. For example, the TACI ECD portion may consist of amino acid residues 67-118, 67-117, 67-116, 67-115, 67-114, 67-113, 67-112, 67-111, 67-110, 67-109, 67-108, 67-107, 67-106, 67-105, or 67-104, based on the amino acid residues listed in SEQ ID NO:709. In some cases, the TACI ECD portion may consist of amino acid residues 68-118, 68-117, 68-116, 68-115, 68-114, 68-113, 68-112, 68-111, 68-110, 68-109, 68-108, 68-107, 68-106, 68-105, or 68-104, based on the residues listed in SEQ ID NO:709. In some cases, the TACI ECD portion may consist of amino acid residues 69-118, 69-117, 69-116, 69-115, 69-114, 69-113, 69-112, 69-111, 69-110, 69-109, 69-108, 69-107, 69-106, 69-105, or 69-104, based on the residues listed in SEQ ID NO:709.In some examples, the TACI ECD portion may consist of amino acid residues 70-118, 70-117, 70-116, 70-115, 70-114, 70-113, 70-112, 70-111, 70-110, 70-109, 70-108, 70-107, 70-106, 70-105, or 70-104, based on the residues listed in SEQ ID NO:709. In some examples, the TACI ECD moiety may consist of amino acid residues 71-118, 71-117, 71-116, 71-115, 71-114, 71-113, 71-112, 71-111, 71-110, 71-109, 71-108, 71-107, 71-106, 71-105, or 71-104, relative to the residues described in SEQ ID NO:709. Any of the above TACI ECD sequences may also be a TACI reference sequence according to TIM, which is a variant TACI in an immunomodulatory protein provided herein, and such an immunomodulatory protein contains a variant TACI polypeptide modified by one or more amino acid modifications (e.g., substitutions) described herein compared to such a TACI reference sequence.

[0325] In particular, some of the BIMs within the provided immunomodulatory proteins have or consist of the sequence described in SEQ ID NO: 528 (a TACI ECD sequence encoded by the nucleotide sequence described in SEQ ID NO: 563). In some embodiments, the reference TACI sequence has or consists of the sequence described in SEQ ID NO: 528, and the provided variant TACI polypeptide is modified by one or more amino acid modifications (e.g., substitutions) described herein compared to such a reference TACI sequence. TACI ECD sequence (CRD2): SEQ ID NO: 528 TIFF2026143547000019.tif4128

[0326] Among the BIMs in the immunomodulatory proteins provided are variant TACI polypeptides. In some embodiments of the provided embodiments, the variant TACI sequence has the sequence of a reference (e.g., unmodified) TACI sequence, e.g., one of the above, but further comprises another amino acid modification, e.g., one or more amino acid substitutions. In particular, so that the variant TACI polypeptide exhibits modified (e.g., increased) binding activity or binding affinity to one or both APRIL or BAFF compared to the reference (e.g., unmodified or wild-type) TACI polypeptide, the BIMs provided herein may be variant TACI polypeptides comprising at least one affinity-modified TD domain (e.g., CRD1 and / or CRD2) or a specific binding fragment thereof, which comprises one or more amino acid substitutions in the TD domain of the reference (e.g., unmodified or wild-type) TACI polypeptide. In some embodiments, the BIM is a variant TACI polypeptide having a binding affinity to APRIL and / or BAFF that differs from the binding affinity of the reference (e.g., unmodified or wild-type) TACI polypeptide control sequence, as determined, e.g., by solid-phase ELISA immunoassay, flow cytometry or Biacore assay. The binding affinity to each of the congeneral binding partners is independent. That is, in some embodiments, the variant TACI polypeptide has increased binding affinity to one or both APRIL and BAFF, and decreased or unchanged binding affinity to the other of APRIL or BAFF, compared to the reference (e.g., unmodified or wild-type) TACI polypeptide.

[0327] In some embodiments, BIM is a variant TACI polypeptide having increased binding affinity to BAFF compared to a reference (unmodified or wild-type) TACI polypeptide. In some embodiments, BIM is a variant TACI polypeptide having increased binding affinity to APRIL compared to a reference (unmodified or wild-type) TACI polypeptide. In some embodiments, BIM is a variant TACI polypeptide having increased binding affinity to both APRIL and BAFF compared to a reference (unmodified or wild-type) TACI polypeptide. The congener ligands BAFF and / or APRIL may be mammalian proteins, e.g., human or mouse proteins. In some embodiments, BIM, which is a variant TACI polypeptide having increased or enhanced binding affinity to APRIL and / or BAFF, has at least about 5%, e.g., at least about 10%, 15%, 20%, 25%, 35%, or 50% increased binding affinity compared to a reference (e.g., unmodified or wild-type) TACI polypeptide control. In some embodiments, the increase in binding affinity compared to a reference (e.g., unmodified or wild-type) TACI polypeptide is greater than 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 times. In any of the examples, the reference (e.g., unmodified or wild-type) TACI polypeptide has the same sequence as the variant TACI polypeptide, except that it does not contain one or more amino acid modifications (e.g., substitutions).

[0328] In some embodiments, the equilibrium dissociation constant (K) of any of the embodiments described above is used for BAFF. d ) is 1 × 10 -5 M, 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M or 1×10 -11 M, or 1 × 10 -12 M may be less than M. In some embodiments, K of any of the above embodiments relative to BAFF dis 1 × 10 -9 M, 1×10 -10 M or 1x10 -11 M, or 1 x 10 -12 Less than M, or approximately 1 × 10 -9 M, 1×10 -10 M or 1x10 -11 M, or 1 x 10 -12 It is less than M. In some embodiments, K of any of the above embodiments relative to BAFF d is 1 × 10 -9 From M, 1 x 10 -12 M or approximately 1 x 10 -12 M is M. In some embodiments, K is one of the aforementioned embodiments relative to BAFF. d is 1 × 10 -9 M or approximately 1 x 10 -9 M, 2×10 -9 M or approximately 2 x 10 -9 M, 4×10 -9 M or approximately 4 x 10 -9 M, 6×10 -9 M or approximately 6 x 10 -9 M, 8×10 -9 M or approximately 8 x 10 -9 M, 1×10 -10 M or approximately 1 x 10 -10 M, 2×10 -10 M or approximately 2 x 10 -10 M, 4×10 -10 M or approximately 4 x 10 -10 M, 6×10 -10 M or approximately 6 x 10 -10 M, 8×10 -10 M or approximately 8 x 10 -10 M, 1×10 -11 M or approximately 1 x 10 -11 M, 2×10 -11 M or approximately 2 x 10 -11 M, 4×10 -11 M or approximately 4 x 10 -11 M, 6×10 -11 M or approximately 6 x 10 -11 M, 8×10 -11 M or approximately 8 x 10 -11 M, or 1 x 10 -12 M or approximately 1 x 10-12 M is any value between any of the above. In some embodiments, BIM in the provided embodiment is the above variant TACI polypeptide and K relative to BAFF. d The binding affinity decreases by more than 1.5 times, or by approximately more than 1.5 times, for example, by more than 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more, or by more than approximately 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more (increase in binding affinity).

[0329] In some embodiments, the equilibrium dissociation constant (K) of any of the embodiments described above for APRIL d ) is 1 × 10 -5 M, 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M or 1×10 -11 M, or 1 × 10 -12 It may be less than M. In some embodiments, K of any of the above embodiments relative to APRIL d is 1 × 10 -9 M, 1×10 -10 M or 1x10 -11 M, or 1 x 10 -12 Less than M, or approximately 1 × 10 -9 M, 1×10 -10 M or 1x10 -11 M, or 1 x 10 -12 It is less than M. In some embodiments, K of any of the above embodiments relative to APRIL d is 1 × 10 -9 From M, 1 x 10 -12 M or approximately 1 x 10 -12 M is M. In some embodiments, K is one of the aforementioned embodiments relative to APRIL. d is 1 × 10 -9 M or approximately 1 x 10 -9 M, 2×10 -9 M or approximately 2 x 10 -9 M, 4×10 -9 M or approximately 4 x 10 -9 M, 6×10-9 M or approximately 6 x 10 -9 M, 8×10 -9 M or approximately 8 x 10 -9 M, 1×10 -10 M or approximately 1 x 10 -10 M, 2×10 -10 M or approximately 2 x 10 -10 M, 4×10 -10 M or approximately 4 x 10 -10 M, 6×10 -10 M or approximately 6 x 10 -10 M, 8×10 -10 M or approximately 8 x 10 -10 M, 1×10 -11 M or approximately 1 x 10 -11 M, 2×10 -11 M or approximately 2 x 10 -11 M, 4×10 -11 M or approximately 4 x 10 -11 M, 6×10 -11 M or approximately 6 x 10 -11 M, 8×10 -11 M or approximately 8 x 10 -11 M, or 1 x 10 -12 M or approximately 1 x 10 -12 M is any value between any of the above. In some embodiments, the BIM of the provided embodiment is the above variant TACI polypeptide and K for APRIL. d The binding affinity decreases by more than 1.5 times, or by approximately more than 1.5 times, for example, by more than 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more, or by more than approximately 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more (increase in binding affinity).

[0330] A reference (e.g., unmodified or wild-type) TACI sequence does not necessarily have to be used as a starting composition for generating BIM, which is a variant TACI polypeptide described herein. Therefore, the use of the term “modified,” e.g., “substitution,” does not imply that this embodiment is limited to a specific method for producing a variant TACI polypeptide or an immunomodulatory protein containing it. A variant TACI polypeptide can be produced, for example, by de novo peptide synthesis, and therefore does not necessarily require modification, such as “substitution,” in the sense of altering codons to encode a substitution. This principle also extends to the terms “addition” and “deletion” of amino acid residues, which similarly do not imply a specific method of production. The means for designing or producing a variant TACI polypeptide are not limited to a specific method. However, in some embodiments, a reference (e.g., unmodified or wild-type) TACI coding nucleic acid is mutagenicated from a reference (e.g., unmodified or wild-type) TACI genetic material and screened for desired specific binding affinity or other functional activity. In some embodiments, variant TACI polypeptides are synthesized de novo using protein or nucleic acid sequences available in any number of publicly available databases and then screened. The National Center for Biotechnology Information provides such information, and its website is publicly accessible via the internet, as is the UniProtKB database mentioned above.

[0331] Unless otherwise indicated, references to amino acid modifications of the variant TACI polypeptide BIM, as shown throughout this disclosure, are specified by amino acid position numbers corresponding to the position numbering of the reference ECD sequence listed in SEQ ID NO:709. Identifying the corresponding positions of modifications, e.g., amino acid substitutions, within the TACI polypeptide, including its TD (e.g., CRD1 and / or CRD2), by, for example, alignment of the reference sequence (e.g., SEQ ID NO:516 or 528) with SEQ ID NO:709, is within the scope of the art. An example of an alignment for identifying corresponding residues is shown in Figure 17A. In the list of modifications throughout this disclosure, the amino acid position is shown in the center, the corresponding reference (e.g., unmodified or wild-type) amino acid is listed before the number, and the identified variant amino acid substitution is listed after the number. If the modification is a deletion at that position, "del" is indicated, and if the modification is an insertion at that position, "ins" is indicated. In some cases, insertions are listed with their centrally located amino acid positions, the corresponding reference amino acids are listed before and after the numbers, and identified variant amino acid insertions are listed after the unmodified (e.g., wild-type) amino acids.

[0332] In some embodiments, BIM is a variant TACI polypeptide having one or more amino acid modifications, e.g., substitutions within a reference (e.g., unmodified or wild-type) TACI sequence, such as any of those described. One or more amino acid modifications, e.g., substitutions, may be located within the ectodomain (extracellular domain) of the reference (e.g., unmodified or wild-type) TACI sequence. In some embodiments, one or more amino acid modifications, e.g., substitutions, are located within the CRD1 domain or its specific binding fragment. In some embodiments, one or more amino acid modifications, e.g., substitutions, are located within the CRD2 domain or its specific binding fragment. In some embodiments of the variant TACI polypeptide, some of the one or more amino acid modifications, e.g., substitutions, are located within the CRD1 domain or its specific binding fragment, and some of the one or more amino acid modifications, e.g., substitutions, are located within the CRD2 domain or its specific binding fragment.

[0333] In some embodiments, the variant TACI polypeptide BIM has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, e.g., substitutions. These modifications, e.g., substitutions, may be located within the CRD1 domain or the CRD2 domain. In some embodiments, the variant TACI polypeptide BIM has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions within the CRD1 domain or its specific binding fragment. In some embodiments, the variant TACI polypeptide BIM has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions within the CRD2 domain or its specific binding fragment. In some embodiments, the variant TACI polypeptide comprising one or more described amino acid modifications (e.g., amino acid substitutions) has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a reference (e.g., unmodified or wild-type) TACI polypeptide or its specific binding fragment having an amino acid sequence such as SEQ ID NO: 516, 528, or 709. In some embodiments, a variant TACI polypeptide comprising one or more described amino acid modifications (e.g., amino acid substitutions) has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 709. In some embodiments, a variant TACI polypeptide comprising one or more described amino acid modifications (e.g., amino acid substitutions) has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 516.In some embodiments, a variant TACI polypeptide comprising one or more described amino acid modifications (e.g., amino acid substitutions) has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO:528.

[0334] In some embodiments, the variant TACI polypeptide BIM has one or more amino acid modifications, such as substitutions, to a reference TACI polypeptide or its specific binding fragment corresponding to positions 40, 59, 60, 61, 74, 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 87, 88, 92, 95, 97, 98, 99, 101, 102, and 103, based on the numbering of SEQ ID NO: 709. In some embodiments, BIM, which is a variant TACI polypeptide, has one or more amino acid modifications, such as substitutions selected from W40R, Q59R, R60G, T61P, E74V, Q75E, Q75R, G76S, K77E, F78Y, Y79F, L82H, L82P, L83S, R84G, R84L, R84Q, D85E, D85V, C86Y, I87L, I87M, S88N, I92V, Q95R, P97S, K98T, Q99E, A101D, Y102D, F103S, F103V, F103Y, or their conserved amino acid substitutions. In some embodiments, the reference TACI polypeptide includes a CRD1 domain or a CRD2 domain, for example, the reference TACI polypeptide is described in SEQ ID NO: 516 or SEQ ID NO: 709.

[0335] In some embodiments, the amino acid substitutions are located only in the CRD2 domain. In some embodiments, the variant TACI polypeptide BIM has one or more amino acid modifications, such as substitutions, in the reference TACI polypeptide or its specific binding fragments corresponding to positions 74, 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 87, 88, 92, 95, 97, 98, 99, 101, 102, and 103, relative to the numbering of SEQ ID NO: 709. In some embodiments, the variant TACI polypeptide BIM has one or more amino acid modifications, such as substitutions selected from E74V, Q75E, Q75R, G76S, K77E, F78Y, Y79F, L82H, L82P, L83S, R84G, R84L, R84Q, D85E, D85V, C86Y, I87L, I87M, S88N, I92V, Q95R, P97S, K98T, Q99E, A101D, Y102D, F103S, F103V, F103Y, or their conserved amino acid substitutions. In some embodiments, the reference TACI polypeptide contains only the CRD2 domain of the CRD domain but lacks the CRD1 domain, for example, the reference TACI polypeptide described in SEQ ID NO:528. Therefore, in some embodiments, the variant TACI polypeptide BIM contains a portion of the ECD sequence of a TACI polypeptide that contains a CRD2 domain but lacks a CRD1 domain.

[0336] Conservative amino acid modifications, such as substitutions, are any amino acid in the same class as the substituted amino acid, other than the reference (e.g., unmodified) or wild-type amino acid. The classes of amino acids are aliphatic (glycine, alanine, valine, leucine, and isoleucine), hydroxyl or sulfur-containing (serine, cysteine, threonine, and methionine), cyclic (proline), aromatic (phenylalanine, tyrosine, and tryptophan), basic (histidine, lysine, and arginine), and acidic / amide (aspartate, glutamate, asparagine, and glutamine).

[0337] In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution at position 75, relative to the numbering SEQ ID NO: 709. In some embodiments, the amino acid substitution at position 75 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain an amino acid substitution. In some embodiments, the substituted amino acid is an acidic amino acid or amide, e.g., a different acidic amino acid or amide compared to a reference (e.g., wild-type or unmodified) TACI polypeptide. In some embodiments, the substituted amino acid at position 75 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 75 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 75 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 75 is glutamine (Gln, Q).

[0338] In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution at position 77, relative to the numbering SEQ ID NO: 709. In some embodiments, the amino acid substitution at position 77 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain an amino acid substitution. In some embodiments, the substituted amino acid at position 77 is an acidic amino acid or amide. In some embodiments, the substituted amino acid at position 77 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 77 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 77 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 77 is glutamine (Gln, Q).

[0339] In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution at position 78, relative to the numbering SEQ ID NO: 709. In some embodiments, the amino acid substitution at position 78 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain the amino acid substitution. In some embodiments, the substituted amino acid at position 78 is an aromatic amino acid, e.g., a different aromatic amino acid compared to a reference (e.g., wild-type or unmodified) TACI polypeptide. In some embodiments, the substituted amino acid at position 78 is phenylalanine (Phe, F). In some embodiments, the substituted amino acid at position 78 is tyrosine (Tyr, Y). In some embodiments, the substituted amino acid at position 78 is tryptophan (Trp, W).

[0340] In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution at position 84 relative to the numbering SEQ ID NO:709. In some embodiments, the amino acid substitution at position 84 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain an amino acid substitution. In some embodiments, the substituted amino acid at position 84 is an acidic amino acid or amide. In some embodiments, the substituted amino acid at position 84 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 84 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 84 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 84 is glutamine (Gln, Q).

[0341] In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution at position 102, relative to the numbering SEQ ID NO:709. In some embodiments, the amino acid substitution at position 102 confers increased binding to BAFF or APRIL compared to a reference (e.g., wild-type or unmodified) TACI polypeptide that does not contain an amino acid substitution. In some embodiments, the substituted amino acid at position 102 is an acidic amino acid or amide. In some embodiments, the substituted amino acid at position 102 is glutamic acid (Glu, E). In some embodiments, the substituted amino acid at position 102 is aspartic acid (Asp, D). In some embodiments, the substituted amino acid at position 102 is asparagine (Asn, N). In some embodiments, the substituted amino acid at position 102 is glutamine (Gln, Q).

[0342] In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution E74V. In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution Q75E. In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution K77E. In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution F78Y. In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution Y79F. In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution L82H. In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution L82P. In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution R84G. In some embodiments, the variant TACI polypeptide BIM contains at least one amino acid substitution R84L. In some embodiments, the variant TACI polypeptide BIM comprises at least one amino acid substitution R84Q. In some embodiments, the variant TACI polypeptide BIM comprises at least one amino acid substitution D85V. In some embodiments, the variant TACI polypeptide BIM comprises at least one amino acid substitution C86Y. In some embodiments, the variant TACI polypeptide BIM comprises at least one amino acid substitution Y102D. In some embodiments, the variant TACI polypeptide BIM comprises two or more amino acid substitutions from any two or more of the aforementioned. In some embodiments, the variant TACI polypeptide BIM comprises one or more amino acid substitutions which are any of the aforementioned conserved amino acid substitutions. In the provided embodiments, the variant TACI polypeptide BIM comprises at least one amino acid substitution in any reference TACI polypeptide sequence described. In some embodiments, at least one amino acid substitution is in the reference TACI sequence described in SEQ ID NO:516.In some embodiments, at least one amino acid substitution is located in the reference TACI sequence described in SEQ ID NO: 528. In some embodiments, at least one amino acid substitution is located in the reference TACI sequence described in SEQ ID NO: 718. In some embodiments, at least one amino acid substitution is located in the reference TACI sequence described in SEQ ID NO: 719.

[0343] In some embodiments, the variant TACI polypeptide BIM contains the amino acid substitution E74V. In some embodiments, the variant TACI polypeptide BIM contains the amino acid substitution Q75E. In some embodiments, the variant TACI polypeptide BIM contains the amino acid substitution K77E. In some embodiments, the variant TACI polypeptide BIM contains the amino acid substitution F78Y. In some embodiments, the variant TACI polypeptide BIM contains the amino acid substitution Y79F. In some embodiments, the variant TACI polypeptide BIM contains the amino acid substitution L82H. In some embodiments, the variant TACI polypeptide BIM contains the amino acid substitution L82P. In some embodiments, the variant TACI polypeptide BIM contains the amino acid substitution R84G. In some embodiments, the variant TACI polypeptide BIM contains the amino acid substitution R84L. In some embodiments, the variant TACI polypeptide BIM contains the amino acid substitution R84Q. In some embodiments, the variant TACI polypeptide BIM contains the amino acid substitution D85V. In some embodiments, the variant TACI polypeptide BIM contains the amino acid substitution C86Y. In some embodiments, the variant TACI polypeptide BIM contains the amino acid substitution Y102D. In some embodiments, the variant TACI polypeptide BIM contains two or more of the two or more amino acid substitutions described above. In some embodiments, the variant TACI polypeptide BIM contains one or more of the amino acid substitutions that are any of the conservative amino acid substitutions described above. In the embodiments provided, the variant TACI polypeptide BIM contains an amino acid substitution in any reference TACI polypeptide sequence described. In some embodiments, the amino acid substitution is in the reference TACI sequence described in SEQ ID NO: 516. In some embodiments, the amino acid substitution is in the reference TACI sequence described in SEQ ID NO: 528. In some embodiments, the amino acid substitution is in the reference TACI sequence described in SEQ ID NO: 718.In some embodiments, the amino acid substitution is in the reference TACI sequence described in SEQ ID NO:719. In some embodiments, the amino acid substitution is D85E / K98T. In some embodiments, the amino acid substitution is I87L / K98T. In some embodiments, the amino acid substitution is R60G / Q75E / L82P. In some embodiments, the amino acid substitution is R60G / C86Y. In some embodiments, the amino acid substitution is W40R / L82P / F103Y. In some embodiments, the amino acid substitution is W40R / Q59R / T61P / K98T. In some embodiments, the amino acid substitution is L82P / I87L. In some embodiments, the amino acid substitution is G76S / P97S. In some embodiments, the amino acid substitution is K77E / R84L / F103Y. In some embodiments, the amino acid substitution is Y79F / Q99E. In some embodiments, the amino acid substitution is L83S / F103S. In some embodiments, the amino acid substitution is K77E / R84Q. In some embodiments, the amino acid substitution is K77E / A101D. In some embodiments, the amino acid substitution is K77E / F78Y / Y102D. In some embodiments, the amino acid substitution is Q75E / R84Q. In some embodiments, the amino acid substitution is Q75R / R84G / I92V. In some embodiments, the amino acid substitution is K77E / A101D / Y102D. In some embodiments, the amino acid substitution is R84Q / S88N / A101D. In some embodiments, the amino acid substitution is R84Q / F103V. In some embodiments, the amino acid substitution is K77E / Q95R / A101D. In some embodiments, the amino acid substitution is I87M / A101D. In the embodiments provided, BIM, which is a variant TACI polypeptide, contains an amino acid substitution in any reference TACI polypeptide sequence described. In some embodiments, the amino acid substitution is in the reference TACI sequence described in SEQ ID NO: 516. In some embodiments, the amino acid substitution is in the reference TACI sequence described in SEQ ID NO: 528. In some embodiments, the amino acid substitution is in the reference TACI sequence described in SEQ ID NO: 718. In some embodiments, the amino acid substitution is in the reference TACI sequence described in SEQ ID NO: 719.

[0344] In some embodiments, the variant TACI polypeptide BIM comprises the amino acid substitutions K77E and F78Y (K77E / F78Y). In the provided embodiments, the variant TACI polypeptide BIM comprises the amino acid substitution in any reference TACI polypeptide sequence described. In some embodiments, the amino acid substitution is located in the reference TACI sequence described in SEQ ID NO: 516. In some embodiments, the amino acid substitution is located in the reference TACI sequence described in SEQ ID NO: 528. In some embodiments, the amino acid substitution is located in the reference TACI sequence described in SEQ ID NO: 718. In some embodiments, the amino acid substitution is located in the reference TACI sequence described in SEQ ID NO: 719.

[0345] In some embodiments, the variant TACI polypeptide BIM comprises the amino acid substitutions K77E and Y102D (K77E / Y102D). In the provided embodiments, the variant TACI polypeptide BIM comprises the amino acid substitution in any reference TACI polypeptide sequence described. In some embodiments, the amino acid substitution is located in the reference TACI sequence described in SEQ ID NO: 516. In some embodiments, the amino acid substitution is located in the reference TACI sequence described in SEQ ID NO: 528. In some embodiments, the amino acid substitution is located in the reference TACI sequence described in SEQ ID NO: 718. In some embodiments, the amino acid substitution is located in the reference TACI sequence described in SEQ ID NO: 719.

[0346] In some embodiments, the variant TACI polypeptide BIM comprises the amino acid substitutions F78Y and Y102D (F78Y / Y012D). In the provided embodiments, the variant TACI polypeptide BIM comprises the amino acid substitution in any reference TACI polypeptide sequence described. In some embodiments, the amino acid substitution is located in the reference TACI sequence described in SEQ ID NO: 516. In some embodiments, the amino acid substitution is located in the reference TACI sequence described in SEQ ID NO: 528. In some embodiments, the amino acid substitution is located in the reference TACI sequence described in SEQ ID NO: 718. In some embodiments, the amino acid substitution is located in the reference TACI sequence described in SEQ ID NO: 719.

[0347] In some embodiments, the variant TACI polypeptide BIM comprises the amino acid substitutions K77E, F78Y, and Y102D (K77E / F78Y / Y102D). In the provided embodiments, the variant TACI polypeptide BIM comprises the amino acid substitutions in any reference TACI polypeptide sequence described. In some embodiments, the amino acid substitutions are located in the reference TACI sequence described in SEQ ID NO: 516. In some embodiments, the amino acid substitutions are located in the reference TACI sequence described in SEQ ID NO: 528. In some embodiments, the amino acid substitutions are located in the reference TACI sequence described in SEQ ID NO: 718. In some embodiments, the amino acid substitutions are located in the reference TACI sequence described in SEQ ID NO: 719.

[0348] In some embodiments, the variant TACI polypeptide BIM contains the amino acid substitution Q75E / R84Q. In the provided embodiments, the variant TACI polypeptide BIM contains the amino acid substitution in any reference TACI polypeptide sequence described. In some embodiments, the amino acid substitution is in the reference TACI sequence described in SEQ ID NO: 516. In some embodiments, the amino acid substitution is in the reference TACI sequence described in SEQ ID NO: 528. In some embodiments, the amino acid substitution is in the reference TACI sequence described in SEQ ID NO: 718. In some embodiments, the amino acid substitution is in the reference TACI sequence described in SEQ ID NO: 719.

[0349] In some embodiments, the variant TACI polypeptide BIM contains one of the mutations listed in Table 2. Table 2 also presents exemplary sequences by referencing the SEQ ID NOs of the reference (e.g., unmodified) TACI polypeptide and the exemplary variant TACI polypeptide. As shown, the exact locus or residues corresponding to a given domain may vary, for example, depending on the method used to identify or classify that domain. Also, in some cases, the adjacent N-terminal and / or C-terminal amino acids of a given domain (e.g., CRD) may also be included in the sequence of the variant TACI polypeptide BIM, for example, to ensure proper folding of the domain when expressed. Thus, it should be understood that the examples of SEQ ID NOs in Table 2 should not be interpreted as limiting. For example, a particular domain of a variant TACI polypeptide, e.g., the ECD domain, or a portion thereof containing CRD1 / CRD2, or CRD2 only, may be several amino acids longer or shorter than the amino acid sequence listed in their respective SEQ ID NOs, e.g., 1 to 10, e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids longer or shorter.

[0350] In some embodiments, the variant TACI polypeptide BIM contains one of the mutations (amino acid substitutions) listed in Table 2. In some examples, the mutation (amino acid substitution) is made within a reference TACI containing the amino acid sequence described in SEQ ID NO:709. In some examples, the mutation (amino acid substitution) is made within a reference TACI containing the CRD1 and CRD2 domains of the TACI, as described, for example, in SEQ ID NO:516. In some examples, the mutation (amino acid substitution) is made within a reference TACI that has been further cleaved by deletions of N-terminal and C-terminal amino acid residues to preserve CRD2, as described, for example, in SEQ ID NO:528.

[0351] The use of the term “modification,” e.g., “substitution” or “mutation,” does not mean that this embodiment is limited to a specific method for producing immunomodulatory proteins. BIM, which is a variant TACI polypeptide, can be produced, for example, by de novo peptide synthesis and therefore does not necessarily require modification, such as “substitution,” in the sense of altering codons to encode substitutions. This principle also extends to the terms “addition” and “deletion” of amino acid residues, which similarly do not imply a specific method of production. The means for designing or producing vTDs are not limited to a specific method. However, in some embodiments, wild-type TD-coding nucleic acids or unmodified TD-coding nucleic acids are mutagenicated from wild-type TD genetic material or unmodified TD genetic material and screened for changes in desired specific binding activity, e.g., binding affinity, and / or NF-κB regulation or other functional activity. In some embodiments, vTDs are de novo synthesized using protein or nucleic acid sequences available in any number of publicly available databases and then screened. The National Center for Biotechnology Information provides such information, and its website is publicly accessible via the internet, such as the UniProtKB database.

[0352] In some embodiments, the variant TACI polypeptide BIM comprises an extracellular domain (ECD) sequence containing CRD1 and CRD2, e.g., a variant TACI polypeptide described in any one of SEQ ID NO: 517-527, 536, 537, 682-701. In some embodiments, the variant TACI polypeptide BIM comprises a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NO: 517-527, 536, 537, 682-701, and retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide BIM comprises one specific binding fragment from among SEQ ID NO: 517-527, 536, 537, 682-701, wherein the specific binding fragment binds to BAFF, APRIL, or BAFF / APRIL heterotrimer and comprises a continuous sequence including amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI.

[0353] In some embodiments, the variant TACI polypeptide BIM consists of or is essentially derived from a variant TACI extracellular domain (ECD) sequence described in any one of SEQ ID NO: 517-527, 536, 537, or 682-701. In some embodiments, the variant TACI polypeptide BIM consists of or is essentially derived from a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NO: 517-527, 536, 537, or 682-701, and retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide BIM consists of or is essentially one of the following specific binding fragments: SEQ ID NO: 517-527, 536, 537, 682-701, the specific binding fragment being bound to BAFF, APRIL, or APRIL / BAFF heterotrimer and comprising a continuous sequence containing amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI.

[0354] In some embodiments, a variant TACI polypeptide, e.g., a BIM that is a variant TACI polypeptide described in any one of SEQ ID NO: 529-535, 538-550, or 673-681, includes an extracellular domain (ECD) sequence containing CRD2 but lacking CRD1 of the reference TACI polypeptide. In some embodiments, a BIM that is a variant TACI polypeptide exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NO: 529-535, 538-550, or 673-681, and includes a polypeptide sequence that retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide BIM comprises one specific binding fragment from among SEQ ID NO: 529-535, 538-550, and 673-681, wherein the specific binding fragment binds to BAFF, APRIL, or a BAFF / APRIL heterotrimer and comprises a continuous sequence including amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI.

[0355] In some embodiments, the variant TACI polypeptide BIM consists of or is essentially derived from the sequence described in any one of SEQ ID NO: 529-535, 538-550, or 673-681. In some embodiments, the variant TACI polypeptide BIM consists of or is essentially derived from the polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NO: 529-535, 538-550, or 673-681, and retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, the variant TACI polypeptide BIM consists of or is essentially composed of one specific binding fragment from SEQ ID NO: 529-535, 538-550, 673-681, the specific binding fragment being bound to BAFF, APRIL, or BAFF / APRIL heterotrimer and comprising a continuous sequence containing amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI.

[0356] In some embodiments, the variant TACI polypeptide BIM is one in which the variant TACI polypeptide contains the sequence described in SEQ ID NO: 535. In some embodiments, the variant TACI polypeptide is essentially derived from the sequence described in SEQ ID NO: 535. In some embodiments, the variant TACI polypeptide consists of the sequence described in SEQ ID NO: 535.

[0357] In some embodiments, the variant TACI polypeptide BIM is one in which the variant TACI polypeptide contains the sequence described in SEQ ID NO: 541. In some embodiments, the variant TACI polypeptide is essentially derived from the sequence described in SEQ ID NO: 541. In some embodiments, the variant TACI polypeptide consists of the sequence described in SEQ ID NO: 541.

[0358] In some embodiments, the variant TACI polypeptide BIM is one in which the variant TACI polypeptide contains the sequence described in SEQ ID NO: 542. In some embodiments, the variant TACI polypeptide is essentially derived from the sequence described in SEQ ID NO: 542. In some embodiments, the variant TACI polypeptide consists of the sequence described in SEQ ID NO: 542.

[0359] In some embodiments, the variant TACI polypeptide BIM is one in which the variant TACI polypeptide contains the sequence described in SEQ ID NO:688. In some embodiments, the variant TACI polypeptide is essentially derived from the sequence described in SEQ ID NO:688. In some embodiments, the variant TACI polypeptide consists of the sequence described in SEQ ID NO:688.

[0360] In some embodiments, the variant TACI polypeptide BIM is one in which the variant TACI polypeptide is encoded by a nucleotide sequence described in any of SEQ ID NO: 552-562, 571, or 572. In some embodiments, the variant TACI polypeptide is encoded by a nucleotide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NO: 552-562, 571, or 572, and that retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI.

[0361] In some embodiments, the variant TACI polypeptide BIM is one in which the variant TACI polypeptide is encoded by a nucleotide sequence described in either SEQ ID NO: 564-570 or 573-585. In some embodiments, the variant TACI polypeptide is encoded by a nucleotide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to one of either SEQ ID NO: 564-570 or 573-585, and retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI.

[0362] (Table 2) Exemplary variant TACI BIM TIFF2026143547000020.tif187165TIFF2026143547000021.tif230165TIFF2026143547000022.tif142165

[0363] In some embodiments, BIM is a wild-type ECD or unmodified ECD of TACI, or a specific binding moiety or specific binding fragment thereof, or includes at least one TD (e.g., at least one CRD, e.g., CRD1 and / or CRD2) that binds to APRIL, BAFF, and / or APRIL / BAFF heterotrimers. In some embodiments, the BIM comprises (ii) an ECD sequence described in SEQ ID NO:709; (ii) an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:709 and binding to APRIL, BAFF, or an APRIL / BAFF heterotrimer; or (iii) a fragment or portion of (i) or (ii) comprising CRD1 and / or CRD2, wherein the portion binds to APRIL, BAFF, or an APRIL / BAFF heterotrimer. In some embodiments, the BIM is a TACI sequence consisting of or essentially derived from the sequence described in SEQ ID NO:709. In some embodiments, BIM is a TACI sequence comprising residues 2–166 of SEQ ID NO:709, lacking the N-terminal methionine described in SEQ ID NO:709. In some embodiments, BIM is a TACI sequence consisting of or essentially derived from the sequence described as amino acids 2–166 of SEQ ID NO:709.

[0364] In some embodiments, BIM is a binding moiety of the wild-type ECD or unmodified ECD of TACI, or a specific binding moiety or specific binding fragment thereof, comprising CRD1 and CRD2 of TACI, and binding to APRIL, BAFF, and / or APRIL / BAFF heterotrimers. In some embodiments, the BIM comprises (ii) the sequence described in SEQ ID NO:719; (ii) an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:719 and binding to APRIL, BAFF, or an APRIL / BAFF heterotrimer; or (iii) a fragment or portion of (i) or (ii) comprising a portion of CRD1 and / or CRD2, wherein the portion binds to APRIL, BAFF, or an APRIL / BAFF heterotrimer. In some embodiments, the BIM comprises the sequence described in SEQ ID NO:719. In some embodiments, the BIM is a TACI sequence consisting of or essentially derived from the sequence described in SEQ ID NO:719. In some embodiments, the BIM comprises (ii) the sequence described in SEQ ID NO:718; (ii) an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:718 and binding to APRIL, BAFF, or an APRIL / BAFF heterotrimer; or (iii) a fragment or portion of (i) or (ii) comprising a portion of CRD1 and / or CRD2, wherein the portion binds to APRIL, BAFF, or an APRIL / BAFF heterotrimer. In some embodiments, the BIM comprises the sequence described in SEQ ID NO:718. In some embodiments, the BIM is a TACI sequence consisting of or essentially derived from the sequence described in SEQ ID NO:718.In some embodiments, the BIM comprises (ii) the sequence described in SEQ ID NO:516; (ii) an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:516 and binding to APRIL, BAFF, or an APRIL / BAFF heterotrimer; or (iii) a fragment or portion of (i) or (ii) comprising a portion of CRD1 and / or CRD2, wherein the portion binds to APRIL, BAFF, or an APRIL / BAFF heterotrimer. In some embodiments, the BIM comprises the sequence described in SEQ ID NO:516. In some embodiments, the BIM is a TACI sequence consisting of or essentially derived from the sequence described in SEQ ID NO:516.

[0365] In some embodiments, BIM comprises only the CRD2 of TACI and is a binding portion of the wild-type ECD or unmodified ECD of TACI, or a specific binding portion or specific binding fragment thereof, that binds to APRIL, BAFF, and / or APRIL / BAFF heterotrimer. In some embodiments, the BIM comprises (ii) the sequence described in SEQ ID NO:528; (ii) an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with respect to SEQ ID NO:528, and binding to APRIL, BAFF, or an APRIL / BAFF heterotrimer; or (iii) a fragment or portion of (i) or (ii) comprising a part of CRD2, wherein the portion binds to APRIL, BAFF, or an APRIL / BAFF heterotrimer. In some embodiments, the BIM comprises the sequence described in SEQ ID NO:528. In some embodiments, the BIM is a TACI sequence consisting of or essentially derived from the sequence described in SEQ ID NO:528.

[0366] In some embodiments, the BIM is a variant TACI comprising an ECD or a specific binding portion or specific binding fragment thereof having a vTD containing one or more amino acid substitutions (mutations or replacements) compared to a reference TACI sequence. The reference TACI sequence may include any of those described in Section II above. The other amino acid substitution may include any of those described in Section II above. For example, the BIM may be a variant TACI comprising an ECD or a specific binding fragment thereof having a vTD containing any of the amino acid substitutions described in Table 2. In some examples, the mutation is made within a reference TACI containing the amino acid sequence described in SEQ ID NO:709. In some examples, the mutation is made within a reference TACI containing the CRD1 and CRD2 domains of the TACI, for example, as described in SEQ ID NO:516. In some examples, the mutation is made within a reference TACI that has been further cleaved by deletions of N-terminal and C-terminal amino acid residues to retain CRD2, for example, as described in SEQ ID NO:528.

[0367] In some embodiments, BIM comprises an extracellular domain (ECD) sequence including CRD1 and CRD2, e.g., a variant TACI polypeptide described in any one of SEQ ID NO: 517-527, 536, 537, 682-701. In some embodiments, BIM is a variant TACI polypeptide comprising a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NO: 517-527, 536, 537, 682-701, and retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, BIM is a variant TACI polypeptide comprising one specific binding fragment from SEQ ID NO: 517-527, 536, 537, 682-701, wherein the specific binding fragment binds to BAFF, APRIL, or a BAFF / APRIL heterotrimer and comprises a continuous sequence including amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI.

[0368] In some embodiments, BIM is a variant TACI polypeptide consisting of or essentially derived from a variant TACI extracellular domain (ECD) sequence described in any one of SEQ ID NO: 517-527, 536, 537, 682-701. In some embodiments, BIM is a variant TACI polypeptide consisting of or essentially derived from a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NO: 517-527, 536, 537, 682-701, and that retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, BIM is a variant TACI polypeptide consisting of or essentially derived from one specific binding fragment among SEQ ID NO: 517-527, 536, 537, 682-701, wherein the specific binding fragment binds to BAFF, APRIL, or APRIL / BAFF heterotrimer and comprises a continuous sequence including amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI.

[0369] In some embodiments, BIM is a variant TACI polypeptide comprising an extracellular domain (ECD) sequence containing CRD2 but lacking CRD1 of a reference TACI polypeptide, e.g., a variant TACI polypeptide described in any one of SEQ ID NO: 529-535, 538-550, 673-681, or 769-794. In some embodiments, BIM is a variant TACI polypeptide comprising a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NO: 529-535, 538-550, or 673-681, and retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, BIM is a variant TACI polypeptide comprising one specific binding fragment from among SEQ ID NO: 529-535, 538-550, 673-681, and 769-794, wherein the specific binding fragment binds to BAFF, APRIL, or a BAFF / APRIL heterotrimer and comprises a continuous sequence including amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI.

[0370] In some embodiments, BIM is a variant TACI polypeptide consisting of or essentially derived from the sequence described in any one of SEQ ID NO: 529-535, 538-550, 673-681, or 769-794. In some embodiments, BIM is a variant TACI polypeptide consisting of or essentially derived from a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NO: 529-535, 538-550, 673-681, or 769-794, and retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI. In some embodiments, BIM is a variant TACI polypeptide consisting of or essentially derived from one specific binding fragment among SEQ ID NO: 529-535, 538-550, 673-681, and 769-794, wherein the specific binding fragment binds to BAFF, APRIL, or a BAFF / APRIL heterotrimer and comprises a continuous sequence including amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) TACI.

[0371] In some embodiments, BIM is a variant TACI polypeptide comprising the sequence described in SEQ ID NO: 535. In some embodiments, BIM is a variant TACI polypeptide essentially consisting of the sequence described in SEQ ID NO: 535. In some embodiments, BIM is a variant TACI polypeptide consisting of the sequence described in SEQ ID NO: 535.

[0372] In some embodiments, BIM is a variant TACI polypeptide comprising the sequence described in SEQ ID NO: 541. In some embodiments, BIM is a variant TACI polypeptide essentially consisting of the sequence described in SEQ ID NO: 541. In some embodiments, BIM is a variant TACI polypeptide consisting of the sequence described in SEQ ID NO: 541.

[0373] In some embodiments, BIM is a variant TACI polypeptide comprising the sequence described in SEQ ID NO: 542. In some embodiments, BIM is a variant TACI polypeptide essentially consisting of the sequence described in SEQ ID NO: 542. In some embodiments, BIM is a variant TACI polypeptide consisting of the sequence described in SEQ ID NO: 542.

[0374] In some embodiments, BIM is a variant TACI polypeptide comprising the sequence described in SEQ ID NO:688. In some embodiments, BIM is a variant TACI polypeptide essentially consisting of the sequence described in SEQ ID NO:688. In some embodiments, BIM is a variant TACI polypeptide consisting of the sequence described in SEQ ID NO:688.

[0375] 2. BCMA In some embodiments, BIM is a wild-type BCMA ECD containing a TD (e.g., CRD) that binds to APRIL, BAFF, and / or APRIL / BAFF heterotrimers, or a specific binding moiety or specific binding fragment thereof, or includes the same. In some embodiments, BIM is a variant BCMA ECD containing a TD (e.g., CRD) that binds to APRIL, BAFF, and / or APRIL / BAFF heterotrimers, or a specific binding moiety or specific binding fragment thereof, or includes the same. In some embodiments, BIM is a BCMA polypeptide or a variant thereof having any of the sequences described in Section II above (e.g., Table 1).

[0376] In some embodiments, BIM is a wild-type ECD or unmodified ECD of BCMA, or a specific binding moiety or specific binding fragment thereof, or includes a TD (e.g., CRD) that binds to APRIL, BAFF, and / or APRIL / BAFF heterotrimers. In some embodiments, the BIM comprises (ii) an ECD sequence described in SEQ ID NO:710; (ii) an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:710 and binding to APRIL, BAFF, or an APRIL / BAFF heterotrimer; or (iii) a fragment or portion of (i) or (ii) comprising a CRD, wherein the portion binds to APRIL, BAFF, or an APRIL / BAFF heterotrimer. In some embodiments, the BIM is a BCMA sequence consisting of or essentially derived from the sequence described in SEQ ID NO:710. In some embodiments, BIM is a BCMA sequence containing residues 2-54 of SEQ ID NO:710, lacking the N-terminal methionine described in SEQ ID NO:710. In some embodiments, BIM is a BCMA sequence consisting of or essentially derived from the sequence described as amino acids 2-54 of SEQ ID NO:710.

[0377] In some embodiments, the BIM comprises (ii) the sequence described in SEQ ID NO:356; (ii) an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with respect to SEQ ID NO:356 and binds to APRIL, BAFF, or an APRIL / BAFF heterotrimer; or (iii) a fragment or portion of (i) or (ii) comprising a part of the CRD, wherein the portion binds to APRIL, BAFF, or an APRIL / BAFF heterotrimer. In some embodiments, the BIM comprises the sequence described in SEQ ID NO:356. In some embodiments, the BIM is a BCMA sequence consisting of or essentially derived from the sequence described in SEQ ID NO:356.

[0378] In some embodiments, the BIM is a variant BCMA comprising an ECD or a specific binding portion or specific binding fragment having a vTD containing one or more amino acid substitutions (mutations or replacements) compared to a reference BCMA sequence. The reference BCMA sequence may include any of those described in Section II above. The other amino acid substitution may include any of those described in Section II above. For example, the BIM may be a variant BCMA comprising an ECD or a specific binding fragment having a vTD containing any of the amino acid substitutions listed in Table 1. In some examples, the mutation is introduced within a reference BCMA containing the amino acid sequence described in SEQ ID NO: 710. In some examples, the mutation is introduced within a reference BCMA containing the CRD domain of the BCMA. In some examples, the mutation is introduced within a reference BCMA described in SEQ ID NO: 356.

[0379] In some embodiments, BIM comprises a variant BCMA polypeptide described in any one of SEQ ID NO:357-435. In some embodiments, BIM is a variant BCMA polypeptide that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, for example, at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NO:357-435, and comprises a polypeptide sequence that retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) BCMA. In some embodiments, BIM is a variant BCMA polypeptide comprising one specific binding fragment from SEQ ID NO:357-435, wherein the specific binding fragment binds to BAFF, APRIL, or a BAFF / APRIL heterotrimer and comprises a continuous sequence including amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) BCMA.

[0380] In some embodiments, BIM is a variant BCMA polypeptide consisting of or essentially derived from a variant BCMA extracellular domain (ECD) sequence described in any one of SEQ ID NO:357-435. In some embodiments, BIM is a variant BCMA polypeptide consisting of or essentially derived from a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, e.g., at least 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NO:357-435, and retains amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) BCMA. In some embodiments, BIM is a variant BCMA polypeptide consisting of or essentially derived from one specific binding fragment of SEQ ID NO:357-435, the specific binding fragment being bound to BAFF, APRIL, or APRIL / BAFF heterotrimer and comprising a continuous sequence including amino acid modifications, e.g., substitutions, that are not present in the reference (e.g., unmodified or wild-type) BCMA.

[0381] In some embodiments, BIM is a variant BCMA polypeptide containing the sequence described in SEQ ID NO:381. In some embodiments, BIM is a variant BCMA polypeptide essentially consisting of the sequence described in SEQ ID NO:381. In some embodiments, BIM is a variant BCMA polypeptide consisting of the sequence described in SEQ ID NO:381.

[0382] In some embodiments, BIM is a variant BCMA polypeptide comprising the sequence described in SEQ ID NO:405. In some embodiments, BIM is a variant BCMA polypeptide essentially consisting of the sequence described in SEQ ID NO:405. In some embodiments, BIM is a variant BCMA polypeptide consisting of the sequence described in SEQ ID NO:405.

[0383] In some embodiments, BIM is a variant BCMA polypeptide comprising the sequence described in SEQ ID NO:406. In some embodiments, BIM is a variant BCMA polypeptide essentially consisting of the sequence described in SEQ ID NO:406. In some embodiments, BIM is a variant BCMA polypeptide consisting of the sequence described in SEQ ID NO:406.

[0384] In some embodiments, BIM is a variant BCMA polypeptide comprising the sequence described in SEQ ID NO:410. In some embodiments, BIM is a variant BCMA polypeptide essentially consisting of the sequence described in SEQ ID NO:410. In some embodiments, BIM is a variant BCMA polypeptide consisting of the sequence described in SEQ ID NO:410.

[0385] In some embodiments, BIM is a variant BCMA polypeptide comprising the sequence described in SEQ ID NO:411. In some embodiments, BIM is a variant BCMA polypeptide essentially consisting of the sequence described in SEQ ID NO:411. In some embodiments, BIM is a variant BCMA polypeptide consisting of the sequence described in SEQ ID NO:411.

[0386] B. T cell inhibitory molecules (TIMs) In some embodiments, the immunomodulatory protein provided comprises a T cell stimulatory receptor (TCR), or a TIM that binds to a ligand of the TCR. In some embodiments, the TCR comprises a cytoplasmic region containing an immunoreceptor-activated tyrosine motif (ITAM), or a cytoplasmic region that interacts with one or more adapter proteins involved in intracellular signaling pathways to induce, mediate, or enhance T cell activation. In some embodiments, the adapter protein comprises a binding domain specific to a phosphotyrosine residue within the cytoplasmic region of the stimulatory receptor. In some embodiments, the TCR comprises a component of the TCR complex, or a co-receptor or co-stimulatory molecule that increases or enhances TCR signaling. In some embodiments, the TCR is TCR, CD3, CD4, CD8, CD28, ICOS, or CD2, including any mammalian orthologue thereof. In some embodiments, the TCR target is human TCR, human CD3, human CD4, human CD8, human CD28, human ICOS, or human CD2. In some embodiments, the TCR is expressed on human T cells.

[0387] In some embodiments, TIMs bind directly to T cell stimulating receptors, for example, components of the TCR complex, or to co-receptors or co-stimulatory molecules that increase or enhance TCR signaling. In some embodiments, TIMs bind to TCR, CD3, CD4, CD8, CD28, ICOS, or CD2, including any mammalian orthologue thereof. In some embodiments, TIMs bind to human TCR, human CD3, human CD4, human CD8, human CD28, human ICOS, or human CD2.

[0388] In some cases, TIMs bind to ligands of T cell stimulatory receptors. In some embodiments, TIMs bind to ligands of components of the TCR complex, or to ligands of co-receptors or co-stimulatory molecules that increase or enhance TCR signaling. In some embodiments, TIMs bind to ligands of TCR molecules, CD3 molecules, CD4 molecules, CD8 molecules, CD28 molecules, ICOS molecules, or CD2 molecules, for example, those expressed on T cells, e.g., human T cells. In some embodiments, TIMs bind to ligands of CD28, e.g., those expressed on T cells, e.g., human T cells. In some embodiments, the ligand is CD80 or CD86, e.g., human CD80 or human CD86. In some embodiments, the ligand is expressed on APCs.

[0389] In some embodiments, the TIM is an antibody or antigen-binding fragment that binds to a T cell stimulating receptor or a ligand for a T cell stimulating receptor. In some embodiments, the TIM is an antibody or antigen-binding fragment that binds to TCR, CD3, CD4, CD8, CD28, ICOS, or CD2, including any mammalian orthologue thereof. In some embodiments, the antibody or antigen-binding fragment binds to human TCR, human CD3, human CD4, human CD8, human CD28, human ICOS, or human CD2, for example, such a molecule expressed on human T cells. In some embodiments, the antibody or antigen-binding fragment binds to CD80 or CD86. In some embodiments, the antibody or antigen-binding fragment binds to human CD80 or human CD86, for example, such a molecule expressed on human APCs.

[0390] In some embodiments, a TIM is a binding partner of a T cell stimulating receptor or a ligand of a T cell stimulating receptor, or includes such a partner. Among the molecules that can be used as a TIM are IgSF protein members, in particular the extracellular domain of an IgSF member that is a T cell stimulating receptor or a ligand thereof.

[0391] In some aspects, TIM is an IgD of an IgSF family member that binds to a T cell stimulating receptor, such as TCR, CD3, CD4, CD8, CD28, ICOS, or CD2, or it is a specific fragment or vIgD of that IgD that binds to a T cell stimulating receptor. In certain embodiments, the T cell stimulating receptor is CD28 or ICOS, and TIM binds to CD28 or ICOS. Exemplary IgSF family members that are binding partners of CD28 or ICOS, or that bind to CD28 or ICOS, include, for example, CD80, CD86, and ICOSL, e.g., human CD80, human CD86, or human ICOSL. In some examples, TIM is an IgD of wild-type CD80, wild-type CD86, or wild-type ICOSL, or it is a vIgD of that IgD, or it is a vIgD of that IgD, and TIM specifically binds to CD28.

[0392] In other contexts, a TIM is an IgD of an IgSF family member that binds to a T cell stimulating receptor ligand, for example, CD80 or CD86, or a specific fragment or vIgD of that IgSF family member that binds to a T cell stimulating receptor ligand. Exemplary IgSF family members that are binding partners of CD80 or CD86, or that bind to CD80 or CD86, include CTLA-4, such as human CTLA-4. In some examples, the TIM is an IgD of wild-type CTLA-4, or that includes it, or its vIgD, and the TIM specifically binds to CD80 or CD86. Exemplary sequences for inclusion as a TIM within a multi-domain immunomodulatory protein provided include molecules described in International PCT Publication Application No. WO2019 / 074983.

[0393] In some embodiments, the multi-domain immunomodulatory proteins provided herein are soluble proteins and / or do not contain a portion containing a transmembrane domain. Those skilled in the art will understand that cell surface proteins containing IgSF proteins typically have an intracellular domain, a transmembrane domain and an extracellular domain (ECD), and that soluble forms of such proteins can be prepared using the extracellular domain or an immunoactive sub-sequence thereof. Therefore, in some embodiments, the TIM lacks the transmembrane domain or a portion of the transmembrane domain of the IgSF member. In some embodiments, the TIM lacks the intracellular (cytoplasmic) domain or a portion of the intracellular domain of the IgSF member. In some embodiments, the TIM contains only the ECD domain or a portion thereof or a specific binding fragment containing an IgSF domain such as an IgV domain.

[0394] For example, in some aspects, the TIM is or includes the ECD of the IgSF receptor, or its specific binding portion or specific binding fragment, containing at least one IgD (e.g., IgV) that binds to the ligand of the T cell stimulating receptor. For example, the TIM may include the ECD of CTLA-4, or the specific binding portion or specific binding fragment of CTLA-4, containing at least one IgD (e.g., IgV) that binds to CD80 or CD86. In some embodiments, the TIM consists of or is essentially the ECD of the IgSF receptor, or its specific binding portion or specific binding fragment, containing at least one IgD (e.g., IgV), for example, the ECD of CTLA-4, or the specific binding portion or specific binding fragment of the ECD of CTLA-4, containing IgD (e.g., IgV). In some embodiments, the TIM is less than the full-length sequence of the receptor ECD of the ligand of the T cell stimulating receptor. In some embodiments, the TIM is a specific binding fragment of one vIgD (e.g., IgV), or only one vIgD (e.g., IgV), or comprises only that. In some embodiments, the TIM consists of or is essentially of the IgV of the receptor of the ligand of the T cell stimulating receptor, e.g., consisting of or is essentially of the IgV of CTLA-4. In some embodiments, the sequence of the TIM containing an ECD containing IgD (e.g., IgV) or its binding portion or binding fragment is a mammalian sequence, including, but not limited to, human, mouse, cynomolgus monkey, or rat. In some embodiments, the TIM sequence is human and / or binds to a human protein.

[0395] In some aspects, vIgD is an affinity-modified domain that exhibits increased binding activity, e.g., increased binding affinity to T cell-stimulating receptors or T cell-stimulating receptor ligands, compared to the binding activity of unmodified or wild-type IgD to the same molecule. In some embodiments, the TIM comprises a vIgD having one or more amino acid substitutions compared to IgD of an IgSF member, e.g., CTLA-4o, where one or more amino acid substitutions confer or result in increased binding affinity to a congenerally binding partner that is a T cell-stimulating receptor or a T cell-stimulating receptor ligand.

[0396] In some embodiments, the TIM is a vIgD containing one or more amino acid modifications, e.g., one or more substitutions (or "mutations" or "replacements"), deletions, or additions to the IgD compared to wild-type or unmodified IgD of the T cell stimulating receptor or the IgSF binding partner of the T cell stimulating receptor. In some aspects, the vIgD contains up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, e.g., amino acid substitutions, deletions, or additions, in the IgD domain of the T cell stimulating receptor or the IgSF binding partner of the T cell stimulating receptor. The modifications (e.g., substitutions) may be present in the IgV domain or the IgC domain. In some embodiments, vIgD has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions) in its IgV domain or its specific binding fragment. In some embodiments, vIgD has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions) in its IgC domain or its specific binding fragment. In some embodiments, vIgD has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with wild-type IgD or unmodified IgD or its specific binding fragment.

[0397] Non-exclusive examples of TIMs in the multi-domain immunomodulatory proteins provided are described in the following subsections. Any of the TIMs described herein may be combined with BIMs described in Section III.A.

[0398] 1. Ligand-binding TIM, e.g., CTLA-4 Immunomodulatory proteins comprising a T cell molecule (TIM) that binds to a ligand of a T cell stimulating receptor are provided herein. In some aspects, the T cell stimulating receptor is CD28, e.g., human CD28, and / or the ligand of the T cell stimulating receptor is CD80 or CD86, e.g., human CD80 or human CD86. In some embodiments, the TIM of the immunomodulatory protein binds to the extracellular portion or ectodomain of CD80 or CD86. In some embodiments, the TIM binds to CD80 or CD86 on the surface of a cell, e.g., on the surface of an APC.

[0399] ...

Claims

1. (1) (i) a T cell stimulating receptor, or (ii) at least one T cell inhibitory molecule (TIM) that binds to a ligand of a T cell stimulating receptor and / or antagonizes the activity of the T cell stimulating receptor; (2) At least one B cell inhibitory molecule (BIM) that binds to a ligand of the B cell stimulating receptor and / or antagonizes the activity of the B cell stimulating receptor Immunomodulatory proteins, including those mentioned above.

2. The immunomodulatory protein according to claim 1, wherein the TIM binds to a ligand of a T cell stimulating receptor.

3. The T cell stimulating receptor is CD28, and / or The ligand for the T cell stimulating receptor is either CD80 or CD86. The immunomodulatory protein according to claim 2.

4. The immunomodulatory protein according to any one of claims 1 to 3, wherein TIM is the extracellular domain of CTLA-4 or its binding portion that binds to CD80 or CD86.

5. The immunomodulatory protein according to claim 4, wherein the extracellular domain of CTLA-4 or its binding portion is (i) the amino acid sequence described in SEQ ID NO:1 or SEQ ID NO:2; (ii) the amino acid sequence of a variant CTLA-4 having at least 85% sequence identity with SEQ ID NO:1 or SEQ ID NO:2; or (iii) a part of (i) or (ii) including an IgV domain.

6. The immunomodulatory protein according to claim 4 or claim 5, wherein the CTLA-4 extracellular domain or its binding portion is described in SEQ ID NO:

1.

7. The immunomodulatory protein according to claim 4 or 5, wherein the CTLA-4 extracellular domain or its binding portion is an amino acid sequence of variant CTLA-4 having at least 85% sequence identity with respect to SEQ ID NO:1, or a portion thereof including an IgV domain, and the variant CTLA-4 sequence comprises one or more amino acid substitutions in SEQ ID NO:1, or a portion thereof including an IgV domain.

8. The immunomodulatory protein according to claim 7, wherein the variant CTLA-4 sequence contains the amino acid substitution C122S.

9. An immunomodulatory protein according to any one of claims 1 to 5, 7, and 8, wherein the CTLA-4 extracellular domain or its binding portion is described in SEQ ID NO:

668.

10. The immunomodulatory protein according to any one of claims 7 to 9, wherein variant CTLA-4 binds to the ectodomains of CD80 and CD86, and optionally, its binding affinity to one or both of CD80 and CD86 is increased compared to the sequence described in SEQ ID NO:1, or a portion thereof including the IgV domain.

11. The immunomodulatory protein according to any one of claims 7-8 and 10, wherein one or more amino acid substitutions include amino acid substitutions selected from L12F, R16H, G29W, T53S, M56T, N58S, L63P, L98Q, or Y105L, or combinations thereof.

12. An immunomodulatory protein according to any one of claims 7-8, 10, and 11, wherein one or more amino acid substitutions include G29W, L98Q, and Y105L.

13. The immunomodulatory protein according to any one of claims 7-8 and 10-12, wherein one or more amino acid substitutions are G29W / N58S / L63P / Q82R / L98Q / Y105L, L12F / R16H / G29W / M56T / L98Q / Y105L, T53S / L63P / L98Q, or G29W / L98Q / Y105L.

14. The immunomodulatory protein according to any one of claims 1 to 8 and 10 to 13, wherein the CTLA-4 extracellular domain or its binding portion is described in any one of SEQ ID NO:92, SEQ ID NO:112, SEQ ID NO:165, or SEQ ID NO:186, or is a part thereof including the IgV domain.

15. The ligand for the B cell stimulating receptor is APRIL or BAFF, and / or The B cell stimulating receptor is either the TACI, BCMA, or BAFF receptor. The immunomodulatory protein according to any one of claims 1 to 14.

16. The immunomodulatory protein according to any one of claims 1 to 15, wherein BIM is a TACI extracellular domain or binding portion that binds to APRIL, BAFF, or a BAFF / APRIL heterotrimer.

17. The immunomodulatory protein according to claim 16, wherein the TACI extracellular domain or its binding portion is an extracellular domain sequence described as (i) the amino acid sequence described as SEQ ID NO:709; (ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO:709; or (iii) a part of (i) or (ii) comprising one or both of the CRD1 domain and the CRD2 domain that bind to APRIL, BAFF, or BAFF / APRIL heterotrimer.

18. The immunomodulatory protein according to claim 16 or claim 17, wherein the TACI extracellular domain or its binding portion comprises a CRD1 domain and a CRD2 domain.

19. The immunomodulatory protein according to any one of claims 16 to 18, wherein the TACI extracellular domain or its binding portion is a cleaved wild-type TACI extracellular domain as described in SEQ ID NO:

516.

20. The immunomodulatory protein according to claim 16 or 17, wherein the TACI extracellular domain or its binding portion is a cleaved wild-type TACI extracellular domain containing cysteine-rich domain 2 (CRD2) but lacking the entirety of cysteine-rich domain 1 (CRD1).

21. The immunomodulatory protein according to claim 16, claim 17, or claim 20, wherein the TACI extracellular domain or its binding portion is a cleaved wild-type TACI extracellular domain consisting of a continuous sequence contained within amino acid residues 67 to 118, including amino acid residues 71 to 104, relative to the position described in SEQ ID NO:

709.

22. The immunomodulatory protein according to claim 20 or claim 21, wherein the cleaved wild-type TACI extracellular domain or its binding portion has an amino acid length of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 59, 50, or 51.

23. An immunomodulatory protein according to any one of claims 16, 17, and 20-22, wherein the TACI extracellular domain or its binding portion is described in SEQ ID NO:

528.

24. The immunomodulatory protein according to claim 16 or claim 17, wherein the TACI extracellular domain or its binding portion is a variant TACI polypeptide comprising one or more amino acid substitutions in the extracellular domain (ECD) or specific binding fragment of a reference TACI polypeptide at a position selected from 40, 59, 60, 61, 74, 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 87, 88, 92, 95, 97, 98, 99, 101, 102, and 103, corresponding to the position numbering described in SEQ ID NO:

709.

25. The immunomodulatory protein according to claim 24, wherein the reference TACI polypeptide is a cleaved polypeptide comprising the extracellular domain of TACI or its specific binding portion that binds to APRIL, BAFF, or a BAFF / APRIL heterotrimer.

26. The immunomodulatory protein according to claim 24 or claim 25, wherein the reference TACI polypeptide comprises the amino acid sequence described in SEQ ID NO:709, or a portion thereof comprising one or both of the CRD1 domain and the CRD2 domain that bind to APRIL, BAFF, or BAFF / APRIL heterotrimer.

27. The immunomodulatory protein according to any one of claims 24 to 26, wherein the reference TACI polypeptide comprises a CRD1 domain and a CRD2 domain.

28. An immunomodulatory protein according to any one of claims 24 to 27, wherein the reference TACI polypeptide is described in SEQ ID NO:

516.

29. The immunomodulatory protein according to any one of claims 24 to 26, wherein the reference TACI polypeptide is a cleaved wild-type TACI extracellular domain containing cysteine-rich domain 2 (CRD2) but lacking the entire cysteine-rich domain 1 (CRD1), and the variant TACI polypeptide comprises one or more amino acid substitutions in the cleaved wild-type TACI extracellular domain.

30. The immunomodulatory protein according to claim 29, wherein the cleaved wild-type TACI extracellular domain consists of a continuous sequence contained within amino acid residues 67 to 118, including amino acid residues 71 to 104, relative to the position described in SEQ ID NO:

122.

31. The immunomodulatory protein according to claim 29 or claim 30, wherein the cleaved wild-type TACI extracellular domain has an amino acid length of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 59, 50, or 51.

32. The immunomodulatory protein according to any one of claims 24-26 and 29-31, wherein the reference TACI polypeptide essentially consists of a CRD2 domain.

33. An immunomodulatory protein according to any one of claims 24-26 and 29-31, wherein the reference TACI polypeptide comprises the sequence described in SEQ ID NO:

528.

34. An immunomodulatory protein according to any one of claims 24-26 and 29-33, wherein the reference TACI polypeptide is described in SEQ ID NO:

528.

35. The immunomodulatory protein according to any one of claims 24 to 34, wherein the variant TACI polypeptide comprises one or more amino acid substitutions at positions selected from 74, 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 87, 88, 92, 95, 97, 98, 99, 101, 102, and 103, corresponding to the numbering described in SEQ ID NO:

709.

36. One or more amino acid substitutions The immunomodulatory protein according to claim 35, or selected from the conservative amino acid substitutions thereof.

37. The immunomodulatory protein according to claim 35 or claim 36, wherein one or more amino acid substitutions include at least one of E74V, K77E, Y79F, L82H, L82P, R84G, R84L, R84Q, D85V, or C86Y.

38. The immunomodulatory protein according to any one of claims 35 to 37, wherein one or more amino acid substitutions are selected from the group consisting of Q75E, K77E, F78Y, R84G, R84Q, A101D, and Y102D, or any combination thereof.

39. One or more amino acid substitutions The immunomodulatory protein according to any one of claims 35 to 38.

40. The immunomodulatory protein according to any one of claims 35 to 39, wherein one or more amino acid substitutions are K77E / F78Y / Y102D, Q75E / R84Q, or R84G.

41. The immunomodulatory protein according to any one of claims 35 to 40, wherein the variant TACI polypeptide has increased binding affinity to one or both of APRIL and BAFF compared to the reference TACI polypeptide.

42. The immunomodulatory protein according to claim 41, wherein the increased binding affinity to BAFF or APRIL is independently increased by more than 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 times.

43. The variant TACI polypeptide contains any one of the sequences described in SEQ ID NO: 517-527, 536, 537, 682-701, or The variant TACI polypeptide contains the sequence described in one of the following SEQ ID NOs: 529-535, 538-550, 673-681, or 760-772. The immunomodulatory protein according to any one of claims 35 to 42.

44. The immunomodulatory protein according to any one of claims 35 to 43, wherein the variant TACI polypeptide is described in any one of SEQ ID NO: 535, SEQ ID NO: 541, SEQ ID NO: 542, or SEQ ID NO:

688.

45. The immunomodulatory protein according to any one of claims 1 to 15, wherein BIM is a BCMA extracellular domain or binding portion that binds to APRIL, BAFF, or a BAFF / APRIL heterotrimer.

46. The immunomodulatory protein according to claim 45, wherein the BCMA extracellular domain or its binding portion is an extracellular domain sequence described as (i) the amino acid sequence described as SEQ ID NO:356; (ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO:356; or (iii) a part of (i) or (ii) including a CRD domain.

47. An immunomodulatory protein according to claim 45 or claim 46, wherein the BCMA polypeptide or its binding site is described in SEQ ID NO:

356.

48. The immunomodulatory protein according to claim 45 or claim 46, wherein the BCMA extracellular domain or its binding portion is a variant BCMA polypeptide comprising one or more amino acid substitutions in the extracellular domain (ECD) of a reference BCMA polypeptide at a position selected from 9, 10, 11, 14, 16, 19, 20, 22, 25, 27, 29, 30, 31, 32, 35, 36, 39, 43, 45, 46, 47, and 48, corresponding to the numbering described in SEQ ID NO:

710.

49. An immunomodulatory protein comprising a variant BCMA polypeptide, wherein the variant BCMA polypeptide comprises one or more amino acid substitutions in the extracellular domain (ECD) of the reference BCMA polypeptide at positions selected from 9, 10, 11, 14, 16, 19, 20, 22, 25, 27, 29, 30, 31, 32, 35, 36, 39, 43, 45, 46, 47, and 48, corresponding to the positional numbering described in SEQ ID NO:

710.

50. An immunomodulatory protein comprising a variant BCMA-Fc fusion protein comprising a variant BCMA polypeptide, an Fc region, and a linker between the BCMA polypeptide and the Fc region, The variant BCMA polypeptide contains one or more amino acid substitutions in the extracellular domain (ECD) of the reference BCMA polypeptide, corresponding to a position selected from among 9, 10, 11, 14, 16, 19, 20, 22, 25, 27, 29, 30, 31, 32, 35, 36, 39, 43, 45, 46, 47, and 48, relative to the position described in SEQ ID NO:

710. Immunomodulatory proteins.

51. The immunomodulatory protein according to any one of claims 48 to 50, wherein the reference BCMA polypeptide is the extracellular domain or binding portion of BCMA that binds to APRIL, BAFF, or a BAFF / APRIL heterotrimer.

52. The immunomodulatory protein according to any one of claims 48 to 51, wherein the reference BCMA lacks N-terminal methionine.

53. The immunomodulatory protein according to any one of claims 48 to 52, wherein the reference BCMA polypeptide comprises the amino acid sequence described in SEQ ID NO:356, or a portion thereof including the CRD domain.

54. An immunomodulatory protein according to any one of claims 48 to 53, wherein the reference BCMA polypeptide is described in SEQ ID NO:

356.

55. One or more amino acid substitutions An immunomodulatory protein according to any one of claims 48 to 54, selected from the conservative amino acid substitutions thereof.

56. The immunomodulatory protein according to any one of claims 48 to 55, wherein one or more amino acid substitutions comprises at least one amino acid substitution selected from H19F, H19K, H19L, H19M, H19R, or H19Y.

57. One or more amino acid substitutions The immunomodulatory protein according to any one of claims 48 to 56.

58. One or more amino acid substitutions is, or An immunomodulatory protein according to any one of claims 48 to 57, comprising:

59. An immunomodulatory protein according to any one of claims 48 to 58, wherein one or more amino acid substitutions are S16A / H19Y / R39Q or contain S16A / H19Y / R39Q.

60. The immunomodulatory protein according to any one of claims 48 to 59, wherein the variant BCMA polypeptide has increased binding affinity to one or both APRIL and BAFF compared to the reference TACI polypeptide.

61. The immunomodulatory protein according to claim 60, wherein the increased binding affinity to BAFF or APRIL is independently increased by more than 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 times.

62. The immunomodulatory protein according to any one of claims 48 to 61, wherein the variant BCMA polypeptide has up to 10 amino acid substitutions compared to the reference BCMA polypeptide.

63. The immunomodulatory protein according to any one of claims 48 to 61, wherein the variant BCMA polypeptide has up to five amino acid substitutions compared to the reference BCMA polypeptide.

64. The immunomodulatory protein according to any one of claims 48 to 63, wherein the variant BCMA polypeptide has at least 90% sequence identity with respect to SEQ ID NO:

356.

65. The immunomodulatory protein according to any one of claims 48 to 64, wherein the variant BCMA polypeptide has at least 95% sequence identity with respect to SEQ ID NO:

356.

66. The immunomodulatory protein according to any one of claims 48 to 65, wherein the variant BCMA polypeptide comprises the sequence described in any one of SEQ ID NO:357 to 435.

67. The immunomodulatory protein according to any one of claims 48 to 66, wherein the variant BCMA polypeptide is described in any one of SEQ ID NO: 357, 377, 380, 381, 390, 391, 396, 402, 405, 406, 407, or 411.

68. An immunomodulatory protein according to any one of claims 49 and 51-67, comprising a heterologous portion linked to a variant BCMA polypeptide.

69. The immunomodulatory protein according to claim 68, wherein the heterogeneous portion is a half-life extension portion, a multimerization domain, a target-directed portion that binds to molecules on the surface of a cell, or a detectable label.

70. The immunomodulatory protein according to claim 69, wherein the half-life extension portion comprises a multimerizing domain, albumin, albumin-binding polypeptide, Pro / Ala / Ser (PAS), the C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), a long unstructured hydrophilic sequence of amino acids (XTEN), hydroxyethyl starch (HES), an albumin-binding small molecule, or a combination thereof.

71. It is a BCMA-Fc fusion protein, The variant BCMA polypeptide is optionally linked to the Fc region of the immunoglobulin via a linker. The immunomodulatory protein according to any one of claims 49 and 51 to 67.

72. The linker contains a peptide linker, and the peptide linker is An immunomodulatory protein according to any one of claims 50 to 71, selected from, or a combination thereof.

73. The immunomodulatory protein according to any one of claims 1 to 48 and 51 to 67, wherein the immunomodulatory protein is a single polypeptide chain comprising at least one TIM and at least one BIM separated by a linker.

74. The immunomodulatory protein according to claim 73, wherein at least one TIM is located amino-terminally relative to at least one BIM within the polypeptide.

75. The immunomodulatory protein according to claim 73, wherein at least one TIM is carboxy-terminal to at least one BIM within the polypeptide.

76. The linker contains a peptide linker, and the peptide linker is An immunomodulatory protein according to any one of claims 73 to 75, selected from, or a combination thereof.

77. An immunomodulatory protein according to any one of claims 1 to 48, 51 to 67, and 73 to 76, comprising an amino acid sequence described in any one of SEQ ID NO: 618 to 623 or 703 to 708, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto and retains activity.

78. The immunomodulatory protein according to any one of claims 1 to 48 and 51 to 67, wherein at least one TIM or at least one BIM is linked to a multimerizing moiety that promotes dimerization, and the immunomodulatory protein is a dimer.

79. The immunomodulatory protein according to claim 78, wherein the multimerization domain is an immunoglobulin Fc region.

80. The immunomodulatory protein according to any one of claims 50 to 72 and 79, wherein the immunoglobulin Fc region is a homodimer Fc region, and the immunomodulatory protein is a homodimer containing two identical copies of the same polypeptide.

81. The immunomodulatory protein according to any one of claims 50 to 72, 79, and 80, wherein the immunoglobulin Fc region is an IgG2 Fc domain, and optionally the IgG2 Fc domain comprises the amino acid sequence described in SEQ ID NO: 726 or 822, or an amino acid sequence exhibiting at least 95% sequence identity to SEQ ID NO: 726 or 822.

82. The immunomodulatory protein according to any one of claims 50 to 72, 79, and 80, wherein the immunoglobulin Fc region is an IgG4 Fc domain containing the amino acid substitution S228P, and optionally, the Fc domain contains the amino acid sequence described in SEQ ID NO: 728 or 823, or an amino acid sequence exhibiting at least 95% sequence identity to SEQ ID NO: 728 or 823.

83. The immunomodulatory protein according to any one of claims 50-72, 79, and 80, wherein the immunoglobulin Fc is an IgG1 Fc domain; or optionally a variant thereof exhibiting reduced binding affinity to the Fc receptor and / or reduced effector function compared to a wild-type IgG1 Fc domain.

84. An immunomodulatory protein according to any one of claims 50-72, 79, 80, and 83, wherein immunoglobulin Fc comprises the amino acid sequence described in SEQ ID NO:

597.

85. The immunomodulatory protein according to any one of claims 50 to 72, 79, 80 and 83, wherein the immunoglobulin Fc is a variant IgG1 Fc domain comprising one or more amino acid substitutions selected from L234A, L234V, L235A, L235E, G237A, S267K, R292C, N297G and V302C according to EU numbering.

86. The immunomodulatory protein according to claim 85, wherein the immunoglobulin Fc region comprises amino acid substitutions L234A, L235E, and G237A according to EU numbering.

87. The immunomodulatory protein according to any one of claims 50 to 72, 79, 80, 83, 85, and 86, wherein the Fc is a variant Fc comprising the amino acid sequence described in SEQ ID NO: 589 or SEQ ID NO:

824.

88. The BCMA-Fc fusion protein has the following structure: BCMA polypeptide (BCMA)-linker-Fc region An immunomodulatory protein according to any one of claims 50-72, 80, and 83-87, comprising:

89. An immunomodulatory protein according to any one of claims 50-72, 80, and 83-88, wherein the BCMA-Fc fusion protein is described in SEQ ID NO:

629.

90. An immunomodulatory BCMA-Fc fusion protein that is a homodimer containing two identical copies of the BCMA-Fc fusion protein described in SEQ ID NO:629, linked by a covalent disulfide bond.

91. The BCMA-Fc fusion protein has the following structure: (BCMA)-linker-Fc region-linker-(BCMA) An immunomodulatory protein according to any one of claims 50-72, 80, and 83-87, comprising:

92. An immunomodulatory protein according to any one of claims 50-72, 80, 83-87, and 91, wherein the BCMA-Fc fusion protein is described in SEQ ID NO:809 or SEQ ID NO:

812.

93. The BCMA-Fc fusion protein has the following structure: (BCMA)-linker-(BCMA)-linker-Fc region An immunomodulatory protein according to any one of claims 50-72, 80, and 83-87, comprising:

94. The BCMA-Fc fusion protein is an immunomodulatory protein according to any one of claims 50-72, 80, 83-87, and 93, as described in SEQ ID NO:

813.

95. The immunomodulatory protein according to any one of claims 50-72, 80, and 83-94, wherein the Fc fusion protein neutralizes APRIL and BAFF.

96. The IC50 for neutralizing APRIL is less than 100 pM, less than 50 pM, less than 40 pM, less than 30 pM, less than 20 pM, less than 10 pM, less than 5 pM, or less than 1 pM, or any value among the above, and / or The IC50 required to neutralize the BAFF is less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 75 pM, less than 50 pM, less than 25 pM, or less than 10 pM, or any value between any of the above. The immunomodulatory protein according to claim 95.

97. The Fc fusion protein blocks the binding of APRIL, BAFF, or APRIL / BAFF heterotrimers to BCMA or TACI, and / or The Fc fusion protein reduces the levels of circulating April, circulating BAFF, or circulating April / BAFF in the target's blood after administration. The immunomodulatory protein according to any one of claims 50 to 72, 80, and 83 to 96.

98. The immunomodulatory protein according to any one of claims 80 to 87, wherein each polypeptide of the homodimer comprises at least one TIM and at least one BIM, and at least one TIM is located at the amino-terminal end relative to at least one BIM within each polypeptide.

99. The immunomodulatory protein according to any one of claims 80 to 87, wherein each polypeptide of the homodimer comprises at least one TIM and at least one BIM, and at least one TIM is carboxy-terminal to at least one BIM within each polypeptide.

100. An immunomodulatory protein according to any one of claims 1 to 44, 78 to 87, 98, and 99, comprising an amino acid sequence described in any one of SEQ ID NO: 610 to 617, 624 to 627, 637, 638, 643, 644, 648, 653, 654, and 759 to 792, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto and retains activity.

101. The immunomodulatory protein according to any one of claims 1-44, 78-87, and 98-100, wherein TIM is the wild-type CTLA-4 extracellular domain or its binding site, and BIM is the TACI extracellular domain or its binding site containing the amino acid substitution K77E / F78Y / Y102D, Q75E / R84Q, or R84G corresponding to the position described in SEQ ID NO:709, and optionally TIM is described in SEQ ID NO:1, and BIM is described in SEQ ID NO:535, 541, 542, or 688.

102. An immunomodulatory protein according to any one of claims 1 to 44, 78 to 87, and 98 to 101, comprising the sequence described in SEQ ID NO:611, SEQ ID NO:788, SEQ ID NO:789, SEQ ID NO:790, or SEQ ID NO:

792.

103. An immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:611, linked by a covalent disulfide bond.

104. An immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:788, linked by a covalent disulfide bond.

105. An immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:789, linked by a covalent disulfide bond.

106. An immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:790, linked by a covalent disulfide bond.

107. An immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:792, linked by a covalent disulfide bond.

108. The immunomodulatory protein according to any one of claims 1-44, 78-87, and 98-100, wherein TIM is a wild-type CTLA-4 extracellular domain or its binding portion, BIM is a cleaved TACI extracellular domain containing a CRD2 domain, and optionally TIM is described in SEQ ID NO:1 and BIM is described in SEQ ID NO:

528.

109. An immunomodulatory protein according to any one of claims 1 to 44, 78 to 87, 98 to 100, and 108, comprising the sequence described in SEQ ID NO: 759, SEQ ID NO: 786, SEQ ID NO: 787, or SEQ ID NO:

791.

110. An immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:759, linked by a covalent disulfide bond.

111. An immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:786, linked by a covalent disulfide bond.

112. An immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:787, linked by a covalent disulfide bond.

113. An immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:791, linked by a covalent disulfide bond.

114. The immunomodulatory protein according to any one of claims 1-44, 78-87, and 98-100, wherein TIM is a CTLA-4 extracellular domain or binding site containing the amino acid substitution G29W / L98Q / Y105L corresponding to the position described in SEQ ID NO:1, and BIM is a TACI extracellular domain or binding site containing the amino acid substitution K77E / F78Y / Y102D, Q75E / R84Q, or R84G corresponding to the position described in SEQ ID NO:709, and optionally TIM is described in SEQ ID NO:186 and BIM is described in SEQ ID NO:535, 541, 542, or 688.

115. An immunomodulatory protein according to any one of claims 1 to 44, 78 to 87, 98 to 100, and 114, comprising the sequence described in SEQ ID NO:

610.

116. An immunomodulatory protein containing two identical copies of the Fc fusion protein described in SEQ ID NO:610, linked by a covalent disulfide bond.

117. An immunomodulatory protein according to any one of claims 1 to 15, 45 to 48, 51 to 67, 78 to 87, 98 and 99, comprising an amino acid sequence described in any one of SEQ ID NO: 601 to 609, 631 to 636, 645 to 647, 649 to 652, or 655 to 659, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto and retains activity.

118. The immunomodulatory protein according to any one of claims 1-15, 45-48, 51-67, 78-87, 98, 99, and 117, wherein TIM is the wild-type CTLA-4 extracellular domain or its binding site, BIM is the BCMA extracellular domain or its binding site containing an amino acid substitution H19L corresponding to the position described in SEQ ID NO:710, optionally TIM is described in SEQ ID NO:1, BIM is described in SEQ ID NO:406, and further optionally the immunomodulatory protein comprises the sequence described in SEQ ID NO:

602.

119. An immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:602, linked by a covalent disulfide bond.

120. The immunomodulatory protein according to any one of claims 1 to 15, 45 to 48, 51 to 67, 78 to 87, 98, 99 and 117, wherein TIM is a CTLA-4 extracellular domain or binding portion containing the amino acid substitution G29W / L98Q / Y105L corresponding to the position described in SEQ ID NO:1, BIM is a BCMA extracellular domain or binding portion containing the amino acid substitution H19L relative to the position described in SEQ ID NO:710, optionally TIM is described in SEQ ID NO:186, BIM is described in SEQ ID NO:406, and further optionally the immunomodulatory protein contains the sequence described in SEQ ID NO:

601.

121. An immunomodulatory protein that is a homodimer containing two identical copies of the Fc fusion protein described in SEQ ID NO:601, linked by a covalent disulfide bond.

122. The immunomodulatory protein according to claim 79, wherein the immunoglobulin Fc region is a heterodimer Fc region, and the immunomodulatory protein is a heterodimer comprising a first and a second polypeptide, wherein the first polypeptide comprises at least one BIM or at least one TIM, and the second polypeptide comprises the other of at least one BIM and at least one TIM.

123. The immunomodulatory protein according to claim 122, wherein the heterodimer Fc comprises one or more amino acid modifications to the wild-type Fc domain to result in heterodimerization between polypeptides, and optionally the wild-type Fc region is an IgG1 Fc region.

124. The immunomodulatory protein according to claim 123, wherein the other amino acid modification is selected from knob-into-hole modifications and charge mutations to reduce or prevent self-association due to charge repulsion.

125. The immunomodulatory protein according to any one of claims 122 to 124, wherein the heterodimeric Fc region further comprises one or more amino acid substitutions for reduced binding affinity to the Fc receptor and / or reduced effector function, optionally compared to the wild-type IgG1 Fc domain.

126. The immunomodulatory protein according to claim 125, wherein one or more amino acid substitutions are selected from L234A, L234V, L235A, L235E, G237A, S267K, R292C, N297G and V302C according to EU numbering.

127. The immunomodulatory protein according to claim 125 or claim 126, wherein the immunoglobulin Fc region comprises amino acid substitutions L234A, L235E, and G237A according to EU numbering.

128. The immunomodulatory protein according to any one of claims 122 to 127, wherein the heterodimer comprises a first polypeptide having the amino acid sequence described in SEQ ID NO: 662 or 663 and a second polypeptide having the amino acid sequence described in SEQ ID NO:

660.

129. Immunomodulatory proteins block the binding of APRIL, BAFF, or APRIL / BAFF heterotrimers to BCMA or TACI, and / or Immunomodulatory proteins reduce the levels of circulating April, circulating BAFF, or circulating April / BAFF in the target's blood after administration. The immunomodulatory protein according to any one of claims 1 to 128.

130. An immunomodulatory protein according to any one of claims 1 to 129, which reduces or inhibits the maturation, differentiation, and / or proliferation of B cells.

131. The immunomodulatory protein blocks the binding of CD80 or CD86 to the costimulatory receptor, and optionally the costimulatory receptor is CD28, and / or Immunomodulatory proteins reduce or inhibit T cell costimulation. An immunomodulatory protein according to any one of claims 1 to 48, 51 to 87, and 98 to 130.

132. A nucleic acid molecule encoding an immunomodulatory protein according to any one of claims 1 to 131.

133. A vector comprising the nucleic acid molecule described in claim 132.

134. The vector according to claim 133, which is an expression vector.

135. The vector according to claim 133 or claim 134, which is a mammalian expression vector or a viral vector.

136. A cell comprising the nucleic acid according to claim 132, or the vector according to any one of claims 133 to 135.

137. A method for producing an immunomodulatory protein, comprising the step of introducing a nucleic acid molecule according to claim 132, or a vector according to any one of claims 133 to 135, into a host cell under conditions that allow the protein to be expressed in the cell.

138. The method according to claim 137, further comprising the step of isolating or purifying immunomodulatory proteins from the cells.

139. An immunomodulatory protein produced by the method described in claim 137 or claim 138.

140. A pharmaceutical composition comprising an immunomodulatory protein according to any one of claims 1 to 131 and 139.

141. The pharmaceutical composition according to claim 140, comprising a pharmaceutically acceptable excipient.

142. A manufactured article comprising a vial or container containing the pharmaceutical composition according to either claim 140 or claim 141.

143. A kit comprising the manufactured article described in claim 142 and an instruction manual.

144. A method for reducing the immune response of a target, comprising the step of administering to a target in need of such reduction an immunomodulatory protein according to any one of claims 1 to 131, or a pharmaceutical composition according to claim 140 or claim 141.

145. The method according to claim 144, wherein the B cell immune response is reduced in the subject, thereby reducing or inhibiting the maturation, differentiation, and / or proliferation of B cells.

146. The method according to claim 144 or claim 145, wherein the circulating levels of APRIL, BAFF, or APRIL / BAFF heterotrimer are reduced in the subject.

147. The method according to any one of claims 144 to 146, wherein the T cell immune response is reduced in the subject, thereby reducing or inhibiting T cell costimulation.

148. The method according to any one of claims 144 to 147, wherein a disease, disorder, or condition in a subject is treated by reducing the immune response.

149. A method for reducing the circulating levels of APRIL, BAFF, or APRIL / BAFF heterotrimer in a subject, comprising the step of administering to the subject an immunomodulatory protein according to any one of claims 1 to 131, or a pharmaceutical composition according to claim 140 or claim 141.

150. A method for treating a disease, disorder, or condition in a subject, comprising the step of administering to a subject in need thereof an immunomodulatory protein according to any one of claims 1 to 131, or a pharmaceutical composition according to claim 140 or claim 141.

151. The method according to claim 148 or claim 150, wherein the disease, disorder or condition is an autoimmune disease, an inflammatory condition, B-cell carcinoma, an antibody-mediated condition, a renal disease, graft rejection, graft-versus-host disease, or a viral infection.

152. The method according to claim 148, claim 150, or claim 151, wherein the disease, disorder, or condition is selected from the group consisting of systemic lupus erythematosus (SLE), Sjögren's syndrome, scleroderma, multiple sclerosis, diabetes mellitus, polymyositis, primary biliary cirrhosis, IgA nephropathy, IgA vasculitis, optic neuritis, amyloidosis, antiphospholipid syndrome (APS), APS II, autoimmune thyroid disease (AITD), Graves' disease, autoimmune adrenalitis, and pemphigus vulgaris.

153. The method according to any one of claims 148 and 150-152, wherein the disease, disorder or condition is B-cell carcinoma, and the cancer is myeloma.

154. A pharmaceutical composition according to claim 140 or claim 141 for use in reducing the immune response of a target.

155. The use of an immunomodulatory protein according to any one of claims 1 to 131, or the use of a pharmaceutical composition according to claim 140 or claim 141, in the manufacture of a pharmacopoeia for reducing the target immune response.

156. A pharmaceutically acceptable composition for use according to claim 154, or for use according to claim 155, wherein the immune response is a B-cell immune response, and reducing the immune response reduces or inhibits the maturation, differentiation, and / or proliferation of B cells.

157. A pharmaceutical composition for use, or use, according to any one of claims 154 to 156, wherein reducing the immune response reduces the circulating levels of APRIL, BAFF, or APRIL / BAFF heterotrimer in the subject.

158. A pharmaceutical composition for use, or use, according to any one of claims 154 to 157, wherein the T cell immune response is reduced in the subject, thereby reducing or inhibiting T cell costimulation.

159. A pharmaceutical composition for use, or use, according to any one of claims 154 to 158, wherein a disease, disorder, or condition in a subject is treated by reducing the immune response.

160. A pharmaceutical composition according to claim 140 or claim 141 for use in treating a disease, disorder, or condition in a subject.

161. The use of an immunomodulatory protein according to any one of claims 1 to 131, or the use of a pharmaceutical composition according to claim 140 or claim 141, in the manufacture of a pharmaceutical for treating a disease, disorder or condition in a subject.

162. A pharmaceutical composition for use according to claim 160, or for use according to claim 161, wherein the disease, disorder or condition is an autoimmune disease, an inflammatory condition, a B-cell cancer, an antibody-mediated condition, a renal disease, graft rejection, graft-versus-host disease, or a viral infection.

163. A pharmaceutical composition for use, or use, according to any one of claims 159 to 162, wherein the disease, disorder, or condition is selected from the group consisting of systemic lupus erythematosus (SLE), Sjögren's syndrome, scleroderma, multiple sclerosis, diabetes mellitus, polymyositis, primary biliary cirrhosis, IgA nephropathy, IgA vasculitis, optic neuritis, amyloidosis, antiphospholipid syndrome (APS), APS II, autoimmune syndrome of polyglandular origin (AITD), Graves' disease, autoimmune adrenalitis, and pemphigus vulgaris.

164. A pharmaceutical composition for use, or use, according to any one of claims 159 to 162, wherein the disease, disorder or condition is B-cell carcinoma, and the cancer is myeloma.

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